Article No. jmbi.1999.3477 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 296, 613±632

Crystal Structures of an N-terminal Fragment fromMoloney Murine Leukemia Virus ReverseTranscriptase Complexed with Nucleic Acid:Functional Implications for Template-primer Bindingto the Fingers Domain

Shabir Najmudin, Marie L. Cote , Dunming Sun, Sarah Yohannan,Sherwin P. Montano, Jun Gu and Millie M. Georgiadis*

Waksman Institute andDepartment of ChemistryRutgers UniversityPiscataway, NJ 08854, USA

Present address: Sarah YohannanChemistry and Biochemistry, MolecInstitute, UCLA, Los Angeles, CA,

Abbreviations used: RT, reverse tMoloney murine leukemia virus; RSvirus; PEG, polyethylene glycol; HIimmunode®ciency virus-type 1.

E-mail addresss of the correspondgeorgiadis@mbcl.rutgers.edu

0022-2836/00/020613±20 $35.00/0

Reverse transcriptase (RT) serves as the replicative polymerase for retro-viruses by using RNA and DNA-directed DNA polymerase activitiescoupled with a ribonuclease H activity to synthesize a double-strandedDNA copy of the single-stranded RNA genome. In an effort to obtaindetailed structural information about nucleic acid interactions with reversetranscriptase, we have determined crystal structures at 2.3 AÊ resolution ofan N-terminal fragment from Moloney murine leukemia virus reverse tran-scriptase complexed to blunt-ended DNA in three distinct lattices. Thisfragment includes the ®ngers and palm domains from Moloney murineleukemia virus reverse transcriptase. We have also determined the crystalstructure at 3.0 AÊ resolution of the fragment complexed to DNA with asingle-stranded template overhang resembling a template-primer substrate.Protein-DNA interactions, which are nearly identical in each of the threelattices, involve four conserved residues in the ®ngers domain, Asp114,Arg116, Asn119 and Gly191. DNA atoms involved in the interactionsinclude the 30-OH group from the primer strand and minor groove baseatoms and sugar atoms from the n ÿ 2 and n ÿ 3 positions of the templatestrand, where n is the template base that would pair with an incomingnucleotide. The single-stranded template overhang adopts two differentconformations in the asymmetric unit interacting with residues in the b4-b5loop (b3-b4 in HIV-1 RT). Our fragment-DNA complexes are distinct frompreviously reported complexes of DNA bound to HIV-1 RT but related inthe types of interactions formed between protein and DNA. In addition,the DNA in all of these complexes is bound in the same cleft of the enzyme.Through site-directed mutagenesis, we have substituted residues that areinvolved in binding DNA in our crystal structures and have characterizedthe resulting enzymes. We now propose that nucleic acid binding to the®ngers domain may play a role in translocation of nucleic acid during pro-cessive DNA synthesis and suggest that our complex may represent anintermediate in this process.

# 2000 Academic Press

Keywords: crystal structure; Moloney murine leukemia virus; reversetranscriptase; protein-DNA complex; processivity

*Corresponding author

, Department ofular BiologyUSA.ranscriptase; MMLV,V, Rous sarcoma

V-1, human

ing author:

Introduction

One of the more interesting and less well-under-stood properties of replicative polymerases is themachine-like property, referred to as processivity,that allows for iterative cycles of nucleotide incor-poration and repositioning of the template-primer

# 2000 Academic Press

614 Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes

substrate in the active site prior to dissociation ofthe template-primer from the enzyme. Reversetranscriptase (RT) is an example of one of the sim-plest replicative enzymes, lacking any associatedcomponents. Through RNA-directed and DNA-directed polymerase activities coupled to a ribonu-clease H activity, RT is able to replicate the retro-viral genome resulting in a double-stranded DNAcopy of the single-stranded RNA genome (Gilboaet al., 1979; Goff, 1990). Mechanisms proposed fortranslocation of template-primer during processiveDNA synthesis include a ratchet-type movementinvolving interactions along a translocation track(Ding et al., 1998; Patel et al., 1995) or with a con-served positively charged area of the ®ngersdomain (Georgiadis et al., 1995). Alternatively,interactions with a minor-groove binding track(Bebenek et al., 1997) have been proposed to con-tribute to translocation of nucleic acid. Details atboth the molecular and biochemical level regardingspeci®c residues and domains that may beinvolved are lacking in our current understandingof translocation of template-primer leading to pro-cessive synthesis of DNA.

RTs are multidomain enzymes including ®ngers,palm, thumb, connection, and ribonuclease(RNase) H domains (Kohlstaedt et al., 1992) thathave variable architectures (Goff, 1990). The Molo-ney murine leukemia virus (MMLV) RT is a mono-meric 75 kDa enzyme as isolated (Moelling, 1974;Roth et al., 1985). The human immunode®ciencyvirus-type 1 (HIV-1) RT contains a p66 subunitincluding all ®ve domains and p51 subunit derivedfrom proteolytic removal of the RNase H domainfrom the p66 subunit (LeGrice, 1993). Similarly, theRous sarcoma virus (RSV) RT is heterodimericwith the smaller 63 kDa subunit resulting fromproteolytic processing of the larger 95 kDa subunit(Leis et al., 1983). Despite architectural differences,the retroviral RTs all perform similar functions(Goff, 1990).

Several crystal structures of DNA polymeraseswith nucleic acid bound in the polymerase activesite have been reported (Doublie et al., 1998; Eomet al., 1996; Huang et al., 1998; Jacobo-Molina et al.,1993; Keifer et al., 1998; Li et al., 1998; Pelletier et al.,1994). These structures have provided details ofthe phosphonucleotidyl transfer reaction, the abil-ity of the enzyme to avoid misincorporation ofnucleotides, and conformational changes that occurduring polymerization. Trapping of a ternary com-plex of nucleic acid and nucleotide with RT wasextremely dif®cult and required cross-linking ofthe substrate to RT in order to obtain a crystalstructure at 3.2 AÊ resolution (Huang et al., 1998).Previously, a report of the structure of HIV-1 RT ina ternary complex with template-primer and Fabprovided the ®rst details of nucleic acid inter-actions with RT in the absence of nucleotide(Jacobo-Molina et al., 1993).

We have obtained complexes of nucleic acidwith an N-terminal fragment from MMLV RT. Thefragment is composed of the ®ngers and palm

domains of MMLV RT and retains polymeraseactivity (Georgiadis et al., 1995). Complexes havebeen crystallized with two different fragment mol-ecules and three different DNA duplexes. Here, wereport three crystal structures of the fragmentbound to blunt-ended DNA duplexes at 2.3 AÊ res-olution and the crystal structure at 3.0 AÊ resolutionfor the fragment complexed to a DNA duplex witha single-stranded overhang mimicking a template-primer. A detailed structural analysis of our com-plexes and comparison to previously reportednucleic acid complexes with HIV-1 RT is presentedhere. In addition, we report mutational studies inthe full-length MMLV RT designed to determinethe role of nucleic acid binding to the ®ngersdomain as seen in our complexes and to provideinsights regarding processive DNA synthesis byRT.

Results

Structure determinations

The structures of the N-terminal fragment ofMMLV RT complexed with blunt-ended DNAduplexes have been determined in three distinctcrystal lattices, form I, form IIa, and form IV (seeTable 1). We previously reported preliminarycharacterizations of form III crystals (Sun et al.,1998), but have not pursued the structure determi-nation of this form for which we have only 4 AÊ

data. Form I, IIa, and IIb crystals are monoclinic,space group P21. Form IV crytals are orthorhombic,in space group P21212. Both form I and IIa crystalsinclude a complex of the fragment with an 8/8-mer DNA duplex. The fragment used in form Icrystals was obtained by limited proteolysis withtrypsin and comprises residues 10-278, while thatused in form II crystals was obtained by bacterialexpression and comprises residues 24-278. Form IVcrystals include a complex of the bacteriallyexpressed fragment and a 6/10-mer, which forms apseudo-hexadecamer DNA duplex. The structureswere determined by an iterative molecular replace-ment procedure starting with the uncomplexedfragment as the search model aided by experimen-tal phasing obtained for form IIa crystals (see theMaterials and Methods). Forms I, IIa and IV crystalstructures have been re®ned to 2.3 AÊ resolution,and re®nement statistics are shown in Table 1.

In order to assess interactions with a single-stranded template overhang, the oligonucleotide(50-CATGCATG-30) found in forms I and IIacrystals was modi®ed to (50-TTTCATGCATG-30)including single-stranded overhangs that mimic atemplate-primer substrate. Thus, in a secondexample of the form II lattice (form IIb), the bac-terially expressed fragment complexed with (50-TTTCATGCATG-30) results in an 8/8-mer duplexwith two single-stranded template overhangs(TTT). This structure has been re®ned to 3.0 AÊ

resolution.

Table 1. Summary of crystallographic and re®nement data

Cell parameters Form I Form IIa Form IIb Form IV

a (AÊ ) 61.14 65.87 66.16 54.74b (AÊ ) 38.43 63.59 63.54 145.49c (AÊ ) 129.75 73.40 73.50 46.74b (�) 100.58 102.91 103.85 -Space group P21 P21 P21 P21212

StatisticsMaximum resolution (AÊ ) 2.3 2.3 3.0 2.3Reflections (unique) 24,281 26,120 11,540 17,117

(total) 74,572 91,393 35,570 32,141Completeness (%) 90.0(84.0) 98.7(97.1) 95.7(92.2) 99.0(98.5)Rsym (%) 4.7 (13.4) 4.8 (17.4) 14.9 (31.4) 6.9 (26.4)I/s 18.3 21.3 5.95 22.8

RefinementResolution range (AÊ ) 20-2.3 20-2.3 20-3.0 20-2.3Rvalue (%) 23.3 20.0 21.5 23.0Rfree (%) 29.6 25.9 29.8 28.6Non-hydrogen atoms 4388 4450 4570 2540Water molecules 281 406 0 175

rmsd from ideal valuesBond lengths (AÊ ) 0.006 0.008 0.011 0.008Bond angles (deg.) 1.32 1.64 1.86 1.29Dihedrals (deg.) 23.39 24.14 24.46 23.81Improper torsions (deg.) 1.19 1.92 1.94 1.13

Ramachandran plot statistics (%/#)Residues in most-favored regions 88.2/374 90.4/394 78.0/340 88.4/190Residues in additional allowed regions 11.3/48 9.2/40 21.6/94 10.2/22Residues in generously allowed regions 0.0/0 0.0/0 0.0/0 0.9/2Residues in disallowed regions 0.5/2 0.5/2 0.5/2 0.5/1

Average B-factor (AÊ 2)Molecule A (all atoms/main-chain) 31.6/30.7 28.5/27.3 19.0/18.6 38.4/37.6Molecule B (all atoms/main-chain) 54.6/53.9 40.4/39.4 28.5/28.5 -DNA: strand 1 (all atoms) 46.6 43.0 44.4 59.8

: strand 2 (all atoms) 51.0 55.9 52.2 68.5

Rsym � {�j(I ÿ hIi)j}/{�(I)}, where hIi refers to the average intensity of multiple measurements of the same re¯ection. Rvalue andRfree � (�jFobs ÿ Fcalcj)/(�jFobsj). Rfree was calculated over 5 % of the amplitudes not used in re®nement. R-values are reported for alldata. Ramachandran plot statistics calculated by PROCHECK (Laskowski, 1993). # Refers to the number of residues in each regionof the Ramachandran plot. Values in parentheses are for the highest resolution shells, 2.38-2.30 AÊ for form I and form IV, 2.34-2.30 AÊ for form IIa, and 3.11-3.00 AÊ for form IIb.

Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes 615

Comparison and description of the structures

Forms I and IIa crystals include two fragmentmolecules (referred to as A and B) and a single 8/8-mer DNA duplex (50-CATGCATG-30) in theasymmetric unit (see Figure 1). Each fragment inthe asymmetric unit provides a binding site for theoligonucleotide. Conversely, the oligonucleotide isitself symmetric and binds to each of the fragmentmolecules in a nearly symmetric manner. As dis-cussed above, form IIb crystals include a modi®ed8/8-mer oligonucleotide resulting in two single-stranded overhangs. Form IV crystals include onefragment molecule and one 6/10-mer (6-mer 50-CTCGTG-30, 10-mer 50-ACGGCACGAG-30) in theasymmetric unit. However, a blunt-ended hexade-camer results in which two 6/10-mers are relatedby crystallographic symmetry and form two G �Amispairs (see Figure 1). Details regarding the G �Amispairs will be discussed elsewhere.

In all crystal structures with two protein mol-ecules in the asymmetric unit (forms I, IIa and IIb),one fragment molecule designated as the A mol-

ecule is better ordered than the other B moleculebased on electron density and overall thermal par-ameters (see Table 1). Residues 10-23, 104-106 and275-278 are completely disordered in both A and Bmolecules of form I. Weak density is seen for boththe N and C termini of form II. The a-helix aA isextended at the N terminus for both molecule A(from residue SerA21) and molecule B (residueHisB22). At the C terminus, residues 276-278 forman extra b-strand extending the end b-sheet foundin the fragment model in form I crystals. We haveused the same secondary structural assignment asfor the uncomplexed fragment (Georgiadis et al.,1995). For form IV, residues 101-108 and 173-178displayed weak electron density. Otherwise, thismodel is quite similar to that found in form II crys-tals. In all four crystal structures of fragment-DNAcomplexes (forms I, IIa, IIb and IV), the averageB-factors for strand 1 (template strand) of the DNAare slightly lower than the average B-factors forstrand 2 (primer strand) as shown in Table 1, pre-sumably due to more extensive interactions withthe protein.

Figure 1. Overall structures of forms IIa, IIb, I, and IV MMLV RT fragment complexed with DNA. All views pre-sent ribbon diagrams for the protein molecules and stick models for the DNA molecules. The proteins are shownwith green b-strands, off-white coils, and yellow a-helices, with the exception of the aD helix, which is shown inwhite. Two MMLV RT fragment molecules and one bound DNA molecule in the asymmetric units of form (a) IIa, (b)IIb, and (c) I are shown in addition to a second symmetry-equivalent DNA molecule (red stick ®gures). (d) The formIV MMLV RT fragment with its bound DNA and the (ÿx ÿ 1, ÿy, z) symmetry-related species, forming a pseudo-hexadecamer DNA molecule (depicted in both red and yellow stick models for emphasis). Tyr64, Asp114, Leu115,Arg116, and Gly191, ®ve principal residues comprising the DNA binding site, are emphasized with dark ball-and-stick models. Note that two conformations of Tyr64 in form IV shown in (d). Figures 1, 2(a), 3, 4(b), 5, and 7 weregenerated using MOLSCRIPT (Kraulis, 1991) and rendered with RASTER3D (Merritt & Bacon, 1997).

616 Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes

Superpositioning of each fragment moleculefrom the complexed (forms I, IIa, IIb and IV) andthe uncomplexed fragment crystal structuresresults in pair-wise rmsd ranging from 0.2-0.7 AÊ

for the best de®ned 160 Ca atomic positions (seeFigure 2). Overall, the fragment structures arequite similar. The largest differences correspond tothe regions in the fragment with the largest

B-factors as seen in Figure 2, including areas onthe surface (particularly in the loop regions) andthe N and C termini where the electron density isweak. The b4-b5 loop (residues 101-109), bestde®ned in molecule A of form IIa, shows the great-est variation. The b-strand (b6) and a-helix (aE)spanning the surface from the ®ngers to the palmdomain (residues 126-140) also show a greater

Figure 2. Comparison of the pro-tein molecules of the different crys-tal forms of MMLV RT fragment.(a) Superpositioning of the frag-ment molecules was done using O(Jones et al., 1991) and was basedupon the overall 160 best-de®nedCa atoms (i.e. for residues 27-61,78-97, 112-125, 147-171, 182-212,218-230 and 235-256). Molecules Aand B of form I are shown in aquaand yellow, respectively, as tracerenderings. Molecules A and B ofform IIa are shown in white andpale green, respectively. Dark bluerepresents form IV, and the uncom-plexed MMLV RT fragment ismagenta. The highly variable b4-b5loop is labeled, as well as the mod-erately variable b8-b9 loop. Theregion labeled 126-142 also variesamong the forms and is thus noted.The amino and carboxy termini aredesignated with N and C, respect-ively. (b) A B-factor plot for main-chain protein atoms, emphasizingthe more disordered regions in theprotein, is shown. The plot is forthe A molecule of form IIa, but ishighly representative of all of thecrystal forms.

Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes 617

¯exibility in the complexed structures. In theuncomplexed monomeric structure, this region iswell ordered and is involved in crystal packinginteractions. A second region in the ®ngers domainincluding residues 172-181 (b8-b9) also has variableconformations in the different structures.

Interactions of the fragment with theDNA duplexes

In form I, IIa, and IV crystal structures, ®ve inde-pendent fragment molecules are complexed toblunt-ended DNA (see Figure 1). In each case,

618 Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes

DNA is bound to the ®ngers domain of the frag-ment as shown in Figure 3. The DNA molecules inform I, IIa, and IIb crystals do not deviate signi®-cantly from B-form DNA in either local or globalparameters as analyzed by 3DNA (Lu & Olson,1999) or CURVES (Lavery & Sklenar, 1997). How-ever, the DNA in the form IV structure does havesome A-like character in the region of interactionswith the protein. The structure of this DNA mol-ecule will be described in detail elsewhere. Asdetailed in Table 2, binding of the fragment to theDNA involves minor groove base atoms N3 andO2 from the n ÿ 2 and n ÿ 3 positions of the tem-plate strand and the 30-OH group from the primerstrand (n denotes the template base that will pairwith an incoming nucleotide). Minor groove baseatoms N3 and O2 are found in every base-pair insimilar positions, as previously noted (Seemanet al., 1976). There are additional hydrogen-bond-

Table 2. Interactions of the N-terminal fragment with DNA

A. DNA sequencesForms I and IIa

Form IIb

Form IV

B. Interactions with duplex DNAProtein atom DNA Atom IA

Y64 OH A2 O40 (T2 O40)(C1 O2)(C1 O50)

P65 O C1 O50Q113 O G8 O30 (G16 O30) 3.76D114 Od1 G8 O40

G8 N3 (G16 N3) 3.36(G16 O30)

Od2 G8 N3 (G16 N3) 3.88G8 N2 (G16 N2) 3.37

L115 N G8 O30 (G16 O30) 2.96R116 N G8 O30 (G16 O30) 3.68Ne (G16 O40)NZ1 T3 O40 (C3 O40) 2.82

A2 N3 3.67T3 O2 (C3 O2) 3.25

(T2 O2)NZ2 T3 O40 (C3 O40) 3.14

A2 N3 (T2 O2) 2.91G8 N3 (G16 N3) 3.92G8 N2 (G16 N2) 3.50

N119 Nd2 (G16 O30)K120 Nz G4 O1P 3.50

G4 O2PG191 O G8 O30 (G16 O30) 3.21

C. Interactions with single-stranded overhangs in form IIb crystalsProtein atom DNA atom IIbA

S67 Og T3* N3 2.61T3* O4 2.45

E69 Oe1 T3* O4 3.43P100 O T3* O2K102 O T2* O40 3.91

T2* N1 3.76Nz T3* O4

Y109 OH T3* O2P 3.90

Bases noted in parentheses are for the form IV crystal structure, Alarly in C.

ing interactions between the fragment and O40sugar atoms and phosphate oxygen atoms. Thus,binding of DNA to the ®ngers domain binding siteinvolves the ®rst three base-pairs in all three crys-tal lattices. Very similar interactions with thedouble-stranded portion of the DNA are found inform IIb crystals in which there are single-strandedoverhangs. Residues in the ®ngers domain thatplay a role in the binding interactions includeAsp114, Arg116, Asn119 and Gly191. All of theseresidues are highly conserved as shown insequence alignments in Figure 4. The residueGly191 was previously identi®ed as belonging tomotif B and is found in all polymerases (Poch et al.,1989). The Asp114, Arg116 and Asn119 residuesare found in region 2 in alignments of RT-relatedpolymerase sequences (Xiong & Eickbush, 1990).

Four hydrogen bonds between the fragment andDNA are found in all ®ve binding sites involving

50-CATGCATG-3030-GTACGTAC-5050-TTTCATGCATG-3030-GTACGTACTTT-5050-CTCGTG-3030-GAGCACGGCA-50

IB IIaA IIaB IV3.24 3.55 3.70 3.62

2.992.72

2.953.57 3.59 3.42 3.733.912.99 3.63 3.52

3.983.55 3.59

3.97 2.77 3.152.87 2.58 2.77 2.823.73 3.48 3.55

3.992.88 2.89 2.93 3.653.63 3.87 3.363.42 3.42 3.36 3.22

3.553.18 3.80 3.59 3.712.85 2.54 2.64 2.79

3.57 3.95 3.403.38 3.46 3.09 3.06

3.912.89

3.193.02 3.46 3.26 3.32

IIbB

3.32

3.24

and B refer to the A and B molecules in forms I and IIa, simi-

Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes 619

side-chain atoms of Arg116 (of the aD helix) andmain-chain atoms of Leu115 and Gly191 (of thetype II b-turn at b9aH) as shown in Figure 3. Informs I and II, bases involved in interactionsinclude G8 at the 30 end of the primer strand, andA2 and T3 in n ÿ 2 and n ÿ 3 positions, respect-ively, from the template strand. The two strands ofthe 8/8-mer are numbered identically. In form IVcrystals, the bases involved in interactions with thefragment include G16 at the 30 end of the primerand T2 and C3 in the n ÿ 2 and n ÿ 3 positions ofthe template strand. The 6/10-mer has been num-bered starting with the 50 end of the 6-mer andcontinuing with the 50 end of 10-mer as A7 to the30 end as G16. (The selection of primer versus tem-plate strand as stated above is based on the formIIb crystal structure with the single-stranded over-hangs). Two hydrogen bonds are formed betweenthe 30-OH group (O30) from G8 (or G16 in form IV)and the main-chain N atom of Leu115 and O atomof Gly191. The other two hydrogen bonds involveArg116 side-chain atoms. The NZ1 atoms of Arg116form hydrogen bonds with O2 from T3 (or C3),and NZ2 atoms form a hydrogen bond with N3 ofA2 (or O2 of T2).

Additional interactions found in all of the ®ngersdomain binding sites involve residues Asp114,Arg116, Asn119 and Gly191, which as previouslynoted are conserved in all retroviral RTs (seeFigure 4). The equivalent residues in HIV-1 RT areAsp76, Arg78, Asn81 and Gly152, respectively. Inour structures, Asp114 positions Arg116 and formsan ion-pair involving NZ2 and Ne atoms of Arg116.In addition, Asp114 forms a hydrogen bond withthe main-chain N of Arg116. Thus, Asp114 appearsto play an important structural role in positioningArg116 in our complexes. The Nd2 atom of Asn119hydrogen bonds to the main-chain O atom ofGly191 and appears to play a critical structuralrole in positioning Gly191.

There are additional hydrogen bonds formed insome but not all of the interacting fragment-DNAmolecules (see Table 2). Each fragment-DNA inter-action involves a total of at least seven hydrogenbonds, including the four mentioned above thatare conserved in all binding sites. For example, inform I crystals Arg116 NZ2 atoms from both A andB molecules hydrogen bond to the O40 atoms of T3in addition to the interaction with N3 of A2. Inform IIa and form IV crystals, the same Arg116NZ2 atoms also hydrogen bond to N2 atoms of G8(or G16 in form IV). Asp114 Od1 forms a hydrogenbond with N3 of G8 in form I crystals, while Od2

forms a hydrogen bond with N2 of G8 (or G16) tothe form I A molecule, form IIa B molecule, andform IV molecule. For the B molecules of bothform I and IIa crystals, there is a hydrogen bondformed between either G4 O1P or G4 O2P with Nz

of Lys120. In addition, Tyr64 OH in the B moleculeof form I forms a hydrogen bond to A2 O40 but inother fragment molecules is hydrogen bondedeither directly or via a bridging water molecule toAsp114. The main-chain O atom of Pro65 in the B

molecule of form I crystals hydrogen bonds to C1O50. There are also non-bonded contacts to theDNA involving residues Leu99, Met66, andGln113.

The residues involved in the interactions withDNA are located in structurally conserved regionsof the fragment (see Figures 1 and 2). In fact, theside-chain conformations of Asp114, Arg116, andGly191 are quite similar in the complex structuresas shown in Figure 5 in which models of the frag-ment have been superimposed based on alpha-car-bon atoms. The conformations of Asp114 andArg116 from form IV and form I fragment mol-ecules are nearly identical. In form II crystals, theconformations of the same residues are again simi-lar but not identical to those found in form IV.Interestingly, Asp114 is not hydrogen-bonded toArg116 in the uncomplexed fragment structure(Georgiadis et al., 1995). The hydrogen-bonding ofanalogous residues Asp76 to Arg78 is similar tothat seen in our complexes for the HIV-1 RT-DNA-TTP structure (Huang et al., 1998) and the uncom-plexed structure of HIV-1 RT (Hsiou et al., 1996)but not in the HIV-1 RT-DNA-Fab structure (Dinget al., 1998; Jacobo-Molina et al., 1993) or the HIV-1RT-nevirapine structure (Ren et al., 1995).

Interactions with the single-stranded overhang

In form IIb crystals, two three-base single-stranded overhangs are present in different confor-mations in the asymmetric unit as shown inFigure 3. Each single-stranded overhang mimics atemplate overhang found in a template-primer sub-strate. Hydrogen-bonding interactions of eachoverhang with A and B molecules in the asym-metric unit are detailed in Table 2. There are threehydrogen bonds formed between the A moleculeand the single-stranded overhang involving resi-dues Ser67 and Glu69 and two longer range inter-actions with Lys102. In contrast, the B moleculeforms two potential hydrogen bonds with Pro100main-chain and Lys102 and one longer contactwith Tyr109. Hydrogen-bonding interactions withthe protein are limited to the ®rst base of the over-hang (T3*), corresponding to the n position, forboth overhangs and involve N3, O4 and O2 atoms.Note that T3* in form IIb is not equivalent to T3discussed in the n ÿ 3 position from form I andform IIa. In form IIb, T6 is equivalent to T3 informs I and IIa.

As expected, the very ¯exible single-strandedoverhangs make different contacts with the A andB molecules that appear to be dictated by the con-formation of the nearby b4-b5 loop in each mol-ecule. As illustrated in Figure 2, the conformationof the b4-b5 loop is quite variable in the differentcrystal structures. The single-stranded overhanginteracting with the A molecule folds back on itselfand forms a single hydrogen bond between then � 1 and n ÿ 1 bases of the template, while thatassociated with the B molecule adopts a moreextended conformation.

Figure 3 (legend opposite)

620 Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes

Figure 3. Stereodiagrams of DNA bound to the ®ngers domain of the N-terminal fragment of MMLV RT in formIIa and form IIb crystal structures. The DNA is shown with red stick models. White broken lines indicate hydrogenbonds ranging from 2.3-3.4 AÊ . (a) A close-up of the hydrogen bonding interactions in the ®ngers domain binding siteof the A molecule of form IIa. Shown are the speci®c contacts to the DNA made by Arg116, the ion-pair formedbetween Asp114 and Arg116, the hydrogen bond between Leu115 N and the 30-OH group of G8, the hydrogen bondbetween Gly191 O and the same 30-OH group of G8, the hydrogen bond between Tyr64 OH group and Asp114 Od2

atom, and the hydrogen bond between Asp114 Od1 and Arg116 N atom. (b) Similar contacts for the A molecule ofform IIb are shown with the addition of contacts from Ser67 Og2 to N3 and O4 of T3*. A black line is drawn from theGly191 label to its main-chain O atom. (c) The single-stranded overhang from the B molecule of form IIb is shownwith the DNA-binding region of the ®ngers domain. The ion-pair between Asp114 and Arg116 is not shown in (c),since the distance from Od1 to Ne is 3.5 AÊ . The alternate conformation of the single-stranded overhang is apparent inthat T3* makes no contacts with Ser67; however, its O4 atom makes a 3.2 AÊ hydrogen bond with Nz of Lys102. (b) Ablack line is drawn from the Gly191 label to its main-chain O atom. (d) A diagram of the hydrogen bonding inter-actions of the protein and DNA atoms is shown for the form IIa model with the 8/8-mer DNA. Short dashes andlong dashes indicate interactions between atoms that are 2.4-3.4 AÊ or 3.5-3.8 AÊ apart, respectively.

Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes 621

Comparison to DNA complexes with HIV-1 RT

We have compared our structures to the tworeported structures of HIV-1 RT complexed withDNA, (1rtd and 2hmi) (Ding et al., 1998; Huanget al., 1998). The relative positions of the DNA withrespect to the active site differ slightly for the twoHIV-1 RT complexes. The position of DNA boundto the fragment is signi®cantly different from DNAbound to HIV-1 RT involving a translation ofapproximately 4-5 AÊ to superimpose the nearestphosphorous coordinates. Thus, the DNA boundto the fragment is farther from the catalyticallyrequired Asp residues in the polymerase active siteand closer to several highly conserved residuessuch as Arg116 (78 in HIV-1 RT) in the ®ngersdomain as shown in Figure 6(c) and (d). Inaddition, we have superimposed the ®ngers andpalm domains of the fragment of MMLV RT, HIV-

1 RT (1rtd) (Huang et al., 1998), and the palmdomain of Taq polymerase (1tau) (Eom et al., 1996)and then compared the positions of the DNA mol-ecules bound to each polymerase shown inFigure 6(a) and (b). The relative positions of theprimer 30 ends and the single-strand overhangs areshown in Figure 6(a) and (b), respectively. The 30end of the primer bound to the fragment and thatbound to Taq DNA polymerase is shifted awayfrom the position seen in the HIV-1 RT-DNA-TTP(1rtd) structure where the nucleotide is trapped inthe polymerase active site. Although the DNA mol-ecules differ in structure and sequence, they arebound similarly to the enzymes in occupying acleft found between the ®ngers, palm, and thumbdomains and thus superimpose approximately asshown in Figure 6(b). DNA bound to the ®ngersdomain of the MMLV RT fragment is quite easily

Figure 4 (legend opposite)

622 Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes

accommodated in the DNA binding cleft pre-viously observed in the HIV-1 RT-DNA-Fab struc-ture (Ding et al., 1998; Jacobo-Molina et al., 1993) asshown in Figure 7. The position of the DNA boundto the MMLV RT fragment would allow it to inter-act with the tip of the thumb as modeled inFigure 7(a). The closest interactions of our DNAwith the HIV-1 RT thumb include atoms from theterminal guanine of the template strand with resi-dues 287 and 254, with only two very close con-

tacts that would easily be accommodated by aslight movement of the thumb. In a similar modelof our complex superimposed with the nevirapinebound structure (1vrt) (Ren et al., 1995) of HIV-1RT, the closest point of contact between our DNAand the thumb is approximately 6 AÊ .

A comparison of the atomic interactions betweenthe ®ngers domain of MMLV RT or equivalentresidues in HIV-1 RT and DNA is shown inTable 3. This analysis is based on a comparison

Table 3. Comparative nucleic acid interactions with the ®ngers domain of MMLV RT and HIV-1 RT

MMLV DNA Distance 1rtd DNA Distance 2hmi DNA Distance

Y64 OH A2 O40 3.24 W24 Cz3 C4 O2P 2.91P65 O C1 O50 2.95 P 25 O G3 N2 3.12L99 Cd2 G8 N2 2.94 F 61 Cz A5 C8 3.59

K65 Nz TTP O2a 2.99TTP O3g 2.67

R72 NZ2 TTP O3a 2.67TTP O2a 3.02TTP O50 3.28

Q113 O G16 O30 3.42 V75 O A5 O40 3.51D114 Od1 G8 O40 3.91 D76 Od1 A5 O40 3.54 D76 Od1 T802 O40 3.73

G8 N3 2.99Od2 G8 N2 3.15

G8 N3 3.55L115 N G8 O30 2.58R116 N G8 O30 3.48 R78 NZ1 A5 O2P 3.81Ne G16 O40 3.99 Cg A5 O30 3.08NZ1 C3 O2 3.22

T3 O40 2.82NZ2 A2 N3 2.54

T3 O40 3.14N119 Nz G16 O30 3.91 N81 Nd2 C6 C50 3.41K120 Nz G4 O1P 2.89

V111 O TTP O2b 3.07D113 N TTP O3b 3.06

TTP O1g 3.60A114 N TTP O2b 3.21Y115 N TTP O30 3.00

OH G22 N2 3.80Q151 Oe1 TTP O30 3.14

TTP O1b 3.56G191 O G8 O30 3.02 G152 O C6 O40 3.20 G152 O T802 O40 3.93

N A5 N3 3.57 N A801 O40 3.35K154 Nz C7 O2P 3.65 K154 Nz G22 N2 3.80

A compilation of DNA interactions with the N-terminal fragment of MMLV RT from Table 2 is included here with the shortestdistances between protein and DNA atoms seen in the different crystalline complexes. The 1rtd refers to the HIV-1RT-DNA-TTPstructure (Huang et al., 1998) and 2hmi refers to the HIV-1 RT-DNA-Fab structure (Ding et al., 1998).

Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes 623

with the HIV-1 RT-DNA-Fab structure (2hmi) andthe HIV-1 RT-DNA-TTP structure (1rtd). There aretwo regions in the ®ngers domain that interactwith nucleic acid substrates (see Table 3). One

Figure 4. Sequence alignments of retroviral reverse transdomain that bind DNA in the complexes with the N-termiwere done using GCG for 251 residues from the ®ngers andwith the Moloney murine leukemia virus RT varied from 29RT. Residues 112-121 and 187-196 (numbering is from MMLVserved residues are boxed and include Asp114, Arg116, Asinclude Asp76, Arg78, Asn81 and Gly152. Box colors correcodes from the SwissProt database all include the pre®x POan abbreviation for the viral source. The viral sources includbon ape leukemia virus; HV1B1, human immunode®ciency vciency virus type 1 (YU-2 isolate); HV2KR, human immundendogenous virus (strain M7); FENV1, feline endogenous viSMRVH, squirrel monkey retrovirus; IPMA, mouse intracisteLK3; RSVP, Rouse sarcoma virus strain Prague C; BIV27, bovMason-P®zer virus; SRV2, simian retrovirus; JSRV, sheepvirus (Japanese isolate BLV-1); SIVM1, simian immunode®cianemia virus (isolate Syoming); FIVSD, feline imunode®ciencphalitis virus; FOAMV, human spumaretrovirus; OMVVS ov(strain 1514). (b) A ribbon rendering is shown highlighting thbonding interactions from the structure of the A molecule frblue, and Asn119 and Gly191 in gray.

includes ®ngertip residues in the vicinity of Arg72(Arg110 in MMLV RT), which hydrogen bond tothe incoming nucleotide TTP in the HIV-1 RT-DNA-TTP structure. The second region includes

criptases including conserved residues from the ®ngersnal fragment from MMLV RT. (a) Sequence alignments

palm domains of the different RTs. Sequence identities% for HIV-1 RT to 77 % for gibbon ape leukemia virusRT) are shown for 25 different retroviral RTs. The con-

n119 and Gly191. Analogous residues in the HIV-1 RTspond to stick model colors used in (b). The sequenceL for the retroviral pol gene and an extension, which ise MLVMO, Moloney murine leukemia virus; GALV, gib-irus type 1 (BH10 isolate); HV1Y2, human immunode®-ode®ciency virus type 2 (isolate KR); BAEVM, baboon

rus ECE1; HTLV2, human T-cell leukemia virus type II;rnal A-particle; SFV3L, simian foamy virus type 3/strainine immunode®ciency virus (isolate 127); MPMV, simian

pulmonary adenomatosis virus; BLVJ, bovine leukemiaency virus (MM142-83 isolate); EIAVY, equine infectiousy virus (isolate San Diego); CAEV, caprine arthritis ence-ine lentivirus (strain SA-OMVV); VILV1, Visna lentiviruse conserved residues in stick models with the hydrogen-

om form IIa crystals. Asp114 is shown in red, Arg116 in

Figure 5. Superpositionings of the higher-resolutionstructures at the DNA binding site. In both views thebackgrounded DNA molecule is that of form IV, andthe ion-pair formed between Asp114 and Arg116 inform IV is shown with black dashes. Both views alsoshow smaller bonds and atoms for the dual confor-mations of Tyr64 of form IV. Superpositionings weredone using the same subset of alpha-carbon atoms listedfor Figure 2. (a) The superpositioning of the A and Bprotein molecules of form I onto that of form IV. Themain-chains and side-chains nearly superimpose withthe exception of the main-chain of the form I B moleculein the region of Tyr64. (b) Superpositioning of the Aand B protein molecules of form IIa onto that of formIV. The main and side-chain superpositionings arenearly identical for the residues shown, and there is anexact mapping of the Asp114 side-chain of the form IIaA molecule and that of form IV.

624 Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes

residues in the vicinity of Arg78 (Arg116), whichinteract with the template-primer extensively in

our complexes and to a much lesser extent in theHIV-1 RT-DNA-TTP structure. Signi®cantly fewerinteractions between the ®ngers domain and theDNA in the HIV-1 RT-DNA-Fab structure are pre-sent than in the related HIV-1 RT-DNA-TTP struc-ture despite similar positioning of the DNA nearthe polymerase active site, possibly due to theaddition of nucleotide in the latter complex. Thepotential to hydrogen bond to the template-primerappears to be conserved based on the structuralcomparison for the following residues: Pro65,Gln113, Asp114, Arg116 and Gly191 (25, 75, 76, 78,and 152 in HIV-1 RT). Of these residues, Pro65,Asp114, Arg116 and Gly191 are highly conserved.The hydrogen-bonding interaction with Gln113(V75) involves the main-chain.

However, the details of the interactions betweenequivalent residues from MMLV RT and HIV-1 RTand DNA differ. The Asp76 Od1 is potentiallyhydrogen bonded to the O40 sugar atom in bothHIV-1 RT-DNA complex structures, while Asp114(MMLV RT) Od1 or Od2 atoms are hydrogenbonded to N3 or N2 minor groove base atoms in atleast one of the ®ve binding sites in our structures.The most striking example is Arg116, which formsfour hydrogen bonds to DNA in some of our struc-tures and two hydrogen bonds to DNA in all ofour structures. The equivalent residue, Arg78, iswithin contact distance (3.81 AÊ ) but not hydrogen-bonding distance of the phosphate backbone forthe n � 1 template base in the HIV-1 RT-DNA-TTPstructure but has no DNA interactions in the HIV-1 RT-DNA-Fab structure. The Gly191 hydrogenbonds to the 30-OH group of the primer strand inour complexes while the equivalent residue inHIV-1 RT, Gly152, hydrogen bonds to O40 sugaratoms in both HIV-1 RT-DNA structures. Thehighly conserved residue Asn119 (Asn81 in HIV-1RT) does not directly interact with nucleic acidbut does hydrogen bond to Gly191(Gly152) inour structures. This hydrogen-bonding interactionis also found in the HIV-1 RT-DNA-Fab structureand potentially in the HIV-1 RT-DNA-TTPstructure.

Site-directed mutagenesis studies

Based on a detailed comparison of the DNAinteractions with Asp114 (76) and Arg116 (78)shown in Table 3, we sought to distinguish bindingof DNA to the ®ngers domain observed in ourstructures from binding in the polymerase activesite observed in the HIV-1 RT structures. Bindingof DNA to the fragment is critically dependent oninteractions with the highly conserved residuesAsp114 and Arg116 in the ®ngers domain. Bindingof DNA in the polymerase active site of HIV-1 RTdoes not appear to depend critically on interactionswith Asp76 and Arg78, involving one possiblehydrogen bond and one longer range interactionwith the DNA, respectively. Based on hydrogen-bonding interactions, we might predict that substi-tution of Arg116 with a Lys residue would not

Figure 6. Comparison of DNA bound to the MMLV RT fragment, HIV-1 RT, and Taq polymerase. SuperimposedDNA molecules bound to the fragment from MMLV RT (form IIb), HIV-1 RT (1rtd), and Taq DNA polymerase (1tau)were rendered in Ribbons (Carson, 1997). Superimpositioning of the palm domains including residues 145-152,196-212, 217-231 and 233-249 from MMLV RT and equivalent residues from HIV-1 RT and Taq polymerase was donein O (Jones et al., 1991) to obtain the positions of the DNA molecules shown in white for 1rtd, red for 1tau, andgreen for the form IIb fragment structure. (a) A direct comparison is shown of the positions of the 30 ends of the pri-mers in each DNA molecule. The 30 end of the template-primer from 1rtd has been trapped in the polymerase activesite poised for a phosphonucleotidyl transfer reaction and is therefore bound to the palm domain. The 30 ends of tem-plate-primers bound in 1tau and in form IIb are captured in the absence of nucleotide and may represent alternatebinding sites for template-primer on the respective polymerases. (b) A view rotated approximately 90 � from that in(a), the differing conformations of the single-stranded overhangs from form IIb (green) and 1rtd (white) are shown aswell as the relative positions of each template-primer. A comparison of template-primer bound to the polymeraseactive site of (c) HIV-1 RT and the ®ngers domain binding site of (d) the N-terminal fragment of MMLV RT. (c) and(d) The electrostatic potential surfaces created in GRASP (Nicholls et al., 1991) of the ®ngers and palm domains fromthe HIV-1 RT-DNA-TTP crystal structure (1rtd) and form IIb MMLV RT fragment structure are shown, respectively,in approximately equivalent orientations. A stick rendering of the bound template-primer is shown in green for theHIV-1 RT structure and gray for the fragment structure. The relative positions of the template-primer and single-stranded overhangs are shown with respect to Arg116 in MMLV RT or Arg78 in HIV-1 RT and the conserved cataly-tic Asp residues of the polymerase active site (PAS). The conformation of the ®ngers domain near the polymeraseactive site in HIV-1 RT is different from that in MMLV RT due to the presence of nucleotide.

Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes 625

seriously impair the ability of RT to form the com-plexes with DNA seen in the HIV-1 RT-DNA com-plex structures (1rtd or 2hmi), which are thoughtto represent the molecule poised for catalysis in thepolymerase active site. However, the same substi-tution should dramatically affect the ability ofDNA to bind to the ®ngers domain site observedin our structures. A similar argument should applyto substitution of Asp114 with a Asn residue,which retains the ability to form a hydrogen bond.Substitution of Arg116 with an Ala residue in thefragment molecule results in loss of DNA bindingin a crystalline complex. Although the R116A frag-ment was preincubated with DNA prior to crystal-lization in order to allow for complex formation,the DNA was not bound to the protein and is not

present in the resultant crystal structure deter-mined at 2.3 AÊ resolution (R.C. & M.M.G., unpub-lished results).

In order to assess the functional signi®cance oftemplate-primer interactions with the ®ngersdomain, we have introduced the following aminoacid substitutions in the full-length MMLV RT:K103A, R110A, D114N, R116 K, R116L,D114NR116 K (DNRK), E117A, and N119A. Sub-stitution of Arg116 with an Ala residue results inan enzyme that behaves very similarly to theR116L enzyme (J.G. et al., unpublished results).The substitutions have been introduced into anN-terminally truncated construct pRT24, expressedin Escherichia coli, and puri®ed. RT24 retains wild-type activity (data not shown). The K103 and R110

Figure 7. Stereodiagrams of DNA bound to the ®ngers domain of the MMLV RT fragment as modeled in the pre-viously de®ned binding cleft in HIV-1 RT. (a) A trace rendering shows the fragment of MMLV RT in blue includingthe ®ngers and palm domains superimposed on the ®ngers, palm, and thumb domains from HIV-1 RT (2hmistructure) (Ding et al., 1998). DNA as bound to the ®ngers domain in form IIb crystals is shown as a stick model inred. The superpositioning of the ®ngers and palm domains from MMLV RT and HIV-1 RT is based on the 160 mostsimilar residues as reported by Georgiadis et al. (1995) and listed in the legend in Figure 2. (b) The same moleculesare superimposed as in (a). The DNA shown in red from the HIV-1 RT-DNA-Fab complex structure (2hmi) is shownfor comparison in a similar view along the cleft formed by the ®ngers, palm, and thumb domains.

626 Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes

are equivalent to K65 and R72 in HIV-1 RT, whichtogether form ®ve hydrogen bonds with theincoming nucleotide. E117A was selected as a con-trol for these studies as it is proximal to Arg116but not involved in DNA binding in any of thestructures. The Asn119 as noted above wouldappear to play a key structural role in positioningGly191, which hydrogen bonds to the primer30-OH group in our structures. The remaining sub-stitutions involve residues 114, 116 or both. Thesubstituted enzymes have been puri®ed andcharacterized using RT assays. The other highlyconserved residue involved in binding DNA in ourcrystal structures is Gly191. We have not madesubstitutions for Gly191 because it would not bepossible to distinguish structural from functionalconsequences.

Homopolymeric assays

In characterizing the substituted enzymes, wehave determined speci®c activities in standardassays using poly(rA)-oligo(dT) as the template-primer. In addition, we have analyzed the lengthof the products synthesized by the substitutedenzymes using poly(rA)-oligo(dT) as the template-primer. The speci®c activities of the substitutedenzymes are shown in Table 4. Substitution ofD114, R116, E117 or N119 results in a modestdecrease in speci®c activity retaining 40-70 % ofwild-type activity. Consistent with a previousreport in which R78 K in HIV-1 RT retained 50 %of wild-type polymerase activity in standardassays, R116K retains 70 % of wild-type activity(Boyer et al., 1992). Substitution of K103 or R110

Table 4. Enzymatic characterizations of substitutedenzymes

Poly(rA) template mRNA template

Substitutionspecific activity

(%)full-length products

(%)

RT24 100 100K103A 9.5 0R110A 1.9 0D114N 58.2 17-25R116 K 70.0 0.7-6D114NR116 K 62.1 0.5-6R116L 40.9 0E117A 73.3 110-120N119A 51.1 0

Speci®c activities for the poly(rA) template assay are given asa percentage of wild-type activity (see Materials and Methods)RT24 has the same speci®c activity as the wild-type enzyme.Activities for the mRNA template assay are given as a percen-tage of wild-type full-length products based on integratedvolumes. The range speci®ed indicates the range observed forthree different time points (see Materials and Methods).

Figure 8. Comparison of the enzymatic properties ofsubstituted MMLV RT enzymes using a mRNA tem-plate assay. An analysis of products for the mRNA tem-plate assay by gel electrophoresis is shown for RT24,D114N, R116K, DNRK (D114N R116K) and E117Aenzymes. The conservative substitutions of Asn forAsp114 or Lys for Arg116 affect the abilities of the sub-stituted enzymes to synthesize full-length products. Thewedges indicate increments in time of 15, 30 and 60minutes for each of the three lanes for products separ-ated on a 2 % agarose gel and visualized followingexposure and processing on a Molecular Dynamicsphosphorimager. Size markers are 1 kb and 50 bp lad-ders in lanes 1 and 2, respectively. Full-length productsranging from 600-650 bases from the gel are shown. Thefull-length products appear to be slightly shorter atlonger time points potentially due to degradation of themRNA template over time.

Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes 627

results in a signi®cant decrease in speci®c activity(10 % and 2 % of wild-type activity, respectively) asexpected. R116K and E117A enzymes have similarspeci®c activities in the standard assay retainingapproximately 70 % of wild-type activity, whereasR116L retains only 41 % of wild-type activity.Based on analysis of the products from the stan-dard assay by gel electrophoresis (see the Materialsand Methods), wild-type, RT24, or E117A enzymesare similar. Products made by D114N, R116K,D114NR116K, R116L, and N119A enzymes weresigni®cantly shorter than wild-type. In addition,these enzymes synthesized qualitatively fewer pro-ducts in the separation range of the agarose gels(results not shown). No discernible long productswere observed for K103A and R110A enzymes.Further product analysis has been done using amRNA template, which should require the substi-tuted enzymes to perform tasks that are more simi-lar to those required in vivo during replication thanthose required in the homopolymeric assay.

Heteropolymeric assays

Using rabbit globin mRNA-oligo(dT) as the tem-plate-primer in assays, products synthesized by thesubstituted enzymes were analyzed. RT24 andE117A enzymes accumulated full-length products(600/650) over time at approximately the samerate as shown in Figure 8. Accumulation of full-length products by D114N, R116K, andD114NR116K enzymes was signi®cantly reducedin comparison to RT24 or E117A. Accumulation offull-length products by D114N was maximally26 % that of RT24, while R116K and DNRK accu-mulated maximally 6 %. A similar result wasobtained using different enzyme concentrationsinstead of time points. Thus, although both R116Kand E117A enzymes retain approximately 70 % ofwild-type activity in standard activity assays, the

R116K enzyme is unable to ef®ciently synthesizeDNA using mRNA as the template. K103A,R110A, R116L, and N119A enzymes made no dis-cernible full-length or other long products in thisassay.

There does not appear to be a direct relationshipbetween the speci®c activities measured in thestandard assay and the relative effectiveness of thesubstituted enzymes to synthesize DNA using amRNA template for enzymes that retain at least40 % of wild-type activity in the standard assay.However, K103A and R110A enzymes, whichretain less than 10 % of wild-type activity in thestandard assay, are also unable to polymerize anydetectable long products using mRNA as a tem-plate. Analysis of the products from the standardassay and the mRNA template assay by gel electro-phoresis is correlated for all the enzymes. All ofthe enzymes that make shorter products than thewild-type enzyme are also unable to make full-length products using mRNA as a template.

Discussion

In an effort to address the biological relevance ofDNA complexes with the fragment from MMLV

628 Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes

RT, we have determined crystal structures in threedistinct lattices with three different oligonucleo-tides. In addition, we have introduced speci®camino acid substitutions for residues involved inbinding nucleic acid in the full-length enzyme andcharacterized the substituted enzymes. Although itis possible that DNA has bound adventitiously tothe fragment in our complexes, it does not seemlikely given the crystallographic and biochemicalresults presented here. Key features of a biologi-cally relevant nucleic acid binding site for RTinclude interactions that are possible for anysequence and that would allow the enzyme toreplicate the retroviral genome. As detailed above,DNA interactions with the fragment involve minorgroove base atoms and sugar OH atoms thatwould be possible for any nucleic acid sequence. Inaddition, the binding site must accommodate atemplate-primer substrate with a single-strandedtemplate overhang. In form IIb crystals, a single-stranded overhang interacts with the b4-b5 loop ofeach fragment molecule in the asymmetric unit.The DNA-fragment interactions with the ®rst threebase-pairs of the duplex remain very similar tothose found in the other crystal structures. Thus,we conclude that the ®ngers domain binding site isnot speci®c for blunt-ended DNA and is able toaccommodate a single-stranded template over-hang. The binding site must also accommodateDNA-DNA as well as RNA-DNA template-primersubstrates. Modeling studies replacing either DNAstrand of the 8/8-mer with RNA show that the ®n-gers domain binding site will accommodate RNAor RNA-DNA hybrids equally well. As shown inFigure 7, DNA bound to the ®ngers domain of theMMLV RT fragment is accommodated in the samecleft de®ned previously in the HIV-1 RT-DNA-Fabcomplex (Ding et al., 1998; Jacobo-Molina et al.,1993) between the ®ngers, palm, and thumbdomains. If the thumb in fact adopts the differentconformations seen in the nevirapine-bound struc-ture (Ren et al., 1995) versus the template-primer-bound structure (Ding et al., 1998; Huang et al.,1998; Jacobo-Molina et al., 1993) during processivesynthesis, the ®ngers domain binding site is quiteattractive as an intermediate binding site thatwould allow the thumb to reposition itself on theDNA. Therefore, based on the crystallographicanalysis, we conclude that the ®ngers domainbinding site for nucleic acid satis®es these criteriafor a biologically relevant binding site in reversetranscriptase.

As noted earlier, trapping of a catalytic complexof HIV-1 RT was accomplished by cross-linkingthe template-primer substrate to the thumbdomain. Unlike other polymerases including ratpol b, phage T7 DNA polymerase, and a largefragment of Taq DNA polymerase I, it has not beenpossible to trap the substrate in the polymeraseactive site of RT simply by providing ddNTPs forincorporation. A possible explanation is that RThas multiple binding sites or modes of bindingnucleic acid that are equally favored especially at

concentrations required for crystallization. Byusing the fragment for our structural studies, wehave eliminated a substantial portion of the DNAbinding site seen in the complexes with HIV-1 RTand have obtained a complex with DNA bound tothe ®ngers domain. We reported that the structureof the uncomplexed fragment was similar to thecorresponding domains in RT structures and con-cluded that truncation of the molecule had notresulted in signi®cant conformational changes inthe ®ngers and palm domains (Georgiadis et al.,1995). We have established in crystallographicexperiments that substitution of Arg116 for an Alaresidue in the fragment results in loss of DNAbinding for the fragment. In addition, we havenow characterized substituted full-length MMLVRT enzymes with the goal of elucidating possiblefunctional roles for template-primer binding to the®ngers domain as seen in our crystal structures.

The substituted enzymes fall into two distinctgroups based on their properties as de®ned in thestandard assay and a mRNA template assay,which are consistent with the structural infor-mation that is currently available. The ®rst groupincludes K103A and R110A, which are unable tosynthesize effectively using either poly(rA) ormRNA as a template. K103 in MMLV RT was pre-viously implicated in binding nucleotide based onsite-directed mutagenesis studies (Basu et al., 1990).Substitution of R110 in MMLV RT or R72 in HIV-1RT has also been previously reported to signi®-cantly reduce activity, and this residue had beenproposed to be involved in binding nucleotide(Chowdhury et al., 1996; Sara®anos et al., 1995). Inthe HIV-1 RT-DNA-TTP (1rtd) structure, K65forms two hydrogen bonds, and R72 forms threehydrogen bonds to the incoming nucleotide. Thus,both of these residues would appear to be crucialfor nucleotide binding.

The second group includes D114N, R116L,R116K, DNRK and N119A, which appear to retainthe ability to synthesize moderately well usingpoly(rA) as the template and much less well usingmRNA as the template. The inability of these sub-stituted enzymes to synthesize effectively from amRNA template suggests that binding of template-primer to the ®ngers domain as observed in ourcrystal structures plays a functional role in polym-erization of DNA by reverse transcriptase. Wehave shown that enzymes with similar reductionsin speci®c activity (E117A and R116K) when com-pared to wild-type are not equally competent atDNA synthesis using a de®ned template such asmRNA. Therefore, the substitutions for D114, R116or N119 are not equivalent to substitution of E117.The de®ned template assays have been performedwith nearly equal enzyme and template-primerconcentrations, which allow the wild-type enzymeor the E117A enzyme to accumulate full-lengthproducts quite effectively. We would not predictthat substitution of D114 or R116 would affect theability of the enzyme to position the template-pri-mer in the polymerase active site. Thus, the bind-

Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes 629

ing of template-primer to the ®ngers domain playssome other functional role. Poly(rA) and mRNAtemplates differ signi®cantly in that mRNA canform loop structures and even tertiary structureswhile poly(rA) cannot. One possible function oftemplate-primer binding to the ®ngers domainmay include melting out loop structures in thetemplate-primer. Other possible roles for the ®n-gers domain binding site may include the initialbinding of template-primer prior to positioningin the polymerase active site, unbinding of thetemplate-primer from the polymerase active sitefollowing an as yet unde®ned number of incorpor-ations, or a role in the mechanism of processiveDNA synthesis. Analysis of the crystal structuresand mutational data is consistent with any of theaforementioned functional roles.

A role in processive synthesis might include amechanistic step involving release from the poly-merase active site following incorporation of adNMP and subsequent trapping of the template-primer in the ®ngers domain binding site prior tothe next phosphonucleotidyl transfer reactionin the polymerase active site. In order to repositionthe template-primer in the polymerase activesite, the enzyme must release the substrate follow-ing an incorporation of nucleotide. Using a ratchet-type of movement between the two binding sites,the enzyme would be able to translocate the tem-plate-primer during processive synthesis. Thismodel would help explain the apparent speci®cityfor the 30-OH group of the primer strand andminor groove of the template strand in our ®ngersdomain binding site. The enzyme might use thisbinding site to keep track of the 30-OH group ofthe primer, which must be precisely repositionedfor the next reaction. We further suggest thatrelease of the template-primer from this site mightbe triggered by the incoming nucleotide. Thesingle-stranded template overhangs interact withthe b4-b5 loop, which include residues K103 andR110. The equivalent residues in HIV-1 RT, K65and R72, interact with the incoming nucleotide inthe HIV-1 RT-DNA-TTP structure. Thus, we mayhave trapped an intermediate in processive DNAsynthesis by RT in our crystal structures.

Materials and Methods

Crystallization

The crystallization and initial characterizations ofmonoclinic forms I and IIa crystals, belonging to spacegroup P21, have been reported (Sun et al., 1998). The bac-terially expressed fragment and oligonucleotides used toobtain form IV crystals were puri®ed as described (Sunet al., 1998). The form IV crystals were obtained using a1:2:8 molar ratio of fragment: 6/10-mer oligonucleotides:ddCTP from 10 % (v/v) polyethylene glycol (PEG) 4000,0.10 M NaCl, and 0.05 M ADA (pH 6.5) as detailed else-where. form IIb crystals were obtained by microseedingwith form IIa crystalline seeds. The complex used forcrystallization included a 1:4 molar ratio of fragment tooligonucleotide (50-TTTCATGCATG-30). The precipitant

included 15 % PEG 4000, 0.1 M NH4Cl, and 0.1 M Hepes(pH 7.5). Drop and reservoir volumes were 2 ml and500 ml, respectively, in vapor diffusion hanging drops.

Determination of the structures

MAD data were collected using a cross-linked form Icrystal derivatized with mercuric acetate forl(1) � 1.0093 AÊ , l(2) � 1.0063 AÊ , and l(3) � 0.99587 AÊ atthe National Synchrotron Light Source, HHMI beamlineX4A, Brookhaven National Labs. Data were recorded onFuji HRIII phosphor imaging plates and digitized usinga Fuji BAS 2000 scanner. Phasing from this experimentproved inadequate due to low occupancy of Hg sites.Data for l(1) were ultimately used for the structuredetermination and re®nement. Details of the data collec-tion have been previously reported for form II crystals inaddition to integration and scaling of data for both formI and IIa crystal forms (Sun et al., 1998). Phasing for cryo-cooled form I crystals was initially obtained by molecu-lar replacement using the uncomplexed fragment modelas the search model. All molecular replacement calcu-lations were done using AMoRe (Navaza, 1994) includ-ing data from 8-4 AÊ resolution. Initial maps for form Iindicated that signi®cant rebuilding would be requiredin order to improve the phasing. We therefore deter-mined the room temperature structure of form I crystalsat 2.8 AÊ resolution ®rst. As expected, initial phasing forthe room temperature form I crystals was signi®cantlybetter than for the cryocooled crystals. (The uncom-plexed fragment structure was determined at room tem-perature). Several cycles of rebuilding in O (Jones et al.,1991) and re®nement with X-PLOR (BruÈ nger, 1992) weredone to improve the model of the fragment complexedto DNA. It was clear at this stage that density for one ofthe two fragment molecules in the asymmetric unit wassigni®cantly better than that for the other one. The bet-ter-de®ned room temperature fragment model was thenused as the search model for molecular replacement, andphasing was improved suf®ciently to interpret the den-sity for the DNA.

We later obtained form IIa crystals and were able tosuccessfully derivatize these crystals with mercuric acet-ate to complement MR phasing. Crystals were soaked ina stabilizing solution including 2 mM mercuric acetateovernight, and data were collected at 108 K on an R-axisIV image plate area detector with source X-rays gener-ated by a Rigaku RU 200 rotating anode and focusedwith the MSC mirrors. The overall phasing power foracentric data to 2.5 AÊ was 1.8. Derivative phasing wasnot necessary but did prove helpful in interpretation ofelectron density for loop regions in the protein, whichdiffered from the search model. The molecular replace-ment problem was solved using the A molecule fromform I as a search model. Electron density for the DNAwas readily interpretable using MR phasing.

Form IIb crystals and form IV crystals were cryo-cooled at 108 K using an Oxford Cryocool System. Datawere collected on an R-axis IV image plate detector withCu Ka radiation, processed with HKL, and scaled withSCALEPACK (Otwinowski, 1993). Form IV crystals werestabilized in 20 % (v/v) ethylene glycol, 16 % PEG 4000,0.1 M NaCl, and 0.05 M ADA (pH 6.5) for cryocooling.Form IIb crystals were stabilized in 20 % (w/v) glycerol,16 % PEG 4000, 0.1 M Hepes (pH 7.5), 0.1 M NH4Cl, and0.05 M NaCl. A molecular replacement solution wasobtained for form IV crystals using the ``A'' fragmentmolecule of the form IIa model as the search model.

630 Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes

Model building and refinement

Model re®nement for both forms I and IIa was carriedout initially with X-PLOR version 3.851 (BruÈ nger, 1992).All models were re®ned using REFMAC (Murshudovet al., 1997) as implemented in CCP4 (CCP4, 1994) andCNS (BruÈ nger et al., 1998) interspersed with modelrebuilding using O (Jones et al., 1991). Models of stan-dard B-form DNA for the 8/8-mer and 6/10-mer DNAduplexes were created using INSIGHTII (1993) andmanually positioned in the Fo ÿ Fc difference electrondensity maps and appropriately rebuilt. Individual B-fac-tor and positional re®nement was performed using allthe data to 2.3 AÊ resolution for forms I, IIa and IV inREFMAC with a bulk solvent and an overall anisotropiccorrection applied. Several models with possible alter-nate conformations in the N and C termini, and in resi-dues 64-69, 101-109, 128-138 and 174-178 of each chainwere re®ned simultaneously.

Form I crystals were derivatized with mercuric acet-ate. Four mercury atoms bound to the sulfur atoms ofCys90 and 157 of the A and B chains were included inthe ®nal model for form I with partial occupancies. Inaddition, Cys236 and Cys262 were de®ned as strainedleft-handed spiral disul®de bridges in both chains. Thereis a large positive peak in the jFo ÿ Fcj maps between thetwo sulfur atoms in both form I molecules. This may rep-resent a partially occupied Hg site. It is possible tomodel both sulfur atoms in alternate conformations, butwe have not done so in this case.

Re®nements of forms I, IIa and IV models were ulti-mately completed using CNS (BruÈ nger et al., 1998) fromwhich improved electron density maps as well as re®ne-ment statistics were obtained (see Table 1). Simulatedannealing procedures were used in addition to positionaland individual B-factor re®nement. The ®nal rounds ofre®nement and rebuilding involved checking modelswith PROCHECK (Laskowski et al., 1993), correctinggeometries, adding new waters manually, and deletingunlikely water molecules.

The starting model for form IIb was the re®ned formIIa model including the 8/8-mer DNA but stripped ofwater molecules. The initial re®nement was done usingREFMAC (Murshudov et al., 1997). Rigid body re®ne-ment was carried out treating molecule A and B andeach DNA strand as a rigid body to allow for differencesin the cell parameters between forms IIa and IIb. Sub-sequent re®nement was carried out with CNS usingsimulated annealing, minimization, and grouped B-factorprocedures. Initial electron density for the single-stranded overhangs was broken requiring several roundsof re®nement and remodeling leading to signi®cantimprovement. The single-stranded overhangs wereextended one base per round of re®nement. IndividualB-factor re®nement was carried out in the ®nal rounds,and the models were checked with PROCHECK(Laskowski et al., 1993). The ®nal model from CNS hasbeen re®ned to 3.0 AÊ resolution. No water moleculeshave been included in the model.

Construction of mutants

Mutations resulting in substitutions in MMLV RThave been made in an N-terminally truncated constructpRT24, which lacks the N-terminal 23 amino acid resi-dues. Construction of pRT24 and E117A will be dis-cussed in detail elsewhere. The resulting RT24 enzymewith wild-type sequence exhibited native behavior in allreverse transcriptase assays that were done. Site-directed

mutagenesis was done using the QuikChange mutagen-esis kit (Stratagene). The desired mutations were ®rstintroduced into a plasmid encoding the ®ngers and palmdomains of MMLV RT residues 24-278 cloned intopET15b (Novagen) (Sun et al., 1998). Substitutionsincluding D114N, R116K, and D114NR116K in MMLVRT were made using appropriate oligonucleotides syn-thesized using an Applied Biosystems 392 DNA/RNAsynthesizer and purifed by gel elecrophoresis prior touse. Plasmids were sequenced and those with the correctsequences were digested with NdeI and MfeI. Plasmidsencoding substitutions for K103A, R110A, R116L andN119A (kindly provided by J. Pfeiffer and A. Telesnits-ky) were digested with KpnI and SalI. The inserts fromthe digestions were ligated into pRT24, and clones weresequenced to verify the mutations.

Expression and purification of RT24 andsubstituted enzymes

BL21 (DE3) cells were used for overexpression of theenzymes. From one liter cultures, pellets were suspendedin 50 mM NaH2PO4/Na2HPO4 (pH 7.8) buffer contain-ing 2 mM n-decyl-b-D-maltopyranoside (Calbiochem),5 % glycerol, 0.3 M NaCl, and 10 mM imidazole. Cellswere lysed using two passes through a French pressurecell at 1000 lb/in2. Lysates were then centrifuged at35,000 rpm for 30 minutes, and crude extracts were thenapplied to 1 ml Ni-NTA (Qiagen) columns, washed, andeluted using standard Qiagen protocols. The partiallypuri®ed samples were then applied to a MonoS column(Pharmacia) following dilution in 50 mM Hepes (pH 7.5),1 mM EDTA, 1 mM DTT, 5 % glycerol and 2 mM n-decyl-b-D-maltopyranoside and eluted by NaCl gradientfractionation. The hexa-His af®nity tag was removedfrom each enzyme following treatment with Thrombin(Novagen). Samples were then reapplied to a MonoS col-umn and fractionated as above, concentrated in Amiconcentricons, and stored at ÿ80 �C. By SDS-PAGE analysis,the samples were homogeneous.

Polymerase assays

Standard reverse transcriptase assays using poly(rA)template and oligo(dT) primer were described (Rothet al., 1985). Enzymes were diluted in 50 mM Hepes(pH 7.5), 1 mM DTT, 1 mM EDTA, 0.2 M NaCl, 5 % gly-cerol and 2 mM n-decyl-b-D-maltopyranoside. Speci®cactivities were determined from duplicate samples ofthree different concentrations of RT (0.13, 0.4, 0.8 nM)within the linear range of the assay. Standard poly(rA)oligo(dT) assays including 0.44, 1.3 or 4 nM RT werealso done in order to compare the lengths of productssynthesized by the substituted enzymes. A unit ofactivity is de®ned as nanomoles of dTTP incorporated at37 �C in 15 minutes. Protein concentrations were deter-mined using the Micro BCA assay from Pierce. Assaysusing rabbit globin mRNA as the template included thefollowing: 50 mM Tris (pH 8.3), 4 mM Hepes (pH 7.5),21 mM DTT, 20 mM dNTPs, 2 mg/ml mRNA, 75 mMNaCl, 0.05 % (v/v) NP40, 0.16 mM n-decyl-b-D-malto-pyranoside, 0.4 % glycerol, 80 nM EDTA, 1.0 mM MgCl2,1 mg/ml oligo dT, 20 mCi of [32P]dATP, and 4.4, 13 or40 nM reverse transcriptase. The rabbit globin mRNAincludes transcripts 600 and 650 bases in length. Reac-tions were stopped by adding SDS and placed on iceprior to electrophoresis. Markers were end-labeled using0.04 unit of T4 polynucleotide kinase and 0.02 mCi of

Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes 631

[g-32P]ATP per 1.5 mg of markers. Products were separ-ated on a 2 % (w/v) agarose gel after 15, 30 and 60 min-ute incubations with 40 nM enzyme at 37 �C or 60minute incubations and visualized following exposureand processing on a Molecular Dynamics phosphorima-ger. Poly(rA) template and oligo(dT) primers wereobtained from Amersham Pharmacia Biotech, rabbit glo-bin mRNA template, 1 kb and 50 bp markers fromGIBCO BRL, and radioactive nucleotides from NENDupont.

Coordinate deposition

Coordinates for form I, form IIa, form IIb and form IVcrystal structures have been deposited in the ProteinData Bank, with accession numbers 1qai, 1qaj, 1d0e, and1d1u, respectively.

Acknowledgments

We thank Wayne Hendrickson (Columbia University),Monica Roth (UMDNJ), Helen Berman and Eddy Arnoldfor helpful discussions, Richard Ebright for critical read-ing of the manuscript, Tax Georgiadis for proofreadingof the manuscript, Julie Pfeiffer and Alice Telesnitsky (U.Mich. Medical School) for mutant proviral constructs,Attilio De Falco for general computing assistance, andDick Leidich and Greg Listner for technical support ofX-ray equipment. This work was supported by a grantGM55026 (M.M.G.) from the National Institutes ofHealth.

References

Basu, A., Basu, S. & Modak, M. J. (1990). Site-directedmutagensis of Moloney murine leukemia virusreverse transcriptase: demonstration of lysine 103 inthe nucleotide binding site. J. Biol. Chem. 265, 17162-17166.

Bebenek, K., Beard, W. A., Darden, T. A., Li, L., Prasad,R., Luxon, B. A., Gorenstein, D. G., Wilson, S. H. &Kunkel, T. A. (1997). A minor groove binding trackin reverse transcriptase. Nature Struct. Biol. 4, 194-197.

Boyer, P. L., Ferris, A. L. & Hughes, S. H. (1992). Cas-sette mutagenesis of the reverse transcriptase ofhuman immunode®ciency virus type 1. J. Virol. 66,1031-1039.

BruÈ nger, A. T. (1992). X-PLOR, Version 3.1: A System forX-Ray Crystallography and NMR, Yale UniversityPress, New Haven, CT.

BruÈ nger, A. T., Adams, P. A., Clore, G. M., Gros, P.,Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J.,Nilges, N., Pannu, N. S., Read, R. J., Rice, L. M.,Simonson, T. & Warren, G. L. (1998). Crystallogra-phy and NMR system (CNS): a new software sys-tem for macromolecular structure determination.Acta Crystallog. sect. D, 54, 905-921.

Carson, M. (1997). Ribbons. Methods Enzymol. 277, 493-505.

CCP4 (1994). The CCP4 suite: programs for protein crys-tallography. Acta Crystallog. sect. D, 50, 760-763.

Chowdhury, K., Kaushik, N., Pandey, V. & Modak, M. J.(1996). Elucidation of the role of Arg110 of murineleukemia virus reverse transcriptase in the catalytic

mechanism: biochemical characterization of itsmutant enzymes. Biochemistry, 35, 16610-16620.

Ding, J., Das, K., Hsiou, Y., Sara®anos, S. G., Clark,A. D., Jr, Jacobo-Molina, A., Tantillo, C., Hughes,S. H. & Arnold, E. (1998). Structure and functionalimplications of the polymerase active site region ina complex of HIV-1 RT with a double-strandedDNA template-primer and an antibody fab frag-ment at 2.8 AÊ resolution. J. Mol. Biol. 284, 1095-1111.

DoublieÂ, S., Tabor, S., Long, A. M., Richardson, C. C. &Ellenberger, T. (1998). Crystal structure of a bac-teriophage T7 DNA replication complex at 2.2 AÊ

resolution. Nature, 391, 251-258.Eom, S. H., Wang, J. & Steitz, T. A. (1996). Structure of

Taq polymerase with DNA at the polymerase activesite. Nature, 382, 278-281.

Georgiadis, M. M., Jessen, S. M., Ogata, C. M.,Telesnitsky, A., Goff, S. P. & Hendrickson, W. A.(1995). Mechanistic implications from the structureof a catalytic fragment of Moloney murine leukemiavirus reverse transcriptase. Structure, 3, 879-892.

Gilboa, E., Mitra, S. W., Goff, S. & Baltimore, D. (1979).A detailed model of reverse transcription and testsof crucial aspects. Cell, 18, 93-100.

Goff, S. P. (1990). Review: retroviral reverse transcrip-tase: synthesis, structure, and function. J. Acquir.Immune De®c. Syndr. 3, 817-831.

Hsiou, Y., Ding, J., Das, K., Clark, A., Jr, Hughes, S. H.& Arnold, E. (1996). Structure of unliganded HIV-1reverse transcriptase at 2.7 AÊ resolution: impli-cations of conformational changes for polymeriz-ation and inhibition mechanisms. Structure, 4,853-860.

Huang, H., Chopra, R., Verdine, G. L. & Harrison, S. C.(1998). Structure of a covalently trapped catalyticcomplex of HIV-1 reverse transcriptase: implicationsfor drug resistance. Science, 282, 1669-1675.

INSIGHTII, (1993). Insight II User Guide 2.3.0 edition,Biosym Technologies, San Diego, CA.

Jacobo-Molina, A., Ding, J., Nanni, R. G., Clark, A. D.,Jr, Lu, X., Tantillo, C., Williams, R. L., Kamer, G.,Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H. &Arnold, E. (1993). Crystal structure of humanimmunode®ciency virus type 1 reverse transcriptasecomplexed with double-stranded DNA at 3.0 AÊ res-olution shows bent DNA. Proc. Natl Acad. Sci. USA,90, 6320-6324.

Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M.(1991). Improved methods for building proteinmodels in electron denisty maps and the location oferrors in these models. Acta Crystallog. sect. A, 47,110-119.

Keifer, J. R., Mao, C., Braman, J. C. & Beese, L. S. (1998).Visualizing DNA replication in a catalytically activeBacillus DNA polymerase crystal. Nature, 391, 304-307.

Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A.& Steitz, T. A. (1992). Crystal structure at 3.5 AÊ res-olution of HIV-1 reverse transcriptase complexedwith an inhibitor. Science, 256, 1783-1790.

Kraulis, P. J. (1991). MOLSCRIPT: a program to produceboth detailed and schematic plots of protein struc-tures. J. Appl. Crystallog. 24, 946-950.

Laskowski, R. A., MacArthur, M. W., Moss, D. S. &Thornton, J. M. (1993). PROCHECK: a program tocheck the stereochemical quality of protein struc-tures. J. Appl. Crystallog. 26, 283-290.

632 Crystal Structures of Reverse Transcriptase-Nucleic Acid Complexes

Lavery, R. & Sklenar, H. (1997). Curves 5.2: helical anal-ysis of irregular nucleic acids.

LeGrice, S. F. J. (1993). Human immunode®ciency virusreverse transcriptase. In Reverse Transcriptase (Goff,A. M. S. A. S. P., ed.), pp. 156-191, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY.

Leis, H., Duyk, G., Johnson, S., Longiaru, M. & Skalka,A. M. (1983). Mechanism of action of the endonu-clease associated with the ab and bb forms of avianRNA tumor virus reverse transcriptase. J. Virol. 45,727-739.

Li, Y., Korolev, S. & Waksman, G. (1998). Crystal struc-tures of open and closed forms of binary and tern-ary complexes of the large fragment of Thermusaquaticus DNA polymerase I: structural basis fornucleotide incorporation. EMBO J. 17, 7514-7525.

Lu, X.-J. & Olson, W. K. (1999). Resolving the discrepan-cies among nucleic acid conformational analyses.J. Mol. Biol. 285, 1563-1575.

Merritt, E. A. & Bacon, D. J. (1997). Raster3d: photo-realistic molecular graphics. Methods Enzymol. 277,505-524.

Moelling, K. (1974). Characterization of reverse tran-scriptase and RNaseH from friend-murine leukemiavirus. Virology, 146, 46-59.

Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997).Re®nement of macromolecular structures by themaximum-likelihood method. Acta Crystallog. sect.D, 53, 240-255.

Navaza, J. (1994). AMoRe: an automated package formolecular replacement. Acta Crystallog. sect. A, 50,157-163.

Nicholls, A., Sharp, K. & Honig, B. (1991). Protein fold-ing and association: insights from the interfacialand thermodynamic properties of hydrocarbons.Proteins: Struct. Funct. Genet. 11, 281-296.

Otwinowski, Z. (1993). Oscillation data reduction pro-gram. In Proceedings of the CCP4 Study Weekend,Data Collection and Processing (Sawyer, L., Isaacs, N.& Bailey, S., eds), pp. 56-62, SERC DaresburyLaboratory, England.

Patel, P. H., Jacobo-Molina, A., Ding, J., Tantillo, C.,Clark, A. D., Jr, Raag, R., Nanni, R. G., Hughes,S. H. & Arnold, E. (1995). Insights into DNA pol-ymerization mechanisms from structure and func-tion analysis of HIV-1 reverse transcriptase.Biochemistry, 34, 5351-5363.

Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H. &Kraut, J. (1994). Structures of ternary complexes ofrat DNA polymerase b, a DNA template-primer,and ddCTP. Science, 264, 1891-1903.

Poch, O., Sauvaget, I., Delarue, M. & Tordo, N. (1989).Identi®cation of four conserved motifs among theRNA-dependent polymerase encoding elements.EMBO J. 8, 3867-3874.

Ren, J., Esnouf, R., Garman, E., Somers, D., Ross, C.,Kirby, I., Keeling, J., Darby, G., Jones, Y., Stuart, D.& Stammers, D. (1995). High resolution structuresof HIV-1 RT from four RT-inhibitor complexes.Nature Struct. Biol. 2, 293-302.

Roth, M. J., Tanese, N. & Goff, S. P. (1985). Puri®cationand characterization of murine retroviral reversetranscriptase expressed in Escherichia coli. J. Biol.Chem. 260, 9326-9335.

Sara®anos, S. G., Pandey, V. N., Kaushik, N. & Modak,M. J. (1995). Site-directed mutagenesis of arginine72 of HIV-1 reverse transcriptase. J. Biol. Chem. 270,19729-19735.

Seeman, N. C., Rosenberg, J. M. & Rich, A. (1976).Sequence-speci®c recognition of double helicalnucleic acids by proteins. Proc. Natl Acad. Sci. USA,73, 804-808.

Sun, D., Jessen, S., Liu, C., Liu, X., Najmudin, S. &Georgiadis, M. M. (1998). Cloning, expression, andpuri®cation of a catalytic fragment of Moloney mur-ine leukemia virus reverse transcriptase: crystalliza-tion of nucleic acid complexes. Protein Sci. 7, 1575-1582.

Xiong, Y. & Eickbush, T. H. (1990). Origin and evolutionof retroelements based upon their reverse transcrip-tase sequences. EMBO J. 9, 3353-3362.

Edited by D. C. Rees

(Received 27 September 1999; received in revised form 16 December 1999; accepted 20 December 1999)