Conserved Water Molecules in a Large Family of MicrobialRibonucleasesRemy Loris,* Ulrike Langhorst, Stefan De Vos, Klaas Decanniere, Julie Bouckaert, Dominique Maes,Thomas R. Transue, and Jan SteyaertLaboratorium voor Ultrastructuur, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Belgium

ABSTRACT We systematically analyzed thecrystallographically determined water molecules ofall known structures of RNase T1 and comparedthem to the ordered solvent in a large number ofrelated microbial nucleases. To assess the crystallog-raphers’ impact on the interpretation of the solventstructure, we independently refined five validationstructures from diffraction data derived from fiveisomorphous crystals of RNase T1. We also com-pared the positions of water molecules found in 11published isomorphous RNase T1 inhibitor com-plexes. These data suggest that the positions of mostof the waters located on the surface of a protein andthat are well-determined in the experimental elec-tron density maps are determined primarily bycrystal packing forces. Water molecules with lesswell-defined electron density are in general uniqueto one or a small number of crystal structures. Onlya small number of the well-defined waters are foundto be independent of the crystal environment. Thesewaters have a low accessible surface area and B-factor, and tend to be conserved in the crystalstructures of a number of evolutionary related ribo-nucleases as well. A single water molecule is foundconserved in all known microbial ribonucleases.Proteins 1999;36:117–134. r 1999 Wiley-Liss, Inc.

Key words: hydration; protein hydration; RNase T1;ribonuclease; barnase

INTRODUCTION

Completely buried waters are often found at conservedsites in homologous protein structures.1 The role of thesewaters in protein structure and function is a much debatedbut still unresolved question. When cavities are geneti-cally engineered inside a protein however, they usuallyremain devoid of water molecules that can be observed byX-ray crystallography.2 In the few cases in which a waterhas been observed within such a cavity, its presence hasbeen found to stabilize3 or to destabilize4 the protein,depending on the case. Similarly, the removal of a buriedwater molecule by cavity-filling mutations resulted inincreased5–8 or decreased conformational stability9–10 de-pending on the protein context. The unpredictability of allthese results illustrates our poor knowledge on proteinhydration.

Many studies have focused on the general features ofprotein-solvent interactions in order to be able to predict

hydration sites, as this gives useful information for molecu-lar dynamics simulations, homology modeling, and proteinstructure determination by X-ray crystallography. More-over, protein hydration is thought to play an importantrole in the folding process.11 Furthermore, chains of watersbetween the active site of a protein and the bulk solventhave been suggested to play a role as a ‘‘proton wire,’’ usedto move hydrogen atoms in and out the active site duringenzymatic action.12 In many cases, water molecules havebeen considered to explain specificity in protein-ligandcomplexes.13–16

An important question that has never been dealt with ina systematic way in the literature, concerns the nature ofthe hydration sites that are observed in a typical crystallo-graphic study of a macromolecule. It is for example notknown to what degree crystal packing forces influence theobserved water positions. Neither is there a consensusamong macromolecular crystallographers on when a peakin an electron density map can safely be regarded asoriginating from an ordered water molecule. Also, theoccurrence of common water molecules within a family ofhomologous proteins has rarely been investigated in de-tail, with the obvious exception of the comparison ofactive-site waters.

The microbial ribonucleases are an ideal model systemfor investigating these problems. Families of crystal struc-tures of identical and homologous proteins in differentliganded states are available. Good expression systemsexist for several of them; RNase T1 has been used in houseas a major work horse for protein engineering over the lastten years.17 Water molecules present in four isomorphousstructures of RNase T1 were first identified by Malin etal.,18 which was the first study aimed at systematicallyidentifying and rationalizing the conservation of hydrationsites in a series of crystal structures. Later this study wasextended19 to include structures from a limited number ofdifferent crystal forms that were solved by 1994.

This work is part of a more elaborate research programthat aims at characterizing and understanding protein-solvent interactions using the microbial RNases as a

Grant sponsor: Vlaams Interuniversitair Instituut voor Biotechnolo-gie; Grant sponsor: the Fonds voor Wetenschappelijk Onderzoek(FWO) Vlaanderen.

*Correspondence to: Remy Loris, Laboratorium voor Ultrastruc-tuur, Vlaams Interuniversitair Instituut voor Biotechnologie, VrijeUniversiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode,Belgium. E-mail: reloris@vub.ac.be

Received 17 December 1998; Accepted 9 March 1999

PROTEINS: Structure, Function, and Genetics 36:117–134 (1999)

r 1999 WILEY-LISS, INC.

model system. In the present paper, we compare in asystematic way the ordered solvent of all known highresolution crystal structures of RNase T1 and barnase andtheir mutants with each other as well as with the crystalstructures of the related ribonucleases from a number ofbacteria and fungi. In a forthcoming study, protein engi-neering and physicochemical methods will be used tocharacterize a particular structurally conserved watermolecule in RNase T1.20

MATERIAL AND METHODSExpression, Purification, Crystallization, and DataCollection of RNase T1

Recombinant RNase T1 was purified and expressed asdescribed previously.33–34 Crystals were grown by theprocedure described by Zegers and coworkers,35 but with 2mM 2’GMP present in the crystallization mixture insteadof the slow substrate cGPS. The crystals belong to spacegroup P212121 and contain a single molecule in theirasymmetric unit. Four crystals, diffracting between 1.9 Aand 1.75 A were used to collect five independent data sets,which we will call 1bu4 to 5bu4 in accordance with the pdbidentifiers of the corresponding refined structures (3bu4and 5bu4 were collected using the same crystal, 5bu4being collected first). The details of these data collectionsare given in Table I. The datasets 1bu4 to 5bu4 were eachgiven to a different investigator, who was asked to refinethe structure using his favorite software and methods. Inorder to avoid as much bias as possible, each structure wasdetermined via molecular replacement using a differentnon-isomorphous starting model. Refinement statisticsand the quality of all refined models are given in Table I.For reasons of comparison, the published statistics on thestructure of the RNase T1–2’GMP complex previouslydetermined in the laboratory of Prof. W. Saenger,21–22

which we used as a sixth independent structure determina-tion, are also included in Table I. The coordinates of theindependently refined RNase T1 structures were submit-ted to the Protein Data Bank.

Refinement of 1bu4

Molecule A of pdb entry 1bvi, stripped of all non-proteinatoms was used as the starting model. Molecular replace-ment and an initial rigid body refinement were done withAMORE36 using data between 8.0 and 3.0 A. The coordi-nates were further refined with REFMAC37 using databetween 20.0 and 1.9 A. A bulk solvent correction calcu-lated with REFMAC and anisotropic scaling, as well asindividual atomic B-factors were used in all followingsteps. Weighting of the X-ray data was modified in order toget a normal variability of the geometry parameters.Initially, the refined model only contained the 104 aminoacid residues of the protein. After a first round of visualinspection, the nucleotide 2’GMP as well as a calcium ionwere added to the model. Waters were added using ARP38

to identify solvent electron density peaks. After the com-bined REFMAC/ARP refinement had converged, the wholeprotein was checked again manually. This was followed byanother round of REFMAC/ARP refinement and visual

inspection. At this point, no further waters could beidentified with ARP, and after convergence of the REFMACrefinement, the structure was considered finished.

Refinement of 2bu4

Molecule A of pdb entry 1rn1 was stripped of all non-protein atoms and used as a starting model. After molecu-lar replacement with AMORE,36 the structure was refinedusing X-plor39 without the use of explicit hydrogens. A bulksolvent correction was used in all refinement steps. Afterthe inhibitor 2’GMP and a calcium ion had been identified,waters were added at sites with Fo-Fc peaks larger than3.0 s if at least one hydrogen-bonding partner was present.Waters were removed in subsequent cycles if the refinedB-factors exceeded 50.0 A2.

Refinement of 3bu4

The structure of the E58A mutant of RNase T1 (pdbentry 1lra), stripped of all non-protein atoms was used as astarting model. Structure determination and refinementwas entirely done with X-plor39 without the use of explicithydrogens. An initial refinement cycle with the startingmodel consisted of a high temperature (4,000 K) slow coolfollowed by 100 cycles of positional and B-factor refine-ment (using the full_refinement script from the X-plortutorial). This resulted in a model with R 5 0.30 andRfree 5 0.36. At this stage, a first electron density map wascalculated, and the inhibitor molecule 2’GMP as well as acalcium ion were fitted into the electron density. This wasfollowed by four alternating rounds of refinement (posi-tional and B-factor) and rebuilding. Water molecules wereidentified using the CCP4 program WATPEAK.40 Aftereach refinement round, the electron density and refinedpositions of the most recently introduced waters werechecked visually. Dual conformations were found for Leu26and Val78.

Refinement of 4bu4

Molecule C of pdb entry 1bvi, stripped of all non-proteinatoms was chosen as starting model. After molecularreplacement with AMORE,36 the structure was refinedwith X-plor.39 The positions of all polar hydrogens weregenerated, and their interactions used in the energy termused for the refinement. Initially a resolution range of10.0–2.5 A was used, and this resolution range wasgradually extended to 10.0–1.80 A. Simulated annealing(1,000K or 3,000K) was used both in refinement and tocalculate omit maps of poorly defined regions of thestructure. Waters were identified from Fo-Fc maps usingthe following criteria: Fo-Fc density larger than 3.0 s andat least one hydrogen-bonding partner. Waters were subse-quently removed if their 2Fo-Fc density was lower than 1.5s, the B-factor refined to a value larger than 60.0 or theyhad no longer at least one hydrogen bonding partner(protein or water). Although the electron density sug-gested multiple conformations for residues Ser17, Val33,Val78, and Ser96, these were not modeled.

118 R. LORIS ET AL.

Refinement of 5bu4

Molecule B from the tetragonal complex of RNase T1with cGPS (pdb entry 7gsp) was used as a starting model.All non-protein atoms were removed from the model, andall temperature factors were put to 20 A2. Molecularreplacement was carried out with AMORE.36 All refine-ment, including a preliminary rigid body refinement usingdata between 8.0 A and 4.0 A (10 cycles) followed by10.0 A and 3.0 A (10 cycles), was done using the programRESTRAIN.41–42 At this point, the R-factor was 0.35 andthe Rfree factor 0.39. From this point on, all data between20.0 and 1.77 A were used. Rounds of restrained positionaland restrained B-factor refinement (15 cycles were usuallynecessary before convergence was reached) were alter-nated with manual inspection of the model. Refinementparameters were chosen according to the suggestions ofthe authors of the program as documented in the write-up.A weighting scheme according to Rees et al.43 as suggestedby the authors in the program documentation and a bulksolvent correction were used throughout the refinement.Water molecules were identified from Fo-Fc differenceelectron density maps. A difference peak was considered asa possible water molecule if it had a height of at least 3times the rms density of the Fo-Fc map, if no steric clasheswere introduced with existing atoms and if at least onehydrogen bond was made (defined as a N...Wat or O...Watdistance between 2.4 A and 3.5 A). A maximum of 15 watermolecules were added in each round, and these werechosen to be the largest electron density peaks meeting thementioned criteria. During the course of refinement, watermolecules were also rejected based on a B-factor criterion.The B-factor above which a water was rejected wasloosened with each further refinement round, and was 65A2 at the point when the structure was considered fin-ished.

Second Round of Refinements

In the first round of refinements (above paragraphs),each investigator was left free as to how many watershe/she wanted to include in his/her structure. Since itturned out that these numbers were overall smaller thanthe number of waters present in the RNase T1 structuresavailable from the Brookhaven database, each investiga-tor was asked to further refine his/her structure up to thepoint where exactly 100 water molecules were included.For each of different structures, this involved just addingextra water molecules interspersed with automatic refine-ment, with the exception of 4bu4. In the case of 4bu4, thesecond round of refinement involved some further manualrebuilding of the structure, as well as the inclusion of anoverall anisotropic B-value correction and a bulk solventcorrection in the calculations.

Alignments and Superpositions.

All crystallographic coordinates were taken from thelatest version of the Protein Data Bank44 or were providedto us directly by the authors. The ribonuclease structuresconsidered in this paper, together with the relevant details

of their crystallographic refinement, are described in TableII. Secondary structure assignments were performed withthe program PROMOTIF,45 which uses the DSSP46 algo-rithm. Sequence alignments were performed manually,taking into account the conserved structural elements ofthese proteins. For structure superposition and visualanalysis the graphics program TURBO was used. Charac-terization of protein-protein contacts was done with X-plor.39 Solvent-accessible surfaces were calculated usingthe program NACCESS47 according the method of Lee andRichards, using a probe sphere with radius 1.4 A.48 Hydro-gen bonds were calculated using HBPLUS49 using D-Adistances of 3.4 or 3.9 A and the default minimum D-H-A,H-A-AA and D-A-AA angles. The side chains of Asn, Gln,and His were swapped if necessary.

Identification of Conserved Water Sites

All comparisons were done by first choosing a referencestructure. The pdb entry 1rga of RNase T150 was used as ageneral reference structure. For comparisons of differentbarnase structures, molecule A of pdb entry 1bni51 waschosen as a reference. For two structures that are com-pared, water molecules are identified that are closer than5.0 A to the nearest protein atom, taking into account thesymmetry elements of the crystal. After superposition ofthe relevant portions of two crystal structures, a hydrationsite was considered conserved between the two structuresif both water molecules were found to be closer than apredefined distance of 1 A. This distance was chosen basedon a preliminary study in which the waters present in pdbentry 1rga were compared with those of several otherRNase T1 structures (both isomorphous and non-isomor-phous, data not shown) using different distance criteria(0.2, 0.4, ... 2.6 A). From this study, it turned out that thenumber of waters in common between two RNase T1structures raises until a distance of 1.0–1.2 is used.Between 1.0 and 2.2 A, relatively few extra waters aredetected. At distances equal or larger than 2.2 A, waters inhydrogen bonding distance to the reference water arebeing detected. Furthermore it turned out that when usinga distance criterion of 1 A or smaller, most hydrogen bondsmade by each detected water and the protein tended to beconserved as well. The results from this control experi-ment are thus similar to what was found in an identicalcontrol experiment in a study on the conservation ofsolvent sites in the legume lectins.23 For multiple superpo-sitions, all structures were superimposed upon the refer-ence structure and a sphere with radius 4 A around allwater molecules belonging to the reference structure wasused.

RESULTS AND DISCUSSIONRefinements of Five Validation Structures of theRNase T1–2’GMP Complex

To evaluate the reliability of the crystallographer’sidentification of water molecules in crystal structuresdetermined at resolution limits as used in our comparativestudy (2.2 A–1.5 A), we collected five sets of data from fourdifferent crystals of an RNase T1–2’GMP complex (space

119CONSERVED WATERS IN MICROBIAL RIBONUCLEASES

group P212121, 1 molecule in the asymmetric unit—seeTable I A). They were refined independently by differentinvestigators involved in this study. Each investigator wasasked to solve his/her structure by molecular replacement(starting with coordinates from different non-isomorphousRNase T1 structures) and refine it using his/her preferredrefinement strategies. These structures (pdb codes 1bu4 to5bu4) will be referred to as the ‘‘validation structures.’’ Thestatistics of these validation structures are summarized inTable IA. The backbone root mean square(rms) deviationsbetween these five structures are small and range from0.127 A to 0.269 A. Any pair of these five structures hasbetween 40 and 60 waters in common.

The overall similarities of the solvent part of these fivevalidation structures and the original structure of theRNase T1–2’GMP complex determined by Arni and co-workers21–22 (pdb code 1rnt) illustrate the reliability of thewaters that are identified in a typical macromolecularcrystal structure. An important difference between our fivevalidation structures and the isomorphous structures thatare available in the protein data bank concerns thenumber of waters that are present in the asymmetricunits. In our independent refinements, this number rangesfrom 50 to 94, while for isomorphous RNase T1 structurespresent in the Brookhaven database this number variesbetween 75 and 162. This difference probably reflects thechange in refinement practice over the last few years. Theintroduction of cross-validation has led to a more conserva-tive approach for the identification of waters in crystalstructures of proteins.

Figure 1A evaluates the reoccurrence of individualwater molecules in our validation structures and in 1rnt.For each individual water molecule in 3bu4, we investi-gated its reappearance in the other five refined structures.Very few waters are only found in a single structuredetermination. It thus appears that most of the watermolecules identified in these independent refinements aretruly present in this particular type of crystalline complexof RNase T1.

In order to be able to make a more direct comparisonwith the RNase T1 structures found in the Brookhavendatabase, all investigators were asked to further refinetheir structure and add a total of exactly 100 waters (TableIC). When comparing the structures obtained from thissecond round of refinement, any two of these structureshad between 56 and 73 waters in common, indicating thatin the first refinement round a number of ‘‘real’’ waterswere indeed missed. A slightly larger number of waterscommon to all or all but one structure are now observed(Fig. 1A), again suggesting that at least some of the newlyadded waters are relevant.

Very similar histograms to the one presented in Figure1A were obtained when other individual validation struc-tures were used as the reference data set. The onlyexception encountered was when using pdb entry 1rnt asthe reference structure (data not shown). This crystalstructure contains 26 waters not found in any other 2’GMPcomplex (compared to 3–17 unique waters in our fivevalidation structures). Inspection of Table I, however,shows that the length of the b-axis of this crystal (46.81 A)

differs by more than 1 A from that found for the other fivecrystals (48.02 6 0.18 A). Similarly, the length of the c-axisof 1rnt (50.11 A) differs significantly from the one of theother five structures (50.80 6 0.04 A). Therefore, 1rnt isnot truly isomorphous to the five validation structures.Subtle differences in crystal packing appear to be reflectedin its solvent structure. The results presented in the nextsection, as well as a comparative study of the solventstructure of a number of closely related lentil lectincrystals,23–24 are in agreement with this observation thatthe solvent structure closely relates to the crystal packing.In conclusion, the results obtained with the independentrefinements of the five validation structures suggest thatmost of the waters identified in these structures are indeedreal and representative for a well-defined crystal packing.

Conserved Water Molecules Within a SinglePacking Environment of RNase T1

In this section, we compare 11 structures of RNase T1,all in the most common crystal form, space group P212121

with one molecule in the asymmetric unit (pdb entries1rga, 1rnt, 2rnt, 3rnt, 6rnt, 8rnt, 9rnt, 7rnt, 2aae, 1rgk,and 1rgl–see Table II A). The structure of a 3’GMP complexof RNase T1 (pdb code 1rga) was used as the referencestructure. The histogram in Figure 1B clearly shows abimodal distribution that distinguishes a large number ofwater sites apparently unique to one or two structures(first group) and a significant number of water sitespresent in all (or all but one) structures (second group). Asmaller number of water molecules displays an intermedi-ate behavior, being present in three to eight structures(third group). The bimodal distribution in Figure 1B is notdue to the large number of waters present in referencestructure 1rga, as this distribution is also observed whenusing other structures with fewer waters as a reference(data not shown).

The waters in the first group are mostly characterized byweaker electron density and relatively high B-values.Although some of them may be the result of spurious peaksin the electron density maps (especially because the refer-ence structure 1rga contains a fairly large number ofwaters), our results obtained with the independently re-fined validation structures of the RNase T1–2’GMP com-plex suggest that many of these waters are indeed real.Hence, their positions must be a consequence of moresubtle long range structural changes due to point muta-tions, other bound ligands, and/or subtle differences incrystal packing.

The second group of water molecules is present in all ormost of these isomorphous structures. They mostly have alow B-value and many of them also have a low solvent-accessible surface in their crystal environment (however,none of them are truly buried, and all of them can still beconsidered as surface waters). These waters are the best-determined waters in the electron density maps. Many ofthem are involved in crystal packing interactions. Thisgroup of waters largely corresponds to the group of 30waters identified by Malin and co-workers in their pioneer-ing study involving only four crystal structures of RNaseT1,18 and also correlates well with the waters found to be

120 R. LORIS ET AL.

conserved in all of our validation structures discussedabove.

The third group of waters with an intermediate behaviorare more difficult to rationalize. They may be common to

different subfamilies within a single space group. Thesedifferent subfamilies of structures can be discerned basedon the rms differences of their Ca backbone coordinates.For example, those structures that contain an inhibitor

TABLE I. Data Collection and Refinement Statistics for the Independent Structure Determinations of RNase T1

A. Data collection 1rntpdb code 1bu4 2bu4 3bu4 4bu4 5bu4 (Arni et al., 1987)21

Detector 30 cm MAR 30 cm MAR 30 cm MAR FAST 30 cm MAR FilmData analysis software Denzo/Scalepack Denzo/Scalepack Denzo/Scalepack Madness/Scala Denzo/ScalepackUnit cell

a (Å) 40.51 40.48 40.48 40.50 40.47 40.44b (Å) 48.00 47.82 48.10 48.12 48.10 46.81c (Å) 50.81 50.79 50.77 50.84 50.78 50.11

Space group P212121 P212121 P212121 P212121 P212121 P212121

Resolution range used in refinement 20.0–1.90 18.0–1.95 20.0–1.77 10.0–1.80 20.0–1.77 6.0–1.90Numbers of observed reflections 44006 38058 38381 57570 380597 Not reportedNumber of unique reflections 7853 7393 9718 9347 9833 6349R-merge (Highest resolution shell) 0.139 (0.583) 0.170 (0.517) 0.081 (0.472) 0.122 (0.544) 0.188 (0.978) 0.088Completeness (%) 95.6 97.3 92.2 96.0 93.8 86.9a

I/SigI (Highest resolution shell) 12.66 (2.47) 10.71 (3.00) 22.96 (3.77) 15.09 (2.51) 23.64 (3.76) Not reportedRedundancy 5.6 5.1 3.9 6.2 38.7 Not reportedb

B. First refinement round 1rntpdb code 1bu4 2bu4 3bu4 4bu4 5bu4 (Arni et al., 1987)21

Resolution range used in refinement 20.0–1.9 18.0–1.95 18.0–1.77 6.0–1.8 20.0–1.77Sigma cut-off used in refinement None None None None None 6.0–1.30R-value 0.180 0.199 0.193 0.200 0.172 0.180R-free value 0.230 0.249 0.236 0.253 0.225 Not usedRefinement program REFMAC X-plor X-plor X-plor RESTRAIN PROLSQBulk solvent correction REFMAC X-plor X-plor None RESTRAIN NoneAnisotropic scaling correction REFMAC None None None None NoneAnisotropic scaling REFMAC Not used Not used Not used Not used —rms bond lengths (Å) 0.009 0.006 0.010 0.010 0.013 0.021rms bond angles (degree) 2.074 1.555 1.732 1.974 2.354 3.292Ramachandran profile

Most favourable (%) 93.0 93.0 94.2 93.0 93.8 93.0Additionally allowed (%) 7.0 7.0 5.8 7.0 6.2 7.0Generously allowed (%) 0.0 0.0 0.0 0.0 0.0 0.0Disallowed (%) 0.0 0.0 0.0 0.0 0.0 0.0

Number of water molecules 89 50 66 82 94 91Number of multiple conformations 1 0 2 0 2 0

C. Second refinement round 1rntpdb code 1bu4 2bu4 3bu4 4bu4 5bu4 (Arni et al., 1987)21

Sigma cut-off used in refinement none none none none none —Resolution range used in refinement 20.0–1.9 18.0–1.95 18.0–1.77 20.0–1.8 20.0–1.77 —R-value 0.180 0.202 0.186 0.160 0.169 —R-free value 0.230 0.246 0.224 0.214 0.222 —Refinement program REFMAC X-plor X-plor X-plor RESTRAIN —Bulk solvent correction REFMAC X-plor X-plor X-plor RESTRAIN —Anisotropic scaling REFMAC none none X-plor none —rms bond lengths (Å) 0.009 0.006 0.008 0.009 0.011 —rms bond angles (degree) 2.032 1.554 1.671 1.805 2.045 —Ramachandran profile

Most favourable (%) 93.0 93.0 93.0 93.0 94.2 —Additionally allowed (%) 7.0 7.0 7.0 7.0 5.8 —Generously allowed (%) 0.0 0.0 0.0 0.0 0.0 —Disallowed (%) 0.0 0.0 0.0 0.0 0.0 —

Number of water molecules 100 100 100 100 100 —Number of multiple conformations 1 0 3 0 2 —

aFor all data with F . s3(F).bFrom the information given in Arni et al. (1987), the redundancy can be estimated to be about 3.5.

121CONSERVED WATERS IN MICROBIAL RIBONUCLEASES

bound in a ‘‘productive’’ conformation in their active siteshow rms differences of 0.14–0.39 A (see Table III). Similarvalues are found among inhibitor-free structures, butlarger differences (0.45–0.54 A) are observed when mem-bers of the two classes of structures are compared witheach other. When comparing all structures, the number ofwater molecules in common between two ‘‘inhibitor-bound’’structures is generally higher (55–93 waters) than thenumber in common between two molecules belonging to adifferent class (inhibitor-bound versus inhibitor-free)(34–64 waters) (Table III).

Figure 2 relates the B-value (Fig. 2A) and the solventaccessibility (Fig. 2B) in the crystal environment for eachwater molecule to the degree of its conservation amongisomorphous crystal structures of RNase T1. Although aclear trend is visible for the B-values (waters present in alarger number of structures tend to have lower B-values),such a trend is essentially absent when the solvent accessi-bility of a water molecule is plotted versus its conservationamong isomorphous structures. There is a significantnumber of unique or quasi-unique waters with low solventaccessibility, but some well conserved water sites havefairly high solvent accessibilities.

Conserved Waters in Different CrystalEnvironments of RNase T1

When comparing solvent structures of different crystalforms of RNase T1, waters whose presence is mainlydetermined by lattice contacts can be separated from thosewhose presence is independent of the crystal packing.Again, the RNase T1–3’GMP complex (pdb entry 1rga) wasused as the reference molecule. In total, nine RNase T1molecules with different packing environments (out of sixcrystal forms - pdb entries 1gsp, 1det, 1rnl, 1rn4, 4rnt, and1lra–see Table II B) were compared with this referencestructure. The histogram in Figure 1C shows that only sixwater molecules are completely conserved in all packingenvironments. Five water molecules were present in all

but one of the structures, and another five in all but two.These 16 waters are described in Table IV, and a number ofthem will be discussed in more detail in the next section.At this point it is interesting to note, however, that there isa clear trend for the most recurrent waters to be involvedin a-helix-to-b-sheet packing and in b-sheet termination(i.e., replace the non-existing ‘‘next strand’’ in the b-lad-der). Interestingly, a preference for conserved water mol-ecules to be involved in helix-helix and helix-sheet interac-tions was also observed by Zegers and co-workers, whostudied protein-solvent interactions in different crystalforms of bovine pancreatic ribonuclease A.25

Obviously, crystal packing is a major determinant in thepositioning of water sites observed by X-ray crystallogra-phy. Those few waters that are nevertheless independentof crystal environment have low solvent accessibilities anddo not contact neighboring protein molecules. We thereforeconclude that water molecules tend to be conserved amongdifferent crystal forms of a protein if they are located in arelatively deep groove or cleft. These waters are thereforeunlikely to be replaced by neighboring molecules in thecrystal lattice. The sizes and shapes of these clefts andgrooves are relatively unaffected by differences in thecrystal environment.

In contrast to the isomorphous structures discussed inthe previous section, a clear trend can be observed for boththe solvent accessibilities (Fig. 2C) and the B-factors (Fig.2D) of the water molecules plotted versus their conserva-tion among different crystal lattices. If a water is highlyexposed on the surface, it has a higher chance of beingreplaced by packing interactions in a different crystalenvironment. The conserved water molecules consistentlyhave a low solvent accessibility, indicating that crystalpacking interactions have a low likelihood to structurallydisplace water molecules or to disturb the structure of agroove or crevice on the surface in such a way that watersare pushed out. There are very few waters with a lowsolvent accessibility that are not conserved among the

Fig. 1. Histograms illustrating the number of wa-ters from a particular crystal structure that are incommon with 0, 1, 2, ... other crystal structures. A.RNase T1–2’GMP validation structures that wereindependently refined (using 3bu4 as a reference).Dark gray bars correspond to the first round ofrefinement, light gray bars to the second round ofrefinement. B. Isomorphous RNase T1 crystal struc-tures in space group P212121 (using 1rga as areference). C. Non-isomorphous RNase T1 crystalstructures (using 1rga as a reference). The choice ofdifferent reference structures was also examined,and was shown not to influence the general trends inthese histograms (data not shown).

122 R. LORIS ET AL.

different space groups indicating that crystal packingforces have little influence on the solvent structure ingrooves or crevices. Identical trends have been observed ina comparison of protein-water interactions in ten crystalforms of T4 lysozyme,26 and may be general behavior ofcrystalline proteins.

In total, we conclude that the inverse correlation be-tween solvent accessibilities in the crystal environmentand the number of structures in which a particular wateris present improves when structures of different spacegroups rather than isomorphous structures are compared,due to differences in crystal packing that influence waterswith otherwise well-defined electron densities. Watershighly exposed on the surface but not involved in directcontact with neighboring molecules are affected to differ-ent degrees by crystal packing. If the crystal environmentdrastically changes (e.g., different space group), exposedwaters are rarely found in common between two struc-

tures. If the two structures that are compared are (essen-tially) isomorphous, the water will in a number of cases becommon to both structures, although subtle differences inthe crystal packing can be enough to generate uniqueexposed hydration sites. In this respect, it is interesting tonote that comparisons between structures belonging to theclassical space group (P212121 such as 1rnt) and structuresbelonging to a related space group (pdb entries 1bir, 1rgc,2aad, and 1trq which have space group P21 with twomolecules in the asymmetric unit and a unit cell andcrystal packing related to the classical space group) show abehavior intermediate between the ‘‘same space group’’ andthe ‘‘different space group’’ comparisons (data not shown).

Structural Classification of the MicrobialRibonucleases

Next, we set out to investigate whether any of the 16structurally conserved water molecules of RNase T1 (listed

TABLE II. Structures Used in Our Comparative Study of Hydration Sites in Microbial RNases

Protein pdb code Organism NMolResol(Å) Rfac

Number of watermolecules/a.u.

Spacegroup Mutant Ligand

A. Structures of RNase T1 in the same crystal packing environment

RNase T1 1rga A. oryzae 1 1.7 0.145 142 P212121 WT 38GMP 1 GRNase T1 1rnt A. oryzae 1 1.9 0.180 91 P212121 WT 28GMPRNase T1 2rnt A. oryzae 1 1.8 0.149 105 P212121 WT 2,5GpGRNase T1 3rnt A. oryzae 1 1.8 0.137 162 P212121 WT VO4

32

RNase T1 6rnt A. oryzae 1 1.8 0.149 104 P212121 WT 28AMPRNase T1 8rnt A. oryzae 1 1.8 0.140 104 P212121 WT Zn21

RNase T1 9rnt A. oryzae 1 1.5 0.143 121 P212121 WT noneRNase T1 7rnt A. oryzae 1 1.9 0.179 75 P212121 Y45W 28AMPRNase T1 2aae A. oryzae 1 1.8 0.145 116 P212121 H40K PO4

32

RNase T1 1rgk A. oryzae 1 2.0 0.142 86 P212121 E46Q 28AMPRNase T1 1rgl A. oryzae 1 2.0 0.148 81 P212121 E46Q 28GMP

B. Structures of RNase T1 in different crystal packing environment

RNase T1 1gsp A. oryzae 2 2.0 0.193 162 P41212 WT cGPSRNase T1 1det A. oryzae 1 1.8 0.194 79 I23 K25Q 28GMP 1 CM-E58RNase T1 1rn1 A. oryzae 3 1.84 0.256 179 P212121 K25Q SO4

22

RNase T1 1rn4 A. oryzae 1 1.8 0.148 82 P212121 H92A noneRNase T1 4rnt A. oryzae 1 2.2 0.162 70 P212121 H92A noneRNase T1 1lra A. oryzae 1 1.9 0.178 90 P21 E58A 28GMP

C. Structures of barnase and binase in different crystal packing environmenta

Barnase 1bni B. amyloliquefaciens 3 2.1 0.179 216 P32 WT noneBarnase 1brn B. amyloliquefaciens 2 1.76 0.190 229 P1 WT d(CpGApC)Barnase 1brs B. amyloliquefaciens 3 2.0 0.172 513 C2 WT barstar (mutant)Barnase 1rnb B. amyloliquefaciens 1 1.9 0.214 96 P3121 WT dCpdCBinase — B. intermedius 2 1.65 0.137 379 C2 WT noneBinase — B. intermedius 2 2.0 0.179 204 P212121 WT 38GMP

D. Structures of other microbial ribonucleases

RNase F1 1fus F. moniliforme 1 1.3 0.187 107 P212121 WT noneRNase Ms 1rds A. saitoi 1 1.8 0.204 31 P212121 WT 28F-3,5GpCRNase U2 1rtu U. sphaerogena 1 1.8 0.143 146 P212121 WT SO4

22

RNase Pb — P. brevicompactum 1 1.4 0.142 146 C2221 WT d(ApCpG)RNase Th — T. harzianum 1 1.67 0.145 154 P3221 WT SO4

22

RNase Ap — A. pallidus 1 1.08 0.120 87 P21 WT SO422

Restrictocin — A. restrictus 2 1.7 0.177 202 P21 WT PO432

RNase Sa 1gmp S. aureofaciens 2 1.7 0.133 492 P212121 WT 28GMPaA number of barnase crystal structures (pdb entries 1ban, 1bao, 1bne, 1bnf, 1bng, 1bnj, 1bns, 1brg, 1brh, 1bri, 1brj, 1brk, 1bsa, 1bsb, 1bsc, 1bsd,and 1bse) isomorphous to entry 1bni have essentially the same water structure as 1bni and are not considered separate.

123CONSERVED WATERS IN MICROBIAL RIBONUCLEASES

in Table IV) are also conserved in the crystal structures ofa number of related microbial ribonucleases. The microbialribonucleases for which a crystal structure has been

determined show different degrees of sequence identityand structural similarity to RNase T1.27–31 They are listedin Table II C,D. The structural conservation of an indi-

TABLE III. Number of Common Waters (Above Diagonal) and Backbone RMS Deviation (Below Diagonal) for allStructures Included in the Comparison of RNase T1 in the Most Common Crystal Form (Space Group P212121 With One

Molecule in theAsymmetric Unit)

Inhibitor-free (no basebound in specificity site) Inhibitor-bound (base bound in specificity site)

1rga 1rnt 2rnt 3rnt 6rnt 8rnt 9rnt 7rnt 2aae 1rgk 1rgl

1rga — 49 79 61 51 55 64 40 51 50 471rnt 0.239 — 49 46 39 51 54 34 38 41 382rnt 0.137 0.216 — 55 47 51 54 35 43 49 483rnt 0.495 0.486 0.482 — 79 82 93 61 76 70 686rnt 0.467 0.469 0.453 0.166 — 78 84 61 68 71 698rnt 0.487 0.487 0.472 0.145 0.140 — 90 63 76 70 709rnt 0.505 0.503 0.493 0.158 0.173 0.098 — 62 80 74 737rnt 0.480 0.497 0.470 0.217 0.161 0.187 0.221 — 56 60 552aae 0.543 0.525 0.531 0.201 0.267 0.229 0.220 0.324 — 64 611rgk 0.521 0.531 0.508 0.316 0.274 0.284 0.314 0.283 0.387 — 641rgl 0.521 0.517 0.506 0.195 0.217 0.170 0.189 0.222 0.254 0.287 —

Fig. 2. Relationships between the degree by which a particular water molecule is conserved indifferent crystal structures, its solvent accessibility and its temperature factor. A,B. Waters presentin isomorphous crystal structures of RNase T1. C,D. Waters present in non-isomorphous crystalstructures of RNase T1.

124 R. LORIS ET AL.

vidual water molecule among these structurally relatedproteins would be an indication for its structural and/orfunctional role in these proteins that cannot be mimickedby amino acid substitutions that occur during evolution.

The structure-based alignment of the amino acid se-quences of the ribonucleases used for this comparison isgiven in Figure 3. From a structural point of view, themicrobial RNases can be divided into two groups: thosethat are structurally most closely related to RNase T1 andthose that are structurally most closely related to barnase.The first group contains only RNases from fungi. Amongthese, the sequence identity between the fungal RNasesT1, Ap, Ms, F1, Pb and Th is high and the sequencesimilarities are obvious upon visual inspection. RNase U2and restrictocin (Re) on the other hand show a much lowersequence identity to RNase T1 (34% and 19% resp.) but

their three-dimensional structures are very similar. Wewill call this group the ‘‘fungal RNases.’’

On the other hand, the bacterial RNases barnase (Ba)and binase (Bi) show little sequence identity with thefungal RNases, except for the active site residues. Theircrystal structures can however be satisfactorily superim-posed on that of RNase T1 based on the coordinates of theirmajor b-sheets in the C-terminal half of their sequences(the structures of their N-terminal halves are very differ-ent and not superimposable). RNase Sa from S. aureofa-ciens is a bacterial RNase, with little sequence identity toeither the RNase T1 group or the barnase group. RNase Sais structurally more closely related to barnase and binasethan to the fungal RNases. In the following sections, wewill refer to the barnase, binase, and RNase Sa as the‘‘bacterial RNases.’’

TABLE IV. Water Sites ConservedAmong Different Microbial Ribonucleases†

SiteBfac(Å2)

Accessibility(Å2) T1 Ap Ms F1 Pb Th U2 Re Ba Sa Ligands (distances)

Located in a crevice formed by three hairpin loops

W1 10.7 5.0 10/10 y y y y y y n n n Cys6-O (2.97) Asn9-ND2 (3.26) Asp76-OD1(2.76) Thr91-OG1 (3.31) Thr93-OG1 (2.69)

Chain of waters in a groove between the a-helix and the major b-sheet, hydrogen bonding to loop L6

W2 7.3 0.9 10/10 y y y y y y y n n Trp59-NE1 (3.02) Pro60-O (2.73) Tyr68-N (3.01)W3 (2.89)

W3 8.0 5.3 10/10 y y y y y n y n y Asn62-N (2.93) Asp66-O (2.77) W2 (2.89) W4(2.78)

W4 11.0 2.1 10/10 y y y y y n n n n Asp15-O (2.86) W3 (2.78) W5 (3.03)W5 12.2 5.2 10/10 y n n y y n n n n Leu62-O (2.74) W4 (3.03)

At divergence of strands S3 and S4 (b-sheet termination)

W6 6.6 4.0 10/10 y y y y y y y n y Ile61-O (2.63) Pro73-O (2.71) Ala75-N (3.35)Asp76-N (2.97) W7 (2.94)

W7 9.3 15.6 8/10 y y y y y y n n n Ser63-N (2.84) Ser63-OG (3.43) W6 (2.94)

Interacting with same loop as W2-W5

W8 17.3 3.1 9/10 y y y y y y y y y Tyr42-O (2.72) Tyr56-N (3.05) Lys41-NZ (2.73)

Connects C-terminus of a-helix with strand S3 and is involved in b-sheet termination

W9 14.2 13.6 9/10 y y y y y n n n n Glu31-O (2.77) W10 (2.63)W10 16.1 17.4 9/10 n n y n n n n n n His27-ND1 (2.71) Pro39-O (3.39) W9 (2.63)

Highly solvent exposed site involved in b-sheet termination

W11 25.9 9.4 9/10 n n n n n y n n n Val89-N (2.83) Val89-O (3.01) Glu102-OE2 (2.97)

Involved in b-sheet termination

W12 13.6 37.8 9/10 y n y y y n n n n Glu102-N (2.82)

Stabilizes a local backbone structure in loop L6

W13 18.6 17.5 8/10 n n y y y n n n n Ser69-OG (3.26) Gly70-O (2.76)

Connects loop L4 and loop L6

W14 14.0 17.7 8/10 n n n y n n n n n Ser35-N (2.82) Ser35-OG (3.31) Gly71-N (3.04)Gly71-O (3.37)

Stabilizes the a-helix by connecting two side chains

W15 14.1 18.3 8/10 n n n y y y y n n Cys2-O (3.01) Thr5-OG1 (2.75) Thr104-OT (2.63)

Stabilizes the a-helix by connecting two side chains

W16 33.1 23.8 8/10 y n n y n n n n n Ser17-OG (3.05) Gln20-NE2 (2.63)†B-factors, solvent accessibility and hydrogen bond distances correspond to pdb entry 1rga.

125CONSERVED WATERS IN MICROBIAL RIBONUCLEASES

Solvent Structure of the Fungal Ribonucleases

We compared the solvent structure of one representativeRNase T1 structure (pdb code 1rga) with one representa-tive structure of all the above mentioned fungal ribonucle-ases. The results are summarized in Table IV and Figure 4.Most intriguingly, three water molecules from 1rga areinvariantly present in all other fungal RNases. If the moredistant RNase U2 and restrictocin are not considered, thisnumber raises to eight.

Of these eight waters, W1 is located in a cleft formed bythe three hairpin loops L5 (Cys6-Asn9), L6 (Ile61-Asp76),and L8 (Thr91-Phe100) of RNase T1 (Figs. 4 and 5). Water

W1 is unique in the sense that it is the only systematicallyconserved water among the fungal ribonucleases whosehydrogen bonding partners are mainly side-chain atoms.This makes W1 the only water whose structural andfunctional role can be assessed by site-directed mutagen-esis experiments. Such a study is currently underway inour laboratory. With the exception of Asn9, all proteinligands of W1 are conserved in the RNases Ap, MS, F1, Pb,Th, and U2. W1 is not present in restrictocin, which hassignificantly different local structure around the positionof W1 due to its much longer hairpin S1-L2-S2 and adifferent conformation of loop L8. Furthermore, the pre-

Fig. 3. Sequence alignment of the different microbial RNases consid-ered in our study. Structurally equivalent segments that were used forsuperpositions of the different RNases are shaded gray. Outside theseshaded areas, the local three-dimensional structures of the proteins canvary considerably, especially when comparing a fungal and a bacterial

RNase. Secondary structural elements are indicated and labeled forRNase T1, barnase, and RNase Sa. The color-coding for the differentelements (helix yellow, major sheet red, minor sheet purple) is usedthroughout the manuscript in Figures 4–7.

126 R. LORIS ET AL.

Fig. 4 (Left). (a) Waters conserved in the family of T1-like fungal RNases (RNases T1; Ap,Ms, F1, Pb, Th, U2, and restrictocin). A ribbon diagram of RNase T1 is shown, with the secondarystructural elements color-coded as in Figure 1 and waters shown as spheres. Waters present inall of these RNases are shown in orange. Those waters present in all RNases except restrictocinare colored yellow and those present in all except U2 are colored green. Dark-blue waters arepresent in RNase T1 and its closest relatives (Ap, Ms, F1, Pb, and Th) but absent in both RNaseU2 and restrictocin. Waters that are conserved in RNase T1 but not in the other T1-like fungalRNases are shown in cyan. The size of the spheres representing the water molecules relates tothe number of RNase T1 packing environments in which the water is present (large sphere 10/10

packing environments, medium 9/10 and small 8/10). For the sake of comparison, the view usedin this figure is also used in Figures 5–7. (b) Close-up view of a cluster of conserved water nearthe N-terminus of the a-helix of RNases T1. Color-coding is as in (a). This Figure, as well asFigures 5–7 are drawn with BOBSCRIPT.52

Fig. 5 (Right). Stereoview of the structural context of W1 in (a) RNase T1 and (b) restrictocin.The replacement of a series of hydrophilic contacts in RNase T1 with hydrophobic contacts inrestrictocin can be clearly seen. In RNases Ap, Ms, F1, Pb, Th, and U2, the situation is essentiallyidentical to that in RNases T1.

127C

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dominantly hydrophilic surrounding of W1 in RNase T1 issubstituted by a series of hydrophobic side chain-sidechain contacts in restrictocin. This is illustrated in Figure 5.

A further set of three waters (W2-W4) is found in agroove between the N-terminal a-helix and the majorb-sheet (Fig. 4), making hydrogen bonds to each other andto backbone atoms of loop L6 that connects strands S4 andS5. These three waters form part of a chain of ten watersthat was identified by Malin and co-workers18 in fourisomorphous inhibitor complexes of RNase T1. In thesestructures, this chain connects Trp59 with a calcium ion.This calcium ion and its water ligands, however, areinvolved in crystal packing and not conserved in differentspace groups. W3 and W4 are not present in RNase U2because of the difference in conformation and length ofloop L6. The three-water cluster is extended by a furtherwater molecule (W5) that is conserved in all RNase T1crystal structures (Fig. 4 ), as well as in RNases Ap, Pb,and Th. The absence of this water in RNase Ms can bereadily explained by the substitution of Tyr64 in RNase Msfor Gly65 in RNase T1. In RNases F1 and U2, the absenceof this water is due to the different conformation of loop L6between strands S4 and S5. Two further water molecules(W6-W7) that are conserved among the T1-like fungalRNases also hydrogen bond to loop L4 (Fig. 4) and can beconsidered to be part of the same cluster as W2-W5 (Fig. 4).Again, the absence of W7 in RNase U2 and restrictocin isexplained by the different conformation of loop L6.

The other two conserved waters among the fungalribonucleases are found isolated, respectively at the hair-pin formed by strands S3 and S4 and loop L5 (W8–seebelow) and at the C-terminus of the a-helix H1 (W9) (Fig. 4).

None of the above mentioned waters are completelyburied, but their solvent-accessible surface is small, rang-ing from 0.9 A2 to 15.6 A2. Not surprisingly, all of thesewaters belong to the 16 waters that we found to be mostconserved in the different packing environments of RNaseT1. Five out of these eight waters are invariant, while theremaining three waters are present in most other crystalforms of RNase T1, except for one or two cases. Inspectionof the crystal structures did not reveal any reason as whythese three waters were absent in those crystal structures.

Solvent Structure of the Bacterial RNases Barnaseand Binase

In contrast to the fungal RNases, where homologousstructures originating from seven organisms could becompared, the crystal structures of only three bacterialRNases are known. These are barnase and binase (from B.amyloliquefaciens and B. intermedius resp.) and RNase Sa(from S. Aureofaciens). Barnase and binase are verysimilar in sequence and structure. In the case of barnase,nine different molecules out of four crystal forms (pdbcodes 1bni, 1brn, 1brs, and 1rnb—Table II) were comparedusing molecule A from pdb entry 1bni as a reference.Eleven water molecules were found conserved in all bar-nase structures (Fig. 6). This is slightly more than the sixwaters found in eight different crystal environments ofRNase T1. Fourteen additional water molecules are found

in all but one barnase structure. These 25 conservedwaters in barnase will be referred to as Wb1 to Wb25. Theyare tabulated in Table V and their distribution on barnaseis shown in Figure 6.

Of these 25 waters, 16 are also present in at least five ofthe six structures of the closely related binase (out of twocrystal forms—Table II).

Most of the waters that are conserved in barnase andbinase occur in groups of two or three, making hydrogenbonds with each other or interacting with the samefunctional groups from the protein. Two groups becomeevident upon visual inspection of the crystal structures: (1)eight waters (Wb12-Wb19, see Table V) are involved inN-capping of the different a-helices and (2) four waters(Wb1-Wb4, see Table V) are found clustered in a networkthat mediates the anchoring of loop L6 upon loop L8 (Fig.6). As loop L8 of barnase is the structural alter ego of loopL6 in RNase T1, it is striking that the only cluster ofconserved waters in the barnase family is found on alocation that is topologically equivalent to the only clusterin the RNase T1 family. The local structures of barnaseand RNase T1 are, however, very different in this regionand no one-to-one relationship can be given between thesebarnase waters and the T1 waters. The remaining watersconserved in barnase and binase are not clustered.

Comparison of the Hydration of the Fungal and theBacterial RNases

Finally, we investigated whether any water is conservedin the fungal RNases (T1, Ap, Ms, F1, Pb, Th, and U2) aswell as in the bacterial RNases (barnase, binase, andRNase Sa). Only one water molecule is common to allmicrobial ribonucleases used in our comparison. This isalso the only water in common between the RNase T1-likefungal RNases and the closely related barnase and binase.Given the very low sequence identity and the large struc-tural divergence among these RNases, it is surprising tofind any. This highly conserved water molecule (W8 inTable IV) is located at the edge of the main b-sheet, wherestrands S3 and S4 of the major five-stranded b-sheet ofRNase T1 diverge (Fig. 7). In RNase T1, it hydrogen bondswith two main-chain and two side-chain partners. Onlythe interactions with the main-chain partners are con-served in the other ribonucleases. The structure of theadjoining hairpin loop connecting strands S3 and S4differs significantly (Fig. 7), which in the case of RNase Saresults in an additional hydrogen bond between the watermolecule and an backbone NH group. A small part of thisloop (at the exit of strand S3), however, has a localstructure that is highly conserved in the whole family ofmicrobial ribonucleases. This part of loop L5 is also theprime determinant of guanine specificity in the microbialRNase family.30 W8 does not contact the bound nucleotidein any of the structures, but may play an important role instabilizing the conformation of the nucleotide binding loop L5.

Although the overall structure of RNase Sa resemblesbarnase more closely than RNase T1, RNase Sa showsseveral local structural similarities with RNase T1 thatare absent in barnase. Two waters are common to all

128 R. LORIS ET AL.

RNase T1-like fungal RNases and RNase Sa (W3 and W6in Table IV, see Figures 7 and 8). These two waters arelinked to main-chain atoms of the loop L6 that followsstrand S4 in RNase T1, and form part of the conservedwater cluster that is found close to the a-helix. The localenvironments of these two waters in RNase T1 and RNaseSa are nevertheless strikingly different. There are fourconserved protein-water hydrogen bonds involving thesetwo waters. In RNase T1 these are W3...Val62(O),W6...Leu61(O), W6...Pro73(O), and W6...Asp76(N), all involv-ing loop L6. The equivalent hydrogen bonds in RNase Sa

are W4...Ile58(O), W28...Val57(N), W28...Arg65(O), andW28...Arg68(N). The other hydrogen bonds made by thesetwo waters are different and in the case of RNase T1involve three conserved waters (W2, W4, and W7). InRNase Sa, W7 is structurally replaced by OG1 of Thr59.Two water ligands of W4 in RNase Sa are also conserved indifferent RNase Sa structures (data not shown), but do notsuperimpose upon waters W2 and W4 of RNase T1.

Nevertheless, from the limited number of crystal struc-tures that are available, it seems that RNase Sa has itsown conserved water cluster in a region of the protein that

TABLE V. Waters ConservedAmong Barnase, Binase, and RNase Sa

SiteBfac(Å2)

Accessibility(Å2) Ba Bi Sa T1 Ligands

Cluster anchoring loop L6 onto loop L8

Wb1 2.0 0.0 9 — — — Val78-NWb2 6.0 2.8 8 6 — — Asn23-ND2 Gly48-O Wb3Wb3 22.3 6.3 9 6 — — Val78-OWb2 Wb4Wb4 27.0 9.6 8 5 — — Thr79-O Wb3

Bridging strands S2 and S3, extending the cluster Wb1-Wb4

Wb5 2.4 0.0 9 6 — — Ile51-N Asp75-OD2Wb6 4.7 0.0 8 — — — Asp75-OD2 Asn84-N Asn84-OD1

In the middle of the exposed side of the main b-sheet, buried under a number of large hydrophilic side chains and bridging strands S2and S3

Wb7 14.8 0.0 9 6 — — Asp54-OD2 Glu73-O Wb8Wb8 9.5 0.0 9 — — — Asp75-OD2 Glu73-OE1 Wb7

Stabilizing hairpin loop L7

Wb9 13.9 13.9 9 6 12 8 Phe56-O Trp71-NWb10 19.4 2.2 8 — 12 — Asn58-OD2Wb11 22.9 12.5 9 — — — Lys66-N Lys66-O Arg69-O

N-cap of Helix 4

Wb12 4.8 1.5 8 6 — — Ile42-N Gly81-O Wb14Wb13 2.0 6.1 8 6 — — Gly40-O Asn43-N Gly81-NWb14 21.0 6.3 9 — — — Ala37-O Wb12

N-cap of Helix 1

Wb15 10.3 19.5 9 6 — — Phe7-N Asn77-OD2 Wb16Wb16 23.5 32.3 8 6 — — Asp8-N Asp8-OD2 Wb15

N-cap of Helix 2

Wb17 10.4 19.0 8 — — — Ser28-N Ser28-OG Asp54-OD1Wb18 40.5 40.1 8 5 12 — Asp54-OD1 Ile55-NWb19 19.9 16.9 9 6 — — Gly52-O

Bound to hairpin loop L9

Wb20 20.8 3.3 9 6 — — Tyr90-O Wb22Wb21 25.8 9.2 8 6 — — Ser91-O Wb21

Bound to loop L8

Wb22 24.2 22.4 8 6 — — Thr79-N Ser80-N Ser80-O

Bound to solvent-exposed surface of Helix 1

Wb23 31.8 32.2 8 6 — — Gly9-O

Bound to an elongated loop L4 at the C-terminus of Helix 2

Wb24 2.4 36.4 8 6 — — Leu20-N

Stabilizing hairpin loop L10

Wb25 9.6 11.6 9 6 12 — Asn100-N Asp101-OD2 Thr105-O Thr105-OG1

129CONSERVED WATERS IN MICROBIAL RIBONUCLEASES

is equivalent to that where RNase T1 has its singleconserved cluster of six waters (data not shown).

In terms of global structure and orientation of the maina-helix relative to the conserved part of the major b-sheetRNase Sa and barnase/binase show greater similarity toeach other than either to T1 (Fig. 7). Three waters areconserved between barnase/binase and RNase Sa thatcannot be found in the T1-like fungal RNases. All threewaters are isolated (Fig. 7), and their local environmentsare much better conserved than is the case for the twowaters common between RNase Sa and the T1-like fungalRNases.

Hydrogen Bonding and Conservation of Water Sites

The identification of conserved water molecules in thepresent study is solely based on a distance criterion: twostructures are considered to have a water site in common ifafter least-squares superposition of the backbone atoms ofthe two proteins, two water molecules are found suffi-ciently close together. During crystallographic refinement,on the other hand, water oxygen atoms are typicallyassigned to blobs of (difference) electron density present atpositions where one or more hydrogen bonds with theprotein or other previously identified water molecules canbe assumed. It is therefore of interest to compare thehydrogen bonds of conserved waters in the different struc-

tures with each other and with the general hydrogenbonding features observed for the bulk of non-conservedwaters.

From a practical point of view, we consider only twoclasses of hydrogen bonds between water and protein :Wat...main chain atom and Wat...side chain atom. Thereason for not distinguishing between donors and accep-tors stems from the observation that in the case of sidechains it is often impossible to determine the donor andthe acceptor (OH groups can be both donor and acceptorwhile in the case of Asn and Gln the positions of side-chainO and N can not be determined from the experimentalelectron density maps). When looking at 1rga, our generalreference protein, the ratio of side-chain to main-chainatoms involved in hydrogen bonds with waters varies from0.71 (when all waters are considered) over 0.58 (for thewaters conserved in all space groups of RNase T1) to 0.46(when only the seven best conserved waters are consideredthat are present in RNases T1, Ap, Ms, F1, Pb, and Th).

Next we looked at the conservation of hydrogen bondsinvolving waters among the different structures. In eachcase, we calculated the hydrogen bonds in which theconserved waters were participating in the reference usinga distance cut-off of 3.4 A. In all other structures, werepeated these calculations, but using both 3.4 A and 3.9 A

Fig. 6. Location of structurally conserved wa-ters in different crystal environments of barnase.(a) A ribbon diagram of barnase is shown, with thesecondary structural elements color-coded as inFigure 3. Waters are shown as spheres, color-coded according to the number of different struc-tures in which they can be found. Orange spherescorrespond to waters present in barnase, binase,and RNase Sa. Green spheres denote waterspresent in barnase and binase, while waters thatare conserved only in the different barnase struc-tures are shown as light-blue spheres. The dark-blue sphere corresponds to a water present inbarnase and RNase Sa, but not in binase. (b)Close-up view of the cluster of waters that interactwith loops L6 and L8 and strands S2 and S3 inbarnase. Waters present in all barnase and binasestructures are colored orange and those present inall barnase structures, but absent in most of thebinase structures yellow. The side chains of resi-dues Asn23, Ser50, Asp54, Asp75, and Arg83 aswell as backbone atoms of residues 48, 49, 51, 56,71, 76, 78, and 79, all of which participate in thishydrogen-bonding network, are shown in ball andstick. Secondary structure elements are labeled.

130 R. LORIS ET AL.

Fig. 7. (Upper) . Water molecules conserved in the three different subfami-lies of ribonucleases examined [RNase T1-like fungal (T1, Ap, Ms, F1, Pb, Th,and U2), bacterial (barnase and binase) and RNase Sa]. Shown are RNaseT1, barnase, and RNase Sa in an identical orientation. Selected waters aredrawn as spheres colored depending on the subfamilies in which they are

conserved: all ribonuclease (blue) RNase T1-like fungal and RNase Sa(orange) or bacterial and RNase Sa (green). (Lower) . Water moleculeconserved in all ribonucleases examined, interacting with a hairpin loop L5connecting strands S3 and S4 (T1 nomenclature).

Fig. 8. Location of two structurally conserved waters common to theRNase T1-like fungal RNases (left) and RNase Sa (right) . These twowaters, which in the case of RNase T1 are part of the major cluster of six

conserved waters, are present in both the T1 family and in RNase Sadespite major differences in the global structure of the proteins as well asthe local environments of these two particular waters.

131CONSERVED WATERS IN MICROBIAL RIBONUCLEASES

as the maximum distance to identify hydrogen bonds. Thesecond criterion of 3.9 A was used to ‘‘rescue’’ hydrogenbonds which are just lower than 3.4 A in some structures,but slightly longer in others (for example due to theexperimental error on the atomic coordinates). For thewaters conserved in the validation structures, as well asfor the waters conserved in the canonical space groupP212121 (isomorphous to 1rga), the majority of hydrogenbonds occur in all structures even when using the strin-gent 3.4 A distance criterion. Little additional conserva-tion of hydrogen bonds can be gained from using therelaxed 3.9 A criterion. This situation does not changedrastically when comparing RNase T1 structures in differ-ent space groups. Between any two structures, most of thehydrogen bonds are still conserved. Hydrogen bonds thatare not conserved are almost invariantly associated withwater–side-chain contacts, where the side chain adopts adifferent conformation, probably due to the different lat-tice environment.

When going to homologous ribonucleases, the number ofconserved hydrogen bonds seems to correlate with theamount of sequence identity between the two proteins thatare compared. Given the very small number of watersconserved between homologous proteins (especially whencomparing distant homologues such as RNase T1 andRNase Sa), it is not possible to draw hard conclusions andthe information becomes anecdotal. In the case of W8,which is present in all ribonucleases examined, two hydro-gen bonds with main-chain atoms are conserved andadditional hydrogen bonds are also observed in somestructures (Fig. 7). In RNase T1, the additional hydrogenbond with the side chain Lys41 is fortuitous, as it is notconserved in different space groups of RNase T1 itself. W3and W6 on the other hand are involved in many hydrogenbonds in both RNase T1 and RNase SA, but in each caseonly one of these hydrogen bonds can be consideredidentical (Fig. 8).

Substrate Mimicry By Solvent

For many proteins, it has been observed that in theabsence of any bound ligand, inhibitor or substrate, theirbinding site(s) are occupied by solvent molecules which toa varying degree mimic the oxygen and/or nitrogen atomsof the natural ligand.32 In the present study, for fiveribonucleases ligand-bound/ligand-free pairs are avail-able: T1, F1, Sa, Ba, and Bi (see Table II). Analysis of thesepairs reveals that there is very little solvent mimicry in thecase of the microbial RNases. No ligand-mimicking watersare observed in barnase and binase, despite the apparentrigidity of their nucleotide binding sites. In the case ofRNase T1, the guanyl base of bound nucleotide inhibitorsdisplace two water molecules that are present in mostinhibitor-free structures. The positions of these two con-served water molecules do not coincide with oxygen ornitrogen atoms of the reaction product 3’GMP bound inpdb entry 1rga. One water is found halfway in between thepositions of N7 and O6 of the guanyl base, while the otherone is found half way in between N2 and N3. On the otherhand, in both RNase F1 and RNase Sa, N7 and O6 of the

bound nucleotide are mimicked by water molecules, whichhydrogen bond to loop L5 (F1) or L4 (Sa) respectively. Inthe case of RNase F1, this mimicry is observed despite asmall conformational change of loop L5.Asimilar conforma-tional change is also observed in RNase T1, but not inRNase Sa.

CONCLUSION

By analyzing the available crystal structures of a largefamily of homologous microbial ribonucleases, we investi-gated the reoccurrence of water molecules observed inmacromolecular crystal structures. A large number ofwaters that generally have a high solvent accessibility andB-factor are unique to only one or a small number ofcrystal structures, even when the structures that arecompared are closely isomorphous. Comparison of a num-ber of independently refined structures of the RNaseT1–2’GMP complex (validation structures) suggests that asignificant fraction of these unique waters are indeed realand not artifacts that are introduced by over-interpreta-tion of the electron density maps, as they are foundindependently by different investigators using differentprotocols and X-ray data. This means that subtle differ-ences in structure and crystal packing due to the bindingof related ligands and/or point mutations affect the overallhydration of the protein in the crystal. Most water mol-ecules therefore adapt themselves to the local protein (orcrystal) environment to optimize their interactions.

In the case of the RNase T1 and barnase crystal struc-tures, the positions of most well-determined water mol-ecules are primarily determined by lattice contacts, andare only conserved among isomorphous crystal structures.Only a small number of waters are conserved amongnon-isomorphous crystal structures. These are indepen-dent of crystal packing, point mutations, and the ligandedstate of the protein. They tend to have a low solventaccessibility and are also found in most of the crystalstructures of closely related RNases. In analogy to aminoacids that are conserved during evolution, one may specu-late that these waters play an important role in thestructure, function, stability, or the folding process of theprotein. This is especially the case for the small number ofwaters that are invariantly present in different subfami-lies which show almost no sequence identity and whosestructures have diverged considerably during evolution. Inthe case of RNase T1 and the subfamily of fungal RNases,several of the conserved waters (W1, W2, W8) are indi-rectly linked to the active site of the enzymes. Theyhydrogen-bond to residues adjacent in sequence to thecatalytic residues or residues involved in base recognition.This suggests that these waters play a role in sustainingthe correct structure of the active site. Alternatively, theuse and conservation of water molecules (instead of forexample amino acid side chains) at these positions may berelated by the ability of these water molecules to exchangewith the bulk solvent and thus providing the necessaryactive site dynamics during catalysis, substrate binding,and/or product release. These ideas are presently being

132 R. LORIS ET AL.

tested by protein engineering and physicochemical meth-ods.

We realize that the results presented in this paper referto only a single family of proteins: the microbial RNases. Itis expected that the number and percentage of structurallyconserved waters may vary in other protein families.Indeed it is obvious that size and shape of the protein,oligomeric structure, general flexibility, and the resolutionat which crystal structures are available influence thenumber of structurally conserved waters that will bedetected. We do, however, believe that the observed trendsregarding the influence of crystal packing and evolution-ary distance are representative for the surface waters onother proteins as well. The proteins investigated span afairly wide range of evolutionary distances, and the num-ber of crystal structures included in the analysis (bothisomorphous and non-isomorphous structures) is signifi-cantly larger that what has been used in other compara-tive studies.

ACKNOWLEDGMENTS

We thank Elke Brosens, Yves Geunes, and Maria Vander-veken for excellent technical assistance. We are grateful toKazuo Nakamura, Jozef Sevcik, Guy Dodson, Yves Mau-guen, Konstantin Panov, and Kostya Polyakov for kindlysupplying coordinates of crystal structures prior to publica-tion and deposition. R. Loris, J. Bouckaert, and D. Maes aresearch associates of the F.W.O.-Vlaanderen. S. De Vosreceived financial support from the I.W.T.

REFERENCES1. Edsall JT, McKenzie HA. Water and proteins. II. The location and

dynamics of water in protein systems and its relation to theirstability and properties. Adv Biophys 1983;16:53–186.

2. Eriksson AE, Baase WA, Zhang XJ et al. Response of a proteinstructure to cavity-creating mutations and its relation to thehydrophobic effect. Science 1992;255:178–183.

3. Buckle AM, Cramer PK, Fersht AR. Structural and energeticresponses to cavity-creating mutations in hydrophobic cores:observation of a buried water molecule and the hydrophilic natureof such hydrophobic cavities. Biochemistry 1996;35:4298–4305.

4. Matthews BW. Structural and genetic analysis of the folding andfunction of T4 lysozyme. FASEB J 1996;10:35–41.

5. Das G, Hickey DR, McLendon D, McLendon G, Sherman F.Dramatic thermostabilization of yeast iso-1-cytochrome c by anasparagine = leucine replacement at position 57. Proc Natl AcadSci USA 1989;86:496–499.

6. Hickey DR, Berghuis AM, Lafond G, Jaefer JA, Cardillo TS,McLendon G. Enhanced thermodynamic stabilities of yeast Iso-1-cytochrome c with amino acid replacements at positions 52 and102. J Biol Chem 1991;266:11686–11694.

7. Vriend G, Berendsen HJC, van der Zee JR, van den Burg B,Venema G, Eijsink VGH. Stabilization of the neutral protease ofBacillus stearothermophilus by removal of a buried water mol-ecule. Protein Eng 1991;4:941–945.

8. Lett CM, Berghuis AM, Frey HE, Lepock JR, Guillemette JG. Therole of a conserved water molecule in the redox-dependent ther-mal stability of Iso-1-cytochrome c. J Biol Chem 1996;271:29088–29093.

9. Berndt KD, Beunink J, Schroder W, Wuthrich K. Designedreplacement of an internal hydration water molecule in BPTI:structural and functional implications of a glycine-to-serine muta-tion. Biochemistry 1993;32:4564–4570.

10. Pedersen JT, Olsen OH, Betzel C, Eschenburg S, Branner S,Hastrup S. Cavity mutants of savinase. Crystal structures anddifferential scanning calorimetry experiments give hints of the

function of buried water molecules in subtilisins. J Mol Biol1994;242:193–202.

11. Rashin AA, Iofin M, Honig B. Internal cavities and buried watersin globular proteins. Biochemistry 1986;25:3619–3625.

12. Meyer E. Internal water molecules and H-bonding in biologicalmacromolecules: a review of structural features with functionalimplications. Protein Sci 1992;1:1543–1562.

13. Gilliland GL, Dill J, Pechik I, Svensson LA, Sjolin L. The activesite of bovine pancreatic ribonuclease: an example of solventmodulated specificity. Protein Pept Lett 1994;1:60–65.

14. Ladbury JE. Just add water! The effect of water on the specificityof protein-ligand binding sites and its potential application todrug design. Chem Biol 1996;3:973–980.

15. Ravishankar R, Ravindran M, Suguna K, Surolia A, Vijayan M.Crystal structure of the peanut lectin – T-antigen complex.Carbohydrate specificity generated by water bridges. Curr Sci1997;72:855–861.

16. Schwabe JWR. The role of water in protein-DNA interactions.Curr Opin Struct Biol 1997;7:126–134.

17. Steyaert J. A decade of protein engineering on ribonuclease T1:Atomic dissection of the enzyme-substrate interactions. Eur JBiochem. 1997;247:1–11.

18. Malin R, Zielenkiewicz P, Saenger W. Structurally conservedwater molecules in ribonuclease T1. J Biol Chem 1991;266:4848–4852.

19. Pletinckx J, Steyaert J, Zegers I, Choe H-W, Heinemann U, WynsL. Crystallographic study of Glu58Ala RNase T1*2’-guanosinemonophosphate at 1.9 A resolution. Biochemistry 1994;33:1654–1662.

20. Langhorst U, Loris R, Denisov VP et al. Dissection of thestructural and functional role of a conserved hydration site inRNase T1. Protein Sci. 1999;8:722–730.

21. Arni R, Heinemann U, Maslowska M, Tokuoka R, Saenger W.Restrained least-squares refinement of the crystal structure of theribonuclease T1–2’-guanylic acid complex at 1.9 A resolution. ActaCrystallogr B 1987;43:548–554.

22. Arni R, Heinemann U, Tokuoka R, Saenger W. Three-dimensionalstructure of the Ribonuclease T1*2’GMP complex at 1.9 A resolu-tion. J Biol Chem 1988;263:15358–15368.

23. Loris R, Stas PPG, Wyns L. Conserved waters in legume lectincrystal structures. J Biol Chem 1994;269:26722–26733.

24. Loris R, Van Overberge D, Dao-Thi MH, Poortmans F, Maene N,Wyns L. Structural analysis of two crystal forms of lentil lectin at1.8 A resolution. Proteins 1994;20:330–346.

25. Zegers I, Maes D, Dao-Thi M, Poortmans F, Palmer R, Wyns L.The structures of RNase A complexed with 3’-CMP and d(CpA):active site conformation and conserved water molecules. ProteinSci 1994;3:2322–2339.

26. Zhang XJ, Matthews BW. Conservation of solvent-binding sites in10 crystal forms of T4 lysozyme. Protein Sci 1994;3:1031–1039.

27. Mauguen Y, Hartley RW, Dodson EJ et al. Molecular structure of anew family of ribonucleases. Nature 1982;297:162–164.

28. Nakamura KT, Iwahashi K, Yamamoto Y, Iitaka Y, Yoshida N,Mitsui N. Crystal structure of a microbial ribonuclease, RNase St.Nature 1982;299:564–566.

29. Hill C, Dodson G, Heinemann U et al. The structural andsequence homology of a family of microbial ribonucleases. TrendsBiochem Sci 1983;8:364–369.

30. Sevcik J, Sanishvili RG, Pavlovsky AG, Polyakov KM. Compari-son of active sites of some microbial RNases: structural basis forguanylic specificity. Trends Biochem Sci 1990;15:158–162.

31. Sevcik J, Dodson EJ, Dodson GG. Determination and least-squares refinement of the structures of ribonuclease Sa and itscomplex with 3’guanylic acid at 1.8 A resolution. Acta CrystallogrB 1991;47:240–253.

32. Frey M. Water structure of crystallized proteins: high-resolutionstudies. In: Westhof E, editor. Water and biological macromol-ecules. London: MacMillan Press; 1993. p 98–101.

33. Steyaert J, Hallenga K, Wyns L, Stanssens P. Histidine 40 ofribonuclease T1 acts as a base catalyst when the true catalyticbase, glutamic acid 58, is replaced by alanine. Biochemistry1990;29:9064–9072.

34. Mayr LH, Schmid FX. A purification method for labile variants ofribonuclease T1. Protein Expr Purif 1993;4:52–58.

35. Zegers I, Loris R, Dehollander G et al. Hydrolysis of a slow cyclic

133CONSERVED WATERS IN MICROBIAL RIBONUCLEASES

thiophosphate substrate of RNase T1 analyzed by time-resolvedcrystallography. Nat Struct Biol 1998;5:280–283.

36. Navaza J. AMORE: an automated package for molecular replace-ment. Acta Crystallogr A 1994;50:157–163.

37. Murshudov GN, VaginAA, Dodson EJ. Refinement of macromolecu-lar structures by the maximum likelihood method. Acta Crystal-logr D 1997;53:240–255.

38. Lamzin VS, Wilson KS. Automated refinement of protein models.Acta Crystallogr D 1993;49:129–147.

39. Brunger AT. X-plor Version 3.1: a system for crystallography andNMR. New Haven: Yale University; 1992.

40. Collaborative Computational Project, Number 4. The CCP4 suite:programs for protein crystallography. Acta Crystallogr D 1994;50:760–763.

41. Haneef I, Moss DS, Stanford MJ, Borkakoti N. Restrained struc-ture-factor least-squares refinement of protein structures using avector processing computer. Acta Crystallogr A 1985;41:426–433.

42. Driessen H, Haneef MIJ, Harris GW, Howlin B, Khan G, Moss DS.RESTRAIN: restrained structure-factor least-squares refinementprogram for macromolecular structures. J Appl Crystallogr 1989;22:510–516.

43. Rees DC. Variance and covariance in experimental electrondensity studies, and the use of chemical equivalence. Acta Crystal-logr A 1979;32:483–488.

44. Bernstein FC, Koetzle TF, Williams GJB et al. The protein data

bank: a computer-based archival file for macromolecular struc-tures. J Mol Biol 1977;112:535–542.

45. Hutchinson EG, Thornton JM. PROMOTIF – a program toidentify and analyze structural motifs in proteins. Protein Sci1996;5:212–220.

46. Kabsch W, Sander C. Dictionary of protein secondary structure:pattern recognition of hydrogen-bonded and geometrical features.Biopolymers 1983;22:2577–2637.

47. Hubbard SJ, Thornton JM. ‘‘NACCESS’’, Computer Program.London: Department of Biochemistry and Molecular Biology,University College London; 1993.

48. Lee B, Richards FM. The interpretation of protein structures:estimation of static accessibility. J Mol Biol 1971;55:397–400.

49. McDonald IK, Thornton JM. Satisfying hydrogen bonding poten-tial in proteins. J Mol Biol 1994;238:777–793.

50. Zegers I, Haikal AF, Palmer R, Wyns L. Crystal structure of RNaseT1 with 3’-guanylic acid and guanosine J Biol Chem 1994;269:127–133.

51. Buckle AM, Henrick K, Fersht AR. Crystal structure analysis ofmutations in the hydrophobic core of barnase. J Mol Biol 1993;234:847–860.

52. Esnouf RM. An extensively modified version of MolScript thatincludes greatly enhanced colouring capabilities. J Mol GraphModel 1997;15:132–134.

134 R. LORIS ET AL.