The refined structure of ribonuclease-A at 1.45 � resolution

Journal of Crystallographic and Spectroscopic Research, Vol. 14, No. 5, 1984

The refined structure of ribonuclease-A at 1.45 ,~ resolution

NIVEDITA BORKAKOT1, DAVID S. MOSS, MICHAEL J. STANFORD, and REX A. PALMER*

Department o f Crystallography, Birkbeck College University o f London, Malet Street

London, WC1E 7HX, UK

(Received Ju(v 17, 1983)

Abstrac t

Features of the refined X-ray crystal structure of bovine pancreatic ribonuclease-A at 1.45 A resolution are described. The positions of the protein atoms have been determined within the range 0.004-0.05 A~, and of solvent atoms, assumed to be oxygens, within the range 0.08-0.13 A. The present model contains 127 solvent molecules, taken to bewater, and a sulfate anion located in the active site. Mean square atomic displacement parameters, /.Aso, refined for each atom, give an indication of the mobility of different parts of the structure. Main-chain Uiso values tend to be less than side-chain values, having an average value of0.15 A 2 compared to 0.25 ,~2. Both main-chain and side-chain average Uiso values tend to increase with distance from the center of gravity of the molecule. Side-chain average Ui~o values also tend to increase with the number of atoms in the side-chain, with different distributions for ring and chain type residues. Side-chain conformations have been analyzed and found on the whole to follow commonly observed distributions. A notable exception to this is the active-site residue His-119 which occupies two distinct sites. Apart from two small clusters of eight and seven atoms respectively, the solvent molecules are distributed in quite small numbers on the protein surface. The solvent clusters occur in the active-site region and, together with the sulfate anion, appear to stabilize residues in this region. Sixty-three solvent atoms have only one identified hydrogen bond contact. Of the rest, 36 form two, 22 form three, and 6 form four hydrogen bonds. There is

467

0277-8068/84/1000-0467503.50/0 �9 1984 Plenum Publishing Corporation

468 Borkakoti et al.

a marked tendency for the mean square displacement parameter, Uiso, for the solvent atoms to be lower for atoms with many hydrogen bond contacts than for those with fewer contacts.

Introduction

The crystal structure of bovine ribonuclease-A was recently refined at 1.45 A resolution (Borkakoti et a l . , 1982) using the least-squares procedure of Morffew and Moss (1982) in conjunction with computer graphics model building (Jones, 1978; Tickle, 1979). This refined model, which supersedes all previous X-ray models of ribonuclease-A, provided details of the main-chain conformation, the secondary structure and its hydrogen bonding, the active- site geometry and sulfate anion, solvent structure in the first coordination sphere, intermolecular packing, and atomic thermal vibrations. A limited discussion of these features was given by Borkakot ie t al. (1982) and we present here further details both of the refined structure and additional features listed below. We have now undertaken an analysis of the thermal motion of the main-chain, side-chains, and solvent atoms, and potential energy func- tions for the active-site residues His-12 and His-119. Further details are also presented in this paper of the main-chain conformational parameters, the helix and /3-sheet structures, side-chain conformations, and the solvent binding.

A detailed analysis of hydrogen bonding, charge distribution, and energy calculations will be published elsewhere.

Experimental

X-ray intensity data for the high-resolution analysis were measured on large crystals (_--<0.7 X 1.0 X 0.7 mm) grown in 30-40% ethanol at pH 5.2-5.7 from type I bovine ribonuclease-A (Sigma) (Rosemeyer, 1961; Gorinsky, 1971; Carlisle et a l . , 1974). Crystal data are listed in Table 1. Intensities were measured on a computer controlled Hilger-Watts Y290 four-circle diffrac- tometer, employing a total of five different crystals for the 1.45 A data set, scaling being facilitated by overlapping measurements. Most intensities were measured twice, either as overlap repeats or symmetry equivalents. After merging, a unique set of 19,238 intensities was available for the analysis. The Lorentz polarization and absorption corrections were applied in the usual way.

Structure of Ribonuclease-A at 1.45 A

Table 1. Crystal data for 1.45 A refinement of ribonuclease-A

469

Space group Unit-cell dimensions

Number of molecules per unit cell Relative atomic mass of protein Number of symmetry-independent reflections Number of residues Number of protein atoms Number of solvent atoms located to date Rms deviations from target geometry

Average mean-square atomic displacement (/Tiso)

R = ~:~,llFol -IFclI/IFol Rms shift in coordinate in final cycle of refinement

P2I a = 30.45 A b = 38.37 c = 53.22 /3 = 105.96 ~ 2 13,685 daltons 19,238 124 951 127 0.018 A (bonds) 0.038 A (bond angle distances) 0.012 A (least-squares planes:

peptide) 0.014 A (least-squares planes:

side-groups) 0.15 A 2 (main-chain atoms) 0.25 A 2 (side-chain atoms) 0.49 A 2 (solvent atoms) 0.23 (all data) 0.024 A

Methods used and general assessment o f results

Refinement and model building

The structure was refined using the program RESTRAIN (Morffew and Moss, 1982) which minimized the funct ion

M = Y~Oov(lFo[ --]Fc[ )2 + Za~d(dT -- de) 2 'b s

where ] Fo[ and [ Fc[ are observed and calculated structure ampli tudes; dT and

dc are target and calculated in tera tomic distances between bonded atoms and be tween a toms across b o n d angles; D are d e t e r m i n a n t s of v a r i a n c e - covariance matrices of coordinates of groups of a toms restrained to be planar. P lanar i ty restraints were applied to the rings of histidine, phenyla lanine , and tyrosine residues and, to a lesser degree, to C ~ - C O - N amide planes of the main-chain.

The parameters refined were the Car tes ian coordinates and a mean- square displacement or isotropic thermal parameter , Uiso, for each nonhydro - gen a tom. All a toms were refined with a fixed occupancy factor of uni ty except for the side group of His-119 for which two posi t ions were refined with separate occupancies, cons t ra ined so that the sum of their occupancies was unity. Restra ints were not applied to either hydrogen bonds or van der Waals


contact distances. No restraints between the Uiso values of neighboring atoms were applied. Some statistics concerning the refinement are given in Table 1.

During the course of the refinement, analysis and rebuilding of the model, as necessary, was undertaken on an Evans and Sutherland Picture System 2 computer graphics system using the program FRODO (Jones, 1978) modified by Tickle (1979). Correction of errors in the model and the location of 127 solvent molecules (water) was carried out using (21Fol - ] F c l) maps.

The geometrical analysis of the refined structure including van der Waals contacts, hydrogen bonding, and torsion angles was carried out using programs written by members of the group (Borkakoti, 1981; Stanford, 1981).

Accuracy and estimation of errors

Assessment of errors in the refined structure was undertaken by two methods.

1. The diagonal elements of the normal matrix (Nii) obtained from the unrestrained refinement were used to derive estimated standard deviations (oi) of the parameters by means of the expression

= rx~o(i F o l - IFcl)21 ''2

where m is the number of reflections and n is the number of parameters. As would be expected, these approximate standard deviations cover a wide range of values and are correlated with Uiso. The range of values may be summarized as follows (Borkakoti et al., 1982):

Protein atoms: a = 0.004-0.050 A with Uiso = 0.04-0.80 •2

Solvent oxygens: a = 0.08"0.130 A with Uiso = 0.10-0.60 A 2.

The neglect of off-diagonal terms of the normal matrix affects the estimates of or, and the above error estimates for the protein atoms are certain to be underestimates due to correlation between positional parameters of neighboring atoms at 1.45/~ resolution. The solvent oxygen atoms are further from other nonhydrogen atoms, and their a estimates should be less seriously affected. The precision of the well-ordered protein atoms may not be much better than that of the best solvent oxygens which is around 0.05 A.

2. Estimated standard deviations obtained by the above method are merely measures of the sharpness of the minimum into which the refinement has converged. The protein crystal actually contains many conformational states, some of which will correspond to other minima in the function M, close to the minimum located by our refinement. In order to explore other local minima, the refined atomic coordinates, model (a), were given random perturbations and then the molecular geometry was re-regularized to give

Structure of Ribonuclease-A at 1.45 ,~ 471

model (b). The mean displacement from the refined model (a) at this stage was 0.11 A. Refinement of this perturbed model was carried out until the atomic coordinate shifts were approximately the same as in the original refinement, yielding model (c): Most atoms refined again toward their original refined positions. However, 13 side-chains mostly of long, external hydrophilic residues remained with some or all of their atoms greater than 0.05 A away from model (a). In some cases (Lys-31, Asn-34, Lys-37, Asp-38, and Lys-41) the side-chains are in poorly defined electron density in 2([ Fo [ - I F c I) maps where gross disorder obviously exists. In other cases (Lys-l, Arg-10, Asn-62, Gln-69, Gin-74, Tyr-76, and Arg-85) the electron density is fairly well defined but some residues may still occupy multiple positions which have not yet been resolved.

Changes in the model as a result o f the refinement

The 1.45 A model of ribonuclease-A was derived from Wlodawer's (1980) 2.5 A X-ray and 2.8 A neutron diffraction studies. Although the rms deviation in atomic position between the current model and the starting model is 1.1 A, this includes some shifts of 7-8 A where extensive rebuilding of side groups was necessary.

Mean-square atomic displacements

Individual isotropic mean-square atomic displacement parameters were refined for all atoms including solvent atoms (assumed to be oxygen). The parameter refined was Ui~o = B~ 8rr 2 A z. Average Uiso values (Uiso) for the main-chain and side-chain atoms are plotted in Fig. 1 for each residue.

Main-chain Uiso distribution

The main-chain Ui~o values are both lower and more evenly distributed than the side-chain values (Fig. 1), indicating a higher degree of ordering in the main-chain. High Ui~o values are observed in the following main-chain regions: Lsy-1, poorly defined in the electron density, externally situated in an environment exposed to the solvent; Ser-21, Glu-101, Asn-113, and Ser-123, all externally situated and having no appreciable H-bond contacts (main- chain or side-chain); Val-124 at the C-terminus, exposed environment. The average over all main-chain Uis o values is 0.15 A 2.

Side-chain Uiso distribution

The average value of Uiso over all side-chains is 0.25 A. In the following discussion a value of U--iso of 0.18 A 2 is considered appreciably lower and of


t,-

t,J

c

- - I - -

I - - o

_ I _ _ - - 0 - -

- i - - - - o _ _ III

[ - - - - o - - I ~ - - 0 0 - -

- - o + " ~

__--<~--

0 - -

- - ' 4 '

<,,l .........

,,+[-- i i I f

0'<' I:~

ed

~

+~ m~

Structure of Ribonuclease-A at 1.45/~ 473

0.34 A 2 appreciably higher than the average value. Most residue types show a wide variation in U--iso values, smaller values often corresponding to tightly packed residues and larger values to externally facing exposed residues. Nonpolar residues forming hydrophobic clusters tend to have less than average Uiso values. The 18 side-chains in this category have an average Uiso of

�9 2 0.16 A 2 compared to the average over all nonpolar residues of 0.22 A . High Uiso values are observed in the following side-chain regions (Fig. 1, Table 2): A (residue 16-24) and B (33-41) both poorly defined in the electron density and externally situated; regions C (61-69), D (74-77), E (91-94) and F (113-115)

Table 2. Average side-group Uis o values in groups or regions a

Region Residues Average Uiso • 102 (A) Situation

a. Regions of high Uis o A 17-24 28 External B 34-39 38 External C 61-69 29 Loop D 74-77 26 External E 91-94 33 Loop F 113-115 34 Loop N-terminal 1 50 C-terminal 122-124 44 b. Regions of low Uiso a 12-17 18 Internal b 42-47 20 Internal c 54-58 15 Pocket d 70-73 20 External e 78-82 17 Pocket f 105-110 17 Pocket g 115-121 22 Internal c. All chain type residues (96 residues) 27

All ring type residues (25 residues) 19 d. Helix regions t~I 3-13 21 a l I 24-33 22 cdll 50-60 19 e. Sheet regions /3I 42-48 19 /3II 71-92 25 /3111 94-110 27 f. Active site (as)

3-8 and 10-16 22 35 22

45-47 23 106-110 16 116-120 22

~The average overall side-chain Uiso is 0.27 A. External and loop residues (Figs. 1 and 6 and Table 3) tend to be appreciably higher, and internal or pocket residues appreciably lower, than average values. Helix, sheet, and active site Uiso values also tend to be low. N- and C-terminal Uiso values are very high.


where C, E, and F form external loops and D is in an externally situated /3-sheet region. Low Uiso values occur in regions: a (12-17), b (44-48), c (54-58), d (70-73), e (78-82),f(I05-110), andg (115-121) where a, b , f , andg are close to the active site (Fig. 4), region c is in a-helix II (Fig. 5), and d and e are in B-sheet regions. At the N-terminus Lys-1 close to the entrance to the active site and poorly defined in the electron density has high main-chain and side-chain Uiso values. Similarly Ser-123 and Val-124 at the C-terminus have high Uiso values. Several other lysines poorly defined in the electron density also appear to be disordered with high Uiso values (Table 2d). Arginines show a great deal more variation in Uiso than do lysines. Arg-33 in c~-helix II has notably small thermal parameters possibly as a consequence of the strong intramolecular H-bond N H . . �9 O=C(Met - 13). There is a tendency for ~ o to increase with the number (N) of atoms in the side-chain for both chain and ring type residues (Fig. 2). This observation is a useful confirmation of the physical significance of the refined Uiso values derived in the analysis. It is interesting to note that prolines (N = 3) fit into the curve for chain type residues and cystines (N = 5) into the curve for ring type residues. The average Pro Uiso is thus larger than might be expected. This is largely due to Ui~o for Cv being high in most cases, an effect possibly due to disorder. Table 2 is a summary of average Uiso values in various specified regions. Figure 3 is a plot of distance of main-chain (C~) and side-chain (terminal atom) from the S atom of the active-site sulfate ion for each residue. As mentioned previously regions A - F (Figs. 1 and 5) tend to have high U--iso values and regions a-g low Ui~o values. Regions A - F as indicated in Fig. 3 tend to be further away, and a-g closer to the active site. These results suggest a possible correlation between Uiso and distance from the active site, an idea which was investigated further as described below.

In order to investigate the radial distribution of Ui~o in the enzyme, plots were prepared of main-chain and side-chain Ui~o values as a function of

Fig. 2. Uis o plotted against number of side- group atoms. The upper graph (dots) is for chain-type residues while the lower graph (open circles) is for ring-type residues. The point for cystine bridges (arrowed) lies on the graph for ring-type residues.

40

3O

Uiso A 2 20

lO

! ~ C h a i n

r, 0 . ~ Ring

Cys

! J i ! I ! I J 1 2 3 4 5 6 7 8 N

s i d e g r o u p a t o m s

Structure of Ribonuclease-A at 1.45 A 475

�9 ~ >

m

~ < ~

o u~ o ~ n ~n o ~n o ~ . ' ~ ~ ' z


.52

.48

144

<U,.o) . 4 0

.315

. 3 2 r

t - . 2 8 L~

- 0 ~ . 2 4

ffl

. 2 0 . 2 8

.16 T f . . . ~ .24 .E

. 1 2 . 2 0 (3

�9 ~ .E IE

. 1 2

i L [ I I t t t i I ~ t t { I I I I l I I I 4 6 8 10 12 14 16 18 2 0 2 2 2 4

rA

Fig. 4. Plot of { Uiso), the local average of Uiso, as a function of r G (,~), the distance from the geometrical center of gravity of the molecule. The upper values (crosses) are for the side-chain averages, and the lower values (dotted) are for the main-chain averages. Bars represent esd's.

d is tance f rom the sulfate g roup and also f rom the cen t ro id of the molecule. The cen t ro id plots show the be t te r l inear co r re l a t ion (Fig. 4). The ma in -cha in values show only a smal l rad ia l increase and the l inear co r re l a t ion precludes molecu la r l ib ra t ion as the p r e d o m i n a n t cause. The larger Uiso values, pa r t i cu la r ly on the outs ide of the molecule, suggest s ignif icant l ib ra t iona l d i so rde r of the s ide-chains a b o u t pos i t ions near the Ca a toms. S imi la r results have been repor ted for lysozyme (S te rnberg et al., 1979; A r t y m u i k et al., 1979).

T h e s t r u c t u r e

General description

A schemat ic r ep resen ta t ion of the r ibonuc lease molecule is shown in Fig. 5, and Fig. 6 shows the po typep t ide chain c o n f o r m a t i o n and water molecules cur ren t ly ident i f ied in the analysis . The qual i ty of the analys is is i l lus t ra ted by

S t r u c t u r e o f R i b o n u c l e a s e - A a t 1.45 A

_ 113

477

5 8

61

HIS12

33

I 00

124

.,.0_0_ HEL IX

. . . . H - B O N D S ( S E L E C T E D )

S - S B R I D G E S

Fig. 5. Schematic diagram ofthe ribonuclease structure. Helixregionsare~I(3-13),c~II(24-33), and ali i(50-60). Sheet regions are /3I(42-48), /3II(1-92), and /31II(94-110). Regions A - F (including loops, C, E, and F) are external regions of high side-chain Uiso and regions a - f a r e internal or pocket regions with low side-chain ~,o (see also Fig. 2 and Table 3).

~ " ~ �9 ~ �9 .'~

. . . . . ! i - . " .

Fig. 6. Stereoview of the ribonuclease-A main-chain conformation and water molecules currently identified in tlae structure.

478 B o r k a k o t i e t al.

t!:. 1 ., A la 19 �9 ~ � 9 1 4 9 1 4 9 1/i e e

A l a 1 2 ; : � 9 * ~ : � 9 �9 �9 �9 1 4 0 .

"�9 �9 �9 1 2 0 - �9 " " G l u 2

�9 e e ! �9 �9 1 0 0 "

80 .

60 .

40 '

. : 20 .

I I t " I =~ ' , ~ : ' - , a 0 -16 0-140 -120 ;1~bf - I . 2 0 - 4 0 - 2'0

Ser21~ �9 ~ - 2 0 .

- 6 0 - p

- 8 0 ,

- 1 0 0 .

- 1 2 0

- 1 4 0 '

Gin 60 - 1 6 0 .

- 1 8 0

A s n 3 4

80 100 120 140 160 180

G l y68

: : 20 40 60

* G ly112

Fig. 7. (~b, ~0) Plot for ribonuclease-A.aRamachandran's allowed regions are indicated: O corresponds to right-handed a-helix, ~ to 3~0 helix, Q) to antiparallel/3-sheet, and �9 to parallel-chain/~ pleated sheet (adapted from Dickerson and Geis, 1969). | indicates observed 310 turn in helix region I (see text). Several glycines lie as might be expected in "unfavorable" regions, and Asn 34 in disordered region B of the structure (Table 2 and Fig. 5) also has an unusual (4~, ~0) conformation marked "k.

examples f rom the electron density in Fig. 7. The general organization of the molecule into two lobes has been described previously (Carlisle et al . , 1974, Borkakoti et al., 1982). Residues involved in the catalytic mechanism are located in the cleft formed between the lobes. The corrected main-chain hydrogen bonding scheme has been described by Borkakoti et al. (1982). There are three sections of helix (a I, a II, and a III; Fig. 5) involving residues 3-12, 24-33, and 50-60, respectively. There are also three stretches of polypeptide chain involved in an extensive sheet format ion (/3I,/3II, and/3 III; Fig. 5) including residues 42-48, 71-92, and 94-110, respectively. Pro-93 and Pro-114 both having cis conformations are involved in reversal of the chain direction in the formation of an antiparallel fl-sheet region linking the two side-lobes of the molecule about the active site cleft.


Secondary structure: 1 helix and B-sheet

A ~b, ~ plot of the main-chain torsion angles is given in Fig. 7. With the exception of Asn-34 in the disordered B region the features of this plot are quite normal.

Helices were identified both f rom the (4~, ~O) values and the hydrogen bonding schemes. The mean (oh, q~) values in helix regions a I , (3-13), a I I (24-33) and a III (50-60) are ( -65 ~ , - 38 ~ compared to the proposed values of ( - 5 7 ~ - 4 7 ~ for a-polyalanine (Arnott and Dover, 1967). Similar average values have been reported for other highly refined protein structures, for example ( -64 ~ - 4 0 ~ in erythrocruorin (Steigemann and Weber, 1979), ( -63 ~ - 4 0 ~ in actinidin (Baker, 1980), ( -64 ~ -41 ~ in human lysozyme (Artymiuk and Blake, 1981), and also ( -67 ~ - 4 7 ~ in bovine pancreatic trypsin inhibitor (Deisenhofer and Steigemann, 1975). Such deviations of the average (4~, $) angles f rom ideal values indicate a tilt of the C = O bonds with respect to the helix axis, as can be seen, for example, in Fig. 9a. This effect is also associated with markedly nonlinear C~-~-O �9 �9 �9 N hydrogen bond angles, having average values of 149 ~ in ribonuclease-A and 154 ~ in actinidin (Baker, 1980). A small 310 twist between Met-30 and Arg-33 in helix region aI , identified by its hydrogen bond pattern (Borkakoti et al., 1982), has even more pronounced outward bending of the C = O bonds. The average C~----O �9 �9 �9 N hydrogen bond angle (four bonds only) of 125 ~ is the same as that in the 310 regions of erythrocruorin (Steigemann and Weber, 1979). There is also a short section of 310 helix in region a I I I f rom Gin-55 to Gln-60.

Figure 9b shows part of the extensive B-sheet structure of ribonuclease-A. Further details of the hydrogen bonding will be published separately.

Geometry o f the proline residues and turns

Of the four prolines in ribonuclease-A, two, Pro-42 and Pro-47, have trans peptide configurations and two, Pro-93 and Pro-114, are cis. The relevant torsion angles are

~ to (deg) Pro-42 - 6 0 -51 -179 t rans Pro-93 -107 14 20 cis Pro-114 - 6 5 -170 8 cis Pro-117 - 7 4 142 -178 trans

The puckering of the proline residues iscurrently under review.The cis prolines 93 and 114 are both involved in sharp turns between/3 regions.

The geometry of bends in ribonuclease-A specified in terms of the scheme of Lewis et al. (1973) is given in Table 3. Bends, reported to occur in the same

1 . . . . . . . The defimtlon of conformation (torsion angle) according to I UPAC convention (1970) was used. For the sequence of atoms PQRS the torsion angle is that required to rotate P into S looking down bond QR. Clockwise rotation is +, the sign being independent of view direction.


regions of ribonuclease-S (Lewis et al., 1973), appear to have different geometrical features f rom those found in ribonuclease-A. Some of these differences may be real, but a true compar ison is not possible wi thout reference to a refined ribonuclease-S structure. Region A in r ibonuclease-A is p robably an extended i to i + 4 loop (Lewis et al., 1973) occurr ing as mentioned previously in a stretch of poorly defined electron density.

Pept ide g e o m e t r y and c o n f o r m a t i o n angle to

Figure 10a shows the geometry derived f rom the 1.45 A refinement of r ibonuclease-A averaged over the 122 trans peptide residues. The geometry of Pauling et al. (1951), shown for compar ison in Fig. 10b, was used for calculation of the amide H positions in r ibonuclease-A employed in the analysis of hydrogen bond goemetry, the results of which will be published elsewhere. The average value of the peptide group torsion angle to for the 121 trans residues is 180 ~ with a s tandard deviation of 9 ~ . The distr ibution of to' = ----- ( 180~ - ]tol), where to' > or < 0 if to > or < 180 ~ , respectively, along the polypeptide chain is given in Fig. 11. We have investigated the possibility but failed to find any evidence in our data of correlation between co' and the Page peptide bond length dcN. Al though the correct functional relationship between to' and dcN may be defined for specific residues b y a b in i t io quan tum

(a) Fig. 8. Examples of electron density in the refined structure of ribonuclease-A: (a) Met-30; (b) Tyr-92; (c) stereoview of location of a water oxygen in the difference electron density (21Fol - Fcl) in ribonuclease-A. O(310) coordinated to Ser-16 and Asp-14.


(b)

~ ~.o..

(c) Fig. 8. (continued)


QN•2 ~ 13

~~176176176 1 ~ ~ %,.

..-" " s

" t. r

5 : 3

(a)

~_2 ~ 13

. t$

] "- 9

"8 0

6

/ ' s

5 : 3

~3

47 48 47 ~8

45 "~" e~ 80 45 " " 88 8~

lg ~1 tg i

IB3 82 /

84 ' ~4 " , 2 i 84 64 ~ ~ 2

~83 ;88 i8~ �9 8~ 18Z 18

(b) F i g . 9. (a) Stereoview illustrating the geometry of a-helix I (residues 3-13). (b) Part of the fl-sheet structure in ribonuclease-A (regions b and e as indicated in Table 2 and Fig . 5). Departure from the ideal/3-sheet structure can be seen (top right) where transition to helix III takes place.

mechan~ical ca lculat ions , intuit ively one would expect dcN to increase with increasing w', being m i n i m u m for oJ' = 0 or oJ = 180 ~ .

Side-chain conformations 2

The conformat ion of each side-chain is described in terms of torsion angles X 1 to X,, where X 1 is always defined by NC~ C~ C~, and n <= 3 except for lysines (n = 4) and arginines (n = 5). 3 This assumes that imidazole and phenyl

ZThe nomenclature for specifying the torsion angle ranges is (e.g., Saenger, 1973): • ( ~ • 1 7 6 t(~- 180~ c(~ 0~

3A sixth torsion angle, X6, is related to X5 by X6 - Xs "~" 180 ~


c- o

, .o

e -

g

g

> > > > > = ~

I

7 , , ~ 7 , 7 ,

7 T777

, 7 1 , , , , 7

o

r~

> O

m + o

+ ~

484 Borkakot i et al.

Fig. 10. Peptide geometry: (a) derived f rom the 1.45 A refinement of ribonuclease-A; (b) values of Pauling et al., (1950, for comparison. These values were sub- sequently used in the calculation of peptide amide H positions.

N 1.471

". -,-,^N ~ - 132., / AT.u'.C " 'N ,,

0 "" Ca (a)

Ca,,,~O., ~, ~ ~ H

,,,~_ttK- 1.32 (~'~7 121 ,t:; N 114

(b)

rings are essentially planar. The conformations of S-S bridges will be described below using the nomenclature of Thornton (1981). Figure 12a shows the distribution of X1 torsion angles, Fig. 12b (in view of the relatively small sample) shows the composite distribution of X2 and X3 values in the refined ribonuclease-A structure. In Fig. 12a commonly occurring X1 values are indicated for each residue type. Values of X1 show a well-defined trimodal distribution (Janin et al., 1978) with average values in the three regions (g ~-- 60 ~ t ~-- 180 ~ - g ~-- - 6 0 ~ of 65, -178, and - 6 4 ~ and corresponding populations of 22, 25, and 42%, respectively. The most populated X regions broken down according to residue type are indicated below (% for each residue is given in parentheses).

X J g t - g

Thr (56%) Val (56%) Met (100%) Ser (60%) Tyr (50%) Tyr (50%)

Lys (50%) His (100%) Arg (50%) Cys (100%)

Glu (60%)

2C le

,.,d 1~

de(; 8

- 4

- 8

- 1 2 - 1 6

- 2 0

, , I] ,I j, t ,Ij j , , o '~ 3,o 4t 51o 8o~Jeo 8o o', ,,60 "o" n

Fig. 11. Bar Plot of o / = _+ (I 80 - J co I ) ~ in ribonuclease-A. The residue numbers refer to the first in sequence. X denotes cis-prolines. The sign of oJ' is positive if o~ < 180 ~ and negative otherwise.


2 5

3TYR 20 [1PHE I

10 ~ SN �9 YS

5 ~,

10 30 50 70 90 110 130 150 170-170-150-130-110-90-70-50-30-lOdeg (a) Xl

1 5

-- 4LYS 3~ I rg 4GLU 5GLN 10 4~lu llLE

I I i I I I i I I I I I 1 I I 1 i 1 0 3 0 5 0 7 0 9 0 110 1 3 0 1 5 0 1 7 0 - 1 7 0 - 1 5 0 - 1 3 0 - 1 1 0 - 9 0 - 7 0 - 5 0 - 3 0 - 1 0 d e g

X2o,X3 (b)

Fig. 12. Side-chain conformations: (a) distribution of X~ side-chain torsion angles in ribonuclease-A. Averages for each of the three regions are indicated by V. The frequencies of residues around the three maxima are indicated. (*) For valines thus indicated the two X ~ values are in the region -60, 180, (**) For valines thus indicated the two X ~ values are in the region -60, 60. ( � 9 For threonines X t = N-C~C/30~I. (b) Composite distribution of X2 and X3 torsion angles in ribonuclease-A, X2 is indicated in upper-case and X3 in lower-case. The numbers of residues involved are indicated in selected regions.

The tors ion angles X2/X3, Fig. 12a, show a less well-defined d is t r ibut ion than X l, with the following values commonly occurring:

60 ~ , 90 ~ , 140 ~ , 180 ~ , - 1 2 0 ~ , - 6 0 ~

C o m m o n l y occurr ing X2 (upper case) and X3 (lower case) values are indicated for each residue type. The most popula ted X2 and X3 regions b roken down according to residue type are indicated below:

x (deg) 60 ~ (g) 90 ~ 140 ~ 180~ ~

X2 His (75%) Arg (75%) Lys (36%) Asn (30%) Glu (80%)

Glu (71%) X3 Gin (71%) Arg (75%)

Glu (80%)

-120 ~ - 6 0 ~ ( -g)

Lys (54%) Tyr (100%) Asn (30%) Phe (75%)

Met (75%)

486 Borkakot i et al.

Noteworthy side-chain conformations are as follows:

75% of methionines have a - g , t, t conformation. 50% of tyrosines and 60% of phenylalanines have a t, - g conformation. 50% of tyrosines and 40% of phenylalanines have a -g , - g conformation.

In view of the small population of values in ribonuclease, no further comment on the above side-chain torsion angles are possible.

Disulfide bridges

The conformation angles (deg) for the four S-S bridges in ribonuclease-A are given below:

Bridge X l X 2 X3 X 2 ' X l t

I Cys-26-Cys-84 -77 -81 -80 -55 -64 II Cys-40-Cys-95 -56 -53 -79 -71 -54

III Cys-58-Cys-110 -75 -54 -91 -123 -58 IV Cys-65-Cys-72 -50 -67 111 -96 -89

These conformations all fall into one of the two common types (Thornton, 1981). Bridges I, II, and III are left-handed (X3 ~ -90~ and IV is right- handed (X3 ~" 90~ being part of a very tight local loop (C) containing only seven residues.

Lysine conformations

There are ten lysine residues in ribonuclease. All exhibit disorder in the form of high mean-square atomic displacement parameters. No definite pattern of conformation is followed except that all torsion angles (X 1-X4) fall approximately in either _+g or t ranges. The conformations observed can be categorized as extended (predominantly t), coiled (predominantly _+g), or intermediate (mixed +g and t). There are accordingly four extended lysines (7, 61, 89, and 104) with an average chain length Ca" �9 �9 N~ of 6.1 A, two coiled (66 and 91) with an average chain length of 4.9 A, and four intermediate (1, 31, 37, and 41) with an average chain length of 5.5 A.

Arginine conformations

The torsion angles X 1 to X 5 for the four arginine residues in ribonuclease- A are given in Table 4. Again, most of the torsion angles are either +g or t, the latter including several values between 120-140 ~ No particular conformation pattern is dominant. Arg-85 is coiled with a chain length of 5.3 A appreciably less than the other three arginines which have an average chain length of 6.8A.

Structure of Ribonuclease-A at 1.45 A. 487

Table 4. Arginine conformation angles (deg) and chain length [Ca" �9 �9 NHI or NHz (,~)]

Chain Residue X l X 2 X ~ X4 X 5 X 6 length (A)

10 179 161 130 -67 -73 128 7.1 33 -64 -72 -176 97 -14 -175 6.7 39 86 128 -175 -67 23 -165 6.7 85 175 140 -47 100 -34 156 5.3

Histidine conformations

There are four his t id ine residues in rib onuc lease -A (12, 48, 105, and 119), two of which (12 and 119) are involved in the cata lyt ic activity. His-119 has been shown to occupy two sites A and B with site occupa t ion factors ref ined in the leas t - squares analysis of 0.80 and 0.20, respect ively (Borkako t i et aL,

1982). The c o n f o r m a t i o n angles of the five his t id ines given in Table 4 are seen to be a p p r o x i m a t e l y - g , - g with the excep t ion of His -119A which is in an in te rmedia te con fo rma t ion and X2 of His-105 ( - 1 0 5 ~ ). The a l ternat ive con- fo rma t ions of His- 119 are thus re la ted by ro ta t ions of 142 ~ (about X 1) and 38 ~ (abou t X2). N o n b o n d e d po ten t ia l energy ca lcula t ions (Pa lmer and Borkako t i , 1983) have conso l ida ted these findings.

X a Torsion angles: general comments

The X 1 values in r ibonuc lease -A largely fo l low the c o m m o n d is t r ibu t ions descr ibed by J a n i n et al. (1978). In ag reemen t with the f indings of these au tho r s the X i d i s t r i bu t ion for Tyr, Phe, His, Met, Leu, Asp, Asn, Glu, Gin, Lys, and Arg residues is p r e d o m i n a n t l y t r imoda l (g, t, - g ) with very few values in the range - 3 0 to 40 ~ and some a r o u n d - 1 2 0 ~ There are, however , some values in the range 80 to 140 ~ a region less occupied accord ing to the f indings of J an in et al. (1978). In the b ranched s ide-chain Val the p r e d o m i n a n t con fo rma t ion repor ted by Jan in et al. (1978) where one of the X1 values is - 6 0 ~ or 180 ~ is observed 4 /9 t imes in r ibonuclease , the less c o m m o n con- f o r m a t i o n with X~ values ~ +60 ~ occurs 3 /9 t imes, and two con fo rma t ions with X~ ~ 120 ~ are observed. All Thr residues, however , do have the - 6 0 ~ 180 ~ c o n f o r m a t i o n pred ic ted by these au thors as being preferred. Values o f x in r ibonuc lease -A for Thr s ide-chains con fo rm to the d i s t r ibu t ion descr ibed by J a n i n et al. (1978). The Ser X ~ d i s t r ibu t ion , p red ic ted by Jan in et al. (1978) to be t r imoda l , has 4 /7 values -~ 60 ~ 1/7 ~ 180 ~ no values ~ - 6 0 ~ but 2 /7 ~ 90 ~ (Ser-16 and Ser-22).

X 2 Torsion angles: general comments

In acco rdance with the obse rva t ions of J an in et al. (1978) values of X 2 for Met , Gin, Glu, Lys, and Arg res idues in r i bonuc l ea se -A show a s t rong

488 Borkakot i et alo

Table 5. Histidine conformation angles (deg) ~

Residue X t X2 X~

12 - 5 5 - 7 0 I l l 48 - 6 0 - 8 0 100

105 - 4 9 - 8 6 94 I19A 149 81 -101 I19B - 6 9 118 -63

aXl= N -Ca - C~- Cv , x 2 = C~-C~-Cv-N ~, and X~ = C~-C#-C~-C~.

preference for the t region. The X l /X2 conformations lie mostly in the five regions predicted by Ponnuswamy and Sasisekharan (1971) indicated below:

Xl, X2 region: 60, t t, t t, 60 -60 , t -60 , 60 Frequency: 1 7 4 10 5

In addition, there are five cases where the X 1, X 2 values lie just outside the above regions: 90, - 1 2 0 ; - 9 0 , 120; 90, -120; a n d - 9 0 , - 1 2 0 (X2). The X1, X2 values of the two Leu residues lie in the preferred regions t, 60 and -60 , t, respectively, and the three Ile in the preferred regions -60 , t and 60, t (two values), respectively. The average X2 torsion angle for the six Tyr and three Phe side groups is 92 + 18 ~ that is, in the preferred region defined by Janin et al. (1978). In the X 1, X2 distribution for these residues four X 1 values are in the - 6 0 ~ position and five in the t position. Similarly values of X~ (defined as in Table 5) occurring in ribonuclease-A have an average value of 109 + 17 ~ Four out of five X l values occur in the - 6 0 ~ position and one (for His-119A) is on the edge of the t region (149 ~ ). All the ring side-groups in ribonuclease-A thus behave very much as expected according to Janin et al. (1978). The X2 distribution for Asn and Asp residues in ribonuclease-A is different f rom that reported by Janin et al. (1978) who report an average value of 156 --_ 38 ~ Ribonuclease-A, however, has a fairly even distribution in three regions: 60 _+ 20 ~ (four values), 100 + 20 ~ (five values), and 160 + 20 ~ (five values).

The water structure in r ibonuclease-A

General c o m m e n t s

Solvent atoms (water oxygens) were recognized in the (21Fol -IFcl) difference electron density both by intensity and definition in the map and from hydrogen bonding propensity. Hydrogen bond contacts were investi-


~ : Ser8~ OG

\ --.-..,/ /2,, , , 0 , 0 2 . 8 , ( . Z o r \ J

...... k Gin 101 . . . .

Fig. 13. Environment of Gin- 101 showing short contacts. There are five short contacts to Oe and 3 to Ne, possibly accompanied by slightly different conformational states of the side-chain not resolved at 1.45 A.

gated between solvent and either main-chain, side-chain, or other solvent atoms. Figure 8c is an example of solvent density and its environment. Potential solvent atoms thus assigned have been retained in the analysis only if Uiso ~ 1.0 A 2 after refinement (in fact, all solvent Uiso values currently included in the analysis are =< 0.78 A2). Other features of solvent geometry are shown in Figs. 6, 13, 14, and 16. To date a to ta l of 127 solvent atoms has been retained. Apart f rom two clusters, one of eight and the other of seven solvent atoms, the solvents identified so far are distributed mainly individually or in pairs, hydrogen bonded on the protein surface. A total of 49 solvent atoms forms hydrogen bonds to main-chain N or O atoms; 46 form hydrogen bonds to side-chain atoms involving 39 different side-chains with up to three solvent hydrogen bonds each; and there are 44 solvent-solvent hydrogen bond contacts involving 67 solvent atoms (Borkakoti et al., (1982).

Of the 63 solvent atoms having only one hydrogen bond contact, 10 are formed to main-chain N or O atoms, 14 to side-chain atoms, and the rest (39) to other solvent atoms. Of the 64 solvent atoms forming more than one hydrogen bond contact, 36 have two such contacts (11 with two solvent-main- chain, 7 with two solvent-side-chain, and 10 with two solvent-solvent contacts); 22 have three such contacts (1 with three solvent-main-chain contacts; 2 with three solvent-side-chain contacts, and 5 with three solvent-solvent contacts); and 6 have four such contacts (1 with four solvent-main-chain contacts).


10

0.1 0 . 2

! ! I !

I I

0.3 0 .4 0 .5 0 . 6 0.7 0 . 8 Uiso A2

(a)

1 5 -

10

5

i

I 0.2 0.3 0 .4 0 .5 0 .6 0.7 0 .8 U isoA 2

(b) Fig. 14, Distribution of Uis o for solvent oxygens hydrogen bonded to (a) main-chain atoms and

(b) side-chain atoms.

Mean-square atomic displacement of solvent atoms and hydrogen bond contacts

The average Uiso over all so lvent a t o m s is 0.49 A 2 ( m i n i m u m 0.16 ~z, m a x i m u m 0.78 A 2) c o m p a r e d to the v a l u e of 0.15 A z for m a i n - c h a i n a n d 0.25 A for s ide -cha in a toms . F o r so lvents h y d r o g e n b o n d e d to m a i n - c h a i n a t o m s

the average Uiso is s l ight ly less wi th a va lue of 0.39 A 2. F igure 14a shows the d i s t r i b u t i o n of U~o values for so lven t a t o ms h y d r o g e n b o n d e d to m a i n - c h a i n

Fig. 15. Plot of Uiso for solvent oxygen atoms versus the number of observed hydrogen bond contacts (1, 2, 3, or 4).

- A2 Uisc 0 .6

0.5

0 . 4

I I I I 1 2 3 4

N

Structure of Ribonuclease-A at 1.45/~ 491

atoms, while Fig. 14b is the corresponding distribution for solvent-side-chain interactions. Both are reasonably well-behaved distributions.

For the solvent oxygen a toms there is a tendency for U~o to decrease according to the number of hydrogen bond contacts it makes. This trend can be seen in Fig. 15 which is a plot of Uiso versus the contact number. This result would be expected in view of the restricted environment associated with increased coord ina t ion number and the general proximity to the protein surface of the solvent a toms thus far identified.

Solvent clusters

Solvent cluster I, Fig. 16a, consists of eight solvent atoms, three of which are hydrogen bonded to main-chain a toms [O(111), O(114), and N(5)] and

:::''" o .a~"o. ................

,, 2'93i ""..~.d~v

" - - ' ~ 0 ""'b

......"

c~ ..... . ? : ~ ........

OS (a)

Fig. 16. (a) Solvent cluster I in ribonuclease-A, consisting of a total of eight interlinked water oxygens; (b) solvent oxygens in the active site cleft of ribonuclease-A.


7

Z ; (b)

Fig. 16. (continued)

two to side-chain atoms (Glu-111 Oe2 and Glu-90e~) . Solvent cluster II contains seven solvent atoms forming hydrogen bonds to two main-chain atoms [0(4) and N(120)] and six side-chain atoms (Gln-11 Oel, Gln-28 OE1, Asn-44 N62, Glu-111 Oel, Asn-71 O6~, and Gin-69 Oe0. The two clusters are thus linked via Glu-111 Oe~ and Oe2 hydrogen bonds.

Solvent molecules and the active site o f ribonuclease-A

The geometry of the active site residues in ribonuclease-A has been discussed by Borkakoti et al. (1982). As shown in Fig. 16b, three solvent atoms play an important role in stabilizing the active site of the native enzyme-- two, 0(202) and O(203), are in solvent cluster II. 0(202) and 0(203) form a bridge across Gin- 111 Oe2 and Lys-7 N 6 via an intermediate solvent atom. Gin- 11 O~2 is further stabilized via solvent 0(282) which bridges Lys-41 N~, Asn-44 N~2 and 0(282) and also acts as a donor in the format ion of a hydrogen bond to a sulfate oxygen atom. Further stabilization is effected by formation of hydro-


gen bonds to sulfate oxygens via histidine imidazole nitrogens (Fig. 16b). The water structure is thus heavily involved in the juxtapositioning of active-site side-chains and the sulfate anion and is therefore vitally important for the functioning of the catalytic mechanism. As discussed above, the two solvent clusters are linked via hydrogen bonds to Glu-111 O~2 (cluster I) and O~ (cluster II), and both are therefore important features in the molecular function.

F u t u r e w o r k

The function of the charged groups, both in determining the tertiary structure of ribonuclease-A and in promoting enzyme-substrate interaction, will be discussed later in a paper on the mechanism of action of the enzyme. At pH 5.7 (at which the crystals used in the X-ray analysis were grown) the Arg, His, and Lys residues are considered to carry positive charge, and Asp and Glu residues a negative charge (Fig. 3). Subdivision of the side-chains (Fig. 3) into a "front surface" (close to the active site) and a "back surface" (away from the active site) reveals the dominance of a net positive charge on the front surface of the molecule as indicated.

The X-ray analysis described in this paper, although now largely com- plete, is still continuing. In particular, studies of the dynamic properties of the molecule have been initiated and the results will be published later. An attempt will also be made to identify solvent molecules further away from the protein surface than has been possible to date. The results of comparisons between the X-ray structure and the structure determined by neutron diffraction (Wlodawer et al., 1982; Wlodawer and Hendrickson, 1982; Wlodawer and Sjolin, 1981) will also be reported later.

Information now available on the active site geometry will be invaluable for the proper assessment of enzyme activity now being attempted by the incorporation of nucleotide and dinucleotide inhibitor molecules into the native crystals. This analysis will be further enhanced by the novel use of a fast, reliable, refinement routine which is now well established in the repertoire of protein crystallography programs (RESTRAIN).

A c k n o w l e d g m e n t s

We gratefully acknowledge the support of the SERC for provision of a research assistantship (NB) and a research studentship (MJS). We also thank Dr. A. Wlodawer for stimulating discussions and the North Atlantic Treaty Organization for grant RG 040.80.


R e f e r e n c e s

Arnott, S., and Dover, D. (1967) J. MoL Biol. 30, 209-212. Artymiuk, P. J., and Blake, C. C. F. (1981) J. Mol. Biol. 152, 737-762. Artymiuk, P. J., Blake, C. C. F., Grace, D. E. P., Oatley, S. J., Phillips, D. C., and Sternberg,

M. J. C. (1979) Nature (London) 280, 563-568. Baker, E. N. (1980) J. Mol. BioL 141, 441-484. B0rkakoti, N. (1981), personal communication. Borkakoti, N., Moss, D. S., and Palmer, R. A. (1982) Acta Cryst. B38, 2210-2217. Carlisle, C. H., Palmer, R. A., Mazumdar, S. K., Gorinsky, B. A., and Yeates, D. G. R. (1974)

J. Mol. Biol. 85, 1-18. Deisenhofer, J., and Steigemann, W. (1975) Acta. Co,st. B 31, 238-250. Diekerson, R. E., and Geis, I. (1969) The Structure and Action o f Proteins (W. A. Benjamin,

Menlo Park, California). Gorinsky, B. A. (1971) Ph.D. Thesis (University of London). IUPAC-IUB Commission on Biochemical Nomenclature (1970) J. Mol. Biol. 52, 1-17. Janin, J , Wodak, S., Levitt, M., and Maigret, B. (1978) J. Mol. Biol. 125, 357-386. Jones, T. A. (1978) J. Appl. Cryst. 11,268-272. Lewis, P. N., Momany, F. A., and Scheraga, H. A. (1973) Biochim. Biophys Acta. 303, 211-229. Morffew, A., and Moss, D. S. (1982) Comput. Chem. 6, I-3. Palmer, R. A., and Borkakoti, N. (1984), to be published. Pauling, L., Corey, R. B., and Branson, H. R. (1951) Proc. Natl. Acad. Sci. U.S.A. 37, 205-210. Ponnuswamy, P. K., and Sasisekheran, V. (1971) Int. J. Protein Res. 3, l-8. Rosemeyer, M. A. (1961) Ph.D. Thesis (University of London). Saenger, W. (1973) Angew. Chem. Int. Ed. Engl. 12, 591-601. Stanford, M. J. (1981), personal communication. Steigemann, W., and Weber, E. (1979) J. Mol. Biol. 127, 309-338. Sternberg, M. J. E., Grace, D. P. E., and Phillips, D. C. (1979) J. Mol. Biol. 130, 231-253. Thornton, J. M. (1981) J. Mol. Biol. 151, 261-287. Tickle, I. J. (1979), personal communication. Wlodawer, A. (1980) Acta Crvst. B36, 1826-1831. Wlodawer, A., Bott, R., and Sjolin, L. (1982) J. Biol. Chem. 257, 1325-1332. Wlodawer, A., and Hendrickson, W. A. (1982) Acta Cryst. A 38, 239-247. Wlodawer, A., and Sj61in, L. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2853-2855.

The refined structure of ribonuclease-A at 1.45 � resolution

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Transcript of The refined structure of ribonuclease-A at 1.45 � resolution