The structure of cytidilyl(2′,5′)adenosine when bound to pancreatic ribonuclease S

J. Mol. Biol.

(1977) 116, 8554375

The Structure of Cytidilyl(2’,5’)Adenosine When Bound to Pancreatic Ribonuclease S SHOSHANAY.

WODAKt

Department of Biological Sciences Columbia University New York, N.Y. 10027, U.S.A. AND MAMIE

Department

Y. LIUf

AND H. w.

of Moleculur Biophysics Yale University

WYCKOFF

and Biochemistry

New Haven, Conn. 06520, U.S.A. (Received 31 January

1977, and in revised form 15 July

1977)

The three-dimensional structure of the RNase S complex with the synthetic dinucleoside monophosphate cytidilyl(2’,5’)adenosine(C,,p,,A) is determined using difference Fourier techniques at 2.0 A resolution in conjunction with computer graphic model-building and energy minimization. The latter has been carried out as a function of the rigid body parameters of the dinucleoside monophosphate and the dihedral angles of the nucleoside portion as well as of relevent, amino acids in the active site of the enzyme. The bound dinucleoside monophosphate is found to assume an extended conformation, with the adenine and cytidine bases nearly perpendicular. The bases form specific hydrogen bonds with groups in the active site. Although the atoms involved in the recognition of the pyrimidine base by the enzyme are the same as in the pyrimidine bases of UMP, CMP and UpcA, the details of the binding are different. The adenosine moiety blocks most of the various positions that His119 occupies in the native enzyme and forces it into one well-defined position. One of the His119 ring protons is in contact with 0(5’) (the leaving group), 0( 1’) of the adenine ribose and with a free phosphoryl oxygen. No strong charge contacts with the phosphate group are observed. We show how combining X-ray data with computer graphic model-building, electron density fitting and energy calculations leads to the model we propose and discuss in detail the enzyme-nucleic acid interactions.

1. Introduction The binding of dinucleotides to bovine pancreatic ribonuclease is of general interest as an example of protein-nucleic acid interaction and specific interest with respect to the mechanism of action of this much-studied enzyme. t Present address: Laboratoire de Chimie Biologique, Facult& des Sciences, Universit& Libra de Bruxelles, 67 Rue des Chevaux, 1640 Rhode-St-Gem%, Belgium. $ Present address: National Institutes of Health, Building 2, Room 314, Bethesda, Md 20014, U.S.A.

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Bovine pancreatic ribonuclease (RNase A) is a member of a class of enzymes that catalyze the hydrolysis of the 3’,5’-phosphodiester linkage in the polynucleotide RNA (Brown & Todd, 1955). The reaction catalyzed by RNase is mainly specific to the linkage in which the 3’-nucleoside contains a pyrimidine base and occurs in two steps : (1) transesterification, leading to the production of oligonucleotides ending in a pyrimidine 2’,3’-cyclic nucleotide; (2) hydrolysis, which produces a terminal 3’pyrimidine nucleotide. In each step of the reaction the O-P bond is split (Limpkin et al., 1954). Pyrimidine 2’,3’-cyclic mononucleotides are also substrates for the hydrolysis step. Ribonuclease S (RNase S) is derived from pancreatic ribonuclease by proteolytic cleavage of the peptide bond between residues 20 and 21 and consists of two chains : the S-peptide, containing residues I through 20, and the S-protein, containing residues 21 through 124. The two chains are held together only by non-covalent interactions and may be reversibly d.issociated (Richards & Vithayathil, 1959). The enzymatic properties of RNase S and RNase A are virtually identical ; however, RNase A crystallizes from an alcoholic solution while RNase S crystallizes from aqueous ammonium sulfate. Only recently have both forms of the enzyme been crystallized from aqueous solution at high pH (Martin et al., 1976). The three-dimensional structure of RNase S, based on a 2.0 A resolution electron density map, has been reported by Wyckoff et al. (1970) and a complete set of stereo drawings have been presented by Richards & Wyckoff (1973). Crystallographic studies of complexes of ribonuclease S with small molecules were mainly limited to the binding of mononucleotides, and a great deal of structural information regarding their interactions with the active site of RNase S has been compiled (Richards & Wyckoff, 1971; Richards et al., 1971). The structure of RNase A has most recently been reported by Carlisle et al. (1974). Kartha et al. (1967) presented the flow of the backbone but have not presented a detailed structure. Crystallographic examination of inhibitor binding to RNase A has been reported by Kartha et al. (1968). The specificity of pancreatic ribonuclease A (RNase A) is such that the nucleotide containing the 3’ linkage to the phosphate must be a pyrimidine, except in a few special cases, while any of the four normal RNA bases can be in the 5’ position. The nature of the 5’ moiety does have an appreciable effect on the rate of the phosphoryl transfer step which cleaves the chain and produces the cyclic phosphate terminal. The relative saturation rates for cleavage of the dinucleotides CpA, CpG, CpC, CpU and the benzyl and methyl esters CpBe and CpMe are 3000, 500, 240, 27, 2, and O-5 s-l (Witzel & Barnard, 1962). The Michaelis constants are 1.0, 3.0, 4.0, 3.7 and 3-O mM, respectively, for the first five of these. One might expect from these rates and binding data that the second base is bound in a specific manner and an adenine binding site has indeed been found. Structural studies of ribonuclease S complexes with dinucleoside monophosphates have not been carried out to the same extent as for mononucleotides. Dinucleoside monophosphates which are substrates for reaction step 1 are turned over rapidly by the enzyme. Their complexes with RNase cannot, therefore, be isolated crystallographically, although the enzyme activity in the crystal is known to be much lower than in solution. Even in the case of e-DNP-lys41-RNase S crystals, which contained approximately 3% of the activity of native RNase S crystals due to contaminat.ion by reactive enzyme (Allewell, 1968; Allewell et al., 1973), the difference Fourier maps

C,,p,,A-RIBONUCLEASE

COMPLEX

x57

for the binding of CpA and UpC as well as calculations based on kinetic arguments indicated that the major contribution to the binding was due to reaction products which interfere with the interpretation of the enzyme-substrate structure. However. if the dinucleoside is an inhibitor that binds the enzyme tightly, the isolation of the complex is possible. These conditions were met by the phosphonate ester analog 01 UpA,CpcA (a dinucleoside monophosphate having a methylene group substituted fol t’he 5’ phosphate oxygen) whose complex with RNase S was studied crystallographitally by Richards & Wyckoff (1973). The mode of interaction of a dinucleoside inhibitor with the enzyme may not be identical to that of the substrate, but it should mimic it fairly well because of similar structural components. However, several enzyme-inhibitor complexes have to be studied in order to attempt to reconstruct the actual enzyme-substrate interactions, or those of the various reaction intermediates. We present here the three-dimensional structure of the RNase S complex with t,he synthetic dinucleoside monophosphate cytidilyl(2’,5’)adenosine, noted as CpA* to indicate that it has a 2’-5’ phosphodiester linkage instead of the normal 3’-5’ linkage. This compound binds to RNase S without being cleaved by the enzyme (unpublished results by Lapidot, and independently by Wodak & Grunberger). Difference Fourier t’echniques at 2-O A resolution were used to obtain the electron densiby profile of the bound dinucleoside, and the electron density map was interpreted using the optical comparator method (Richards, 1968) and an interactive computer graphic display system. Relatively noisy maps precluded unambiguous and detailed interpretation. Therefore, model building was combined with energy calculations of the enzyme-inhibitor complex to discriminate between and refine trial st’ruct’ures.

2. Materials and Methods (a) X-ray

crystallograghy

The dinucleoside monophosphate CpA* was synthesized by Y. Lapidot and kindly given to us. The RNase S enzyme was obtained from Sigma Chemical Corporation. The crystalline complex of RNase S and CPA* was prepared by diffusing 3mM-CpA* into enzyme crystals of space group P3,21. The soaking was done in 75% saturated ammoniuln sulfate for 48 h at pH 5.5. The unit cell parameters of the complex are a = b = 44.75 a, c = 96.74 A, y = 120”. They differ by an average of 0.2% from those for the native enzyme. The intensity data of the native RNase S and the enzyme--CPA* complex crystals were Ineasured on a rnodified 4-circle Picker diffractometer having 2 sources and 2 detectors and using a dynamically controlled w stepscan mode (Wyckoff et al., 1967; Wyckoff, unpublished results). Three crystals were sufficient to obtain a 2.0 A resolution data set since the radiation damage produced less than a 5% fall-off in intensity. Different sets of data belonging to 4 equivalent asymmetric units for t,he derivative crystal were collected. An absorption correction was applied using the method of North et al. (1968). Scaling, merging and editing of the redundant data was performed using the programs written b3 (:. M. Reeke, Jr (197la). The difference amplitudes were calculated using the program DIFF3D written by J. Greer and modified for the P3,21 space group by S. Wodak &. M. Liu. The average difference in structure factor magnitude Rx between native and derivative crystals relative to the native F was about 16% RF where 1FF,l is the structure the native crystal. A total

= IlFdl

factor amplitude of 6881 difference

- l2”rdl I IFNI

of the derivative crystal, and 1F,I is that of terms, representing 92% of the data to 2.0 .!!

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resolution, were combined with RNase S phases calculated in a refinement (Powers, 1976) (based on the structure previously determined by Wyckoff et al. (1970)) to compute the difference Fourier synthesis using the program GNRFOURI (Reeke, 19715). AF values for general reflections were given double weight in the Fourier summation to compensate for the inherent amplitude and phase errors which would on the average, reduce the value map was contoured at 30% of the of the electron density by a factor of 2. The difference maximum electron density level, corresponding to the value of 20 where D is the error (noise level) of the difference map evaluated according to Henderson & Moffat (1971). The interpretation of the difference electron density maps was performed as follows: a model of CpA* having fixed bond-distances and bond-angles whose values were compiled from the crystal structures of the dinucleoside monophosphates (2’-5’)ApU (Shefter et al., 1969) was built, using a model-building computer program linked to an interactive Adage model-50 graphic display terminal, as described by Katz & Levinthal (1972). The initial conformation of the CpA* model was obtained by setting the dihedral angles to the values obtained for a Kendrew model of the dinucleoside monophosphate after it was matched to the difference electron density map using an optical comparator (Richards, 1968). This device was also used in the initial stages of the interpretation to search for the most appropriate sugar conformation, to determine qualitatively the interactions of the CpA* molecule with the enzyme, and to record specific side-chain movements around the active site caused by the binding of CPA*. The initial conformation was then refined by visual fitting of the CpA* model to the contours of the difference electron density map using the interactive computer graphic display system instead of the real space refinement as described by Diamond ( 1971). Using the graphics computer as an optical comparator has several appealing features: the contouring of the electron density levels is automatic. The contour lines can be drawn in planes parallel to any of the 3 faces of the crystallographic unit cell and displayed simultaneously so that the electron density profile is clearly defined to the viewer from all angles. Moreover, the parameters used to match the model to the electron density map, namely the dihedral angles and the rigid body rotations and translations, can be easily changed, distances between non-bonded atoms can be checked repeatedly during the fitting procedure, and final atomic parameters can be obtained with great precision. Figure 1 illustrates the best fit of the CpA* model to the difference electron density map as obtained by the procedure described above. Table 1 lists the corresponding dihedral angles of the dinucleoside and Figure 2 illustrates the nomenclature convention. Throughout the fitting, the sugar rings and the bases were treated as rigid bodies. However, no limits were set on the ranges of other dihedral angles in the dinucleoside. While it has been observed that the preferred conformation of nucleotides is restricted to a much more limited range of dihedral angles (Sundaralingam, 1966; Arnott & Hukins, 1969; Kim et al., 1973) than in proteins, it is not clear that these restrictions would apply to a dinucleoside bound to an enzyme. Moreover, CpA* contains a 2’-5’ phosphodiester linkage for which the preferred conformation is less well established. Fitting an atomic model to a difference Fourier map presents certain inherent problems which stem from the fact that peaks in such maps do not correspond to actual atomic positions but rather to shifts and differences in these positions. Other problems due to the particular experiment, such as noise brought about either by error in the phase data and/or low occupancy, can also hamper correct interpretation. Therefore, in order to obtain a reasonable molecular model for the bound CpA* we did not rely solely on the fitting to the difference electron density map but we performed additional refinement using conformational energy calculations of the enzyme-inhibitor complex. The difference electron density due to movements of the protein was not interpreted in this study. Only the movements of specific side-chains in the active site have been studied in conjunction with the enzyme-inhibitor energy calculations, and will be discussed in the next section. (b) Energy calculations The conformational energy using the protein manipulation

of the RNase S-CPA* complex was computed and minimized program (PMP) (Levinthal et aE., 1975; Honig et al., 1976).

C,,p,,A-RIBONUCLEASE

COMPLEX

FIG. 1. A stereo view showing the best fit of the CpA* model to tho difference electron densit) map contoured perpendicular to the ac plane of the crystallographic unit cells, at a contour level of l/3 the height of the maximum density level. The arrows indicate t,he locat,ion of the cytosiw (Cl), and aclenosinc (A) groups.

TABLE

Dihedral Optical comparator

-70

1

angles? for the CpA* Model-building, graphic fitting

-77

1GO

160

170

170

-30

-35

model Energy minimization

-82 153 173

-4”

125

138

121

200

220

230

2GO -90

280 -99

270 -107

t Angles in degrees, as obtained: (a) Using Richards’ optical comparator. (b) After the model-building and fitting procedure. (c) At the end of the energy minimization procedure. p and p’, as well as all other torsional angles of the ribose ring, have not been refined indepontlmtly, since the ribose ring was fitted to the electron density as a unit. For detailed angle conventions, see Fig. 2. 55

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-N(b)

(a)

C2‘-Endo

C3’-Enda (bl

Side view of bond bhg rotated

View along the bono being rotated (cl

FIQ. 2. Nomenclature conventions for the dinucleoside CpA* following the rules of Sundaralingam (1969). (a) Drawing representing the numbering scheme and dihedral angle nomenclature for the 2’-5’ CPA*. (b) Sign convention and definition of the dihedral angles of CPA*. (c) The 2 main puckering modes of the ribose ring. The angle x about the glycosidic bond is defined by the atoms O(l’)-C(l’)-N(9)-C(S) for purines and by the atoms O(l’)-C(l’)-N(l)-C(6) for pyrimidines. The 0” position for both cases corresponds to the cis conformations of the bonds involved. The angle is measured positive if the N( 1)-C(6) bond is rotated counterclockwise relative to the 0( l’)-C( 1’) bond when viewed along the C( 1’)-N( 1) direction.

The crucial aspects of this computerized molecular model-building system are the real time coupling between calculations in the IBM 360/91 computer and the interactive display of the Adage graphic computer. By typing instructions at the Adage terminal, it is possible to measure distances between atom pairs, bond angles, and dihedral angles. It is also possible to change the conformation of the molecule by rotating about bonds and to minimize the energy of the molecule with respect to any set of torsional angles specified. These operations are all carried out in the 360/91 computer, and their effect on the conformation is displayed on the graphics CRT. Only non-bonded (Gibson & Scheraga, 1967) and Coulombic energy terms were included in the calculations. The dielectric constant was taken to be 3.5 and formal charges of 1-0.4 + 0.3 for all charged hydrogens and - 0.3 for for a carbonyl carbon, - 0.4 for all oxygens, all nitrogens were used for the protein portion of the comp1ex.t Partial charges for the nucleoside portion were modified slightly from those reported by Renugopalakrishnan et al. (1971), to account for the presence of the 2’ phosphoester linkage. Hydrogen bonds t Except for the following residues: Asn and Gln side-chain nitrogens and charged hydrogens were given the formal charge of -0.4 and +0.2, respectively. The net charges on Glu, Asp and Lys, Arg were taken as -0.2 and +0.2, respectively. The His side-chains were considered as neutral.

C,,p,,A-RIBONUCLEASE

COMPLEX

861

were obtained using the non-bonded H---O and H---N interactions suggested by McGuire et al. (1972) together with the standard electrostatic interactions. In order to limit computational time and since we are only interested in the active-site region, all pair-wise interactions for interatomic distances greater than 7 A were ignored. The Davidon algorithm (Davidon, 1959; Fletcher & Powell, 1966) was used to minimize the conformational energy. The start,ing conformation of the enzyme-inhibitor complex consisted of a model of CpA* based on co-ordinates obtained from the model-building and fitting procedure described above, and a model of RNase S built using the refined atomic co-ordinates of Powers (1976). Side-chain positions of residues Thr45, Asn69, Gln69, Asn71 and His119 were adjusted to match the conformations recorded from a preliminary fitting of the complex obtained with the Richards’ optical comparator. The adjustments were made using the interactive graphics system. In addition an artificial potential of the form 200 (T - r0)2 was used to obtain a reasonable starting conformation for the enzyme-inhibitor hydrogen bonds. The value of r,, was taken to be 3.0 A for an X--X atom pair, where X is either an oxygen or a nitrogen, and 2.0 A for an X---H atom pair. The conformation of the complex was then refined using an energy minimization procedure which was applied in 2 steps and led to an alleviation of several close contacts and to a drop of 344 kcal/mol in the conformational energy. First, the energy of the complex was minimized with respect to the 6 parameters that control the rigid body motion of CpA* relative to the enzyme. Next, the conformational energy of the entire complex was minimized by varying simultaneously the position and orientation of the CPA* molecule and the dihedral angles of selected protein side-chains and those of the dinucleoside monophosphate. A total of 40 parameters were varied simultaneously in this step. These included 7 torsional angles, x~,$,#,w,w’,~‘,#‘,x~ of the CpA* molecule and the side-chain dihedral angles of Lys41, Thr45, Asn67, Gln69, Asn71 and His1 19. The side-chain of Lys41 was allowed to move freely during the minimization procedure in order to permit possible close interactions with the dinucleoside with thr hope that these would clarify the role of Lys41 in the enzymatic activity of RNase S. The CpA* torsional angles at the outcome of the energy minimization are listed in Table 1, and the final enzyme-inhibitor interactions are summarized in Table 2.

TABLE 2

Comparison between the dihedral angles of CPA*, as they were obtained by graphic$tting and energy calculations, and those obtained for the Y-5’ dinucleoside Ap U whose structure was determined crystallographicallyt

(2’,6’)-ApU (Shefter et al., 1969) (2’,6’)-CpA

*

4

w

w’

4

57

170

313

232

244

163

173

328

121

230

t See Shefter et al. (1969).

The final co-ordinates of CpA* bound to RNase S and shifted side-chain atoms in the active site are listed in Tables 3 and 4, respectively. It was observed that the energy refinement did not bring about significant changes to the nucleoside conformation or position in the active site. This can be attributed to the fact that the nucleoside is severely constrained by non-bonded interactions with the enzyme. The particular distribution of these interactions strongly suggests only one possible fit. The energy refinement was most useful however, in the detailed characterization of the nucleic acid-protein interactions. 55:

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TABLE 3

Close contacts and other relevent interaction distances in A between atoms of the active site of RNase X and CpA* as well as between atoms of the His119 side-chain and other atoms of the RNase X-CpA* complex at the outcome of the Jitting and energy minimization procedure Residues on RNase X

CpA*

Distance (4

Cytidine

N

O(2) N(3) N(3) C(4) C(4) C(4) N(4) N(4) N(4)

-0Y CY Oy C6 Cf Oy CY Cf

Thr45 Thr45 Thr45 Thr45 Phe120 Phel20 Thr45 Thr45 Phel20

4.0 3.2 3.5 3.3 4.4 4.5 3.4 4.5 4.4

O(1’) C(2’)

-NE 0

Lys41 Phe120

2.6 4.5

P O(I) O(I) O(I) O(I) O(I) O(I) O(I) O(I) WI) O(II)WI) O(5’) O(5’) O(5’) O(5’)

N6 CY C6 NE -N CL7 CP C 0 NE NE CE C/3 CY N6 Cf

His119 His119 His119 His119 Phe120 Phel20 PhelXO PhelPO Phe120 Glnll His12 His12 His1 19 His119 His119 His119

3.9 3.3 3.8 4.0 3.0 3.5 3.5 3.5 2.9 2.8 3.2 3.8 3.8 3.6 3.1 3.9

O(l’) O(1’) O(l’) C(3’) C(3’) (34’) C(4’) C(5’) C(5’) C(5’)

CY -N6 CE N6 Cf N6 CE CY N6 CC

His119 His119 His119 His119 His119 His119 His119 His119 His119 His119

4.0 3.0 4.0 3.6 3.8 3.3 3.9 3.9 2.9 3.5

-0c CS Of Of OS CP -0e N(6) ----OS N(6) CP

GM11 Glulll Glulll Glu69 As1171 Ala109 Gln69 Asn71 Ala109

2.7 3.8 2.9 4.0 4.0 3.7 3.1 2.9 4.5

2’ Ribose

Phosphate

5’ Ribose

Adenine

N(l)

-

(72) C(2) C(6) (26) (36) N(6) -

c,,p,,A-RIBONUCLEASE TABLE

3 (continued) Distance

Residues on RNase S

His119 Nt

----0 Or

NE

X63

COMPLEX

(4

Phe120

2.9

Asp121

3.5

The Ltmino acid nomenclature corresponds to the accepted convention (IUPAC-IUB Commission 1970). The dinucleoside nomenclature corresponds to the one in Fig, 2. Broken lines indicate possihle hydrogen bonds.

on Biochemical Nomenclature,

3. Results and their Interpretation (a) Conformation

of CpA*

when bound to RNase S

Figure 3 shows the three-dimensional conformation of CpA* when bound to RNase S and Table 5 presents a comparison of the model’s torsional angles winith values obtained from the crystal structure of A(2’,5’)U (Shefter et al., 1969). The sugars in the bound CpA* occur both in the C3’-endo conformation. The sugar-base conformation as defined by the angle about the glycosidic bond is xA = -82” for for cytosine. Both angles are on the edge of the Ant,i adenosine and xc = -107” range (-75” to + 15”) as defined by Donohue & Trueblood (1960), and as depicted in Figure 4(a). It is not possible to conclude on the basis of this study that these angles represent a strained nucleoside conformation since no attempts have been made here to relax the structure of the complex or the free nucleoside as a function of bond distances and bond angles. The conformation of the phosphodiester linkage determined by the angles, w and conformational energ> DJ’: corresponds to an allowed region of the phosphodiester

N FIG. 3. A stereo view of the 3.dimensional of RNase S.

conformation

of CpA* when hound to the active site

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TABLE 4

A. Co-ordinates of the bound CpA* in a right-handed orthogonal system, in .k N&XIX?

X

Y

z

Adenine N(l) (76)

N(6) C(6) C(4) N(3)

WV N(7) C(8) N(g) Adenine

-2.8 -2.4 - 1.7 -2.9 -3.6 -3.9 -3.6 -2.8 -3.4 -3.9

-3.6 -4.3 -6.3 -3.6 -2.4 -1.8 -2.6 -3.9 -3.0 -2.0

12.9 11.8 11.8 10.7 10.8 11.9 13.0 9.3 8.7 9.6

-4.7 -6.0 -4.1 -4.4 -6.0 -6.0 -6.3 -7.3

-0.9 - 1.2 -0.3 1.1 - 1.0 -0.3 -1.0 -2.1

9-l 8.9 7.8 7.9 6.8 6.6 7.6 7.1

-8.3 -9.4 -8.8 - 10.7 -9.6

-2.6 -3.6 -6.0 -3.6 -3.1

7.9 7.3 7.2 7.9 6.8

-9.2 -8.2 -7.2 -9.1 -9.0 - 8.0 - 10.3 - 10.4

-4.0 -3.3 -2.7 -2.4 -2.3 -1.4 -3.1 -4.1

4.7 3.7 4.4 2.9 1.4 0.8 2.7 3.7

- 10.3 -11.3 -12.4 -11.3 - 10.3 - 10.3

-6.6 -6.1 -6.6 -7.3 -8.2 -9.4 -7.6 -6.3

3.1 2.4 2.2 1.9 1.9 1.4 2.6 3.1

ribose

ccl’) O(l’) C(2’)

W’) C(3’) O(3’) C(4’) C(6’) Phosphate (x5’) P O(I) WI)

W’) Cytosine

ribose

C(2’) C(3’) O(3’) C(4’) C(6’) O(f) O(l’) C(l’l Cytidine N(l)

f-72) WV N(3) C(4) N(4)

C(5) (26)

-9.1 -9.3

&,p,,A-RIBONUCLEASE TABLE

COMPLEX

xti.5

4 (continued)

R. Co-ordinates (in a right-handed orthogonal system, in A) of the new positions qf the amino acids in the active site of RNase X whose dihedral angles were varied during the energy mini&ion procedure

1.8 0.8 0.2 0.2 -- 0.5 0.1 -- 0.8

- 0.9 -0.6 0.7 1.7 1.3 1.9 1.8

15.2 15.6 14.4 13.8 13.4 16.2 15.7

- 7.9 - 9.3 - 10.1 - 10.3 -9.7 -9.3 -8.7

3.6 4.0 3.9 5.1 2.8 5.4 6.3

4.5 4.7 3.9 2.5 1.7 0.4 2.3 4.1 3.2

-8.7 - 8.3 -7.0 -- 7.2 -5.9 -6.0 -5.0 -9.4 - 10.2

8.0 9.5 9.7 9.4 9.7 9.6 10.3 10.4 IWO

1.9 0.9 0.9 0.9 0.0 1.6 -0.4 -1.5

-9.8 -9.8 - 8.4 -7.2 -7.2 -6.1 - 10.2 -9.9

13.5 14.6 15.2 14.2 13.3 14.4 14.0 14.6

-7.6 -- 7.8 -6.7 --6.1 -5.7 -6.2 -- 5.6 -5.0 -7.8 -7.6

-- 5.8 -5.8 -4.8 -. 5.2 -4.2 -4.8 -6.4 -6.1 -7.2 -8.2

12.5 11.0 10.5 9.2 8.3 7.2 8.6 7.5 10.4 11.0

- 16.4 - 15.5 - 16.0 - 14.9 - 13.7 - 12.4 -11.2

Gln69 s CE CP C’Y cs oe NE C 0

His119 N CCC CP CY N6 CE C6 NC c 0 These new positions are supported amino acids listed but for Lysll.

by electron

density

in the difference

FowGr

map for all the

866

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(cl

(0)

(a)

W’ (bl

FIG. 4.(a). Schematic drawing of the Syn and Anti ranges of the glycosidic torsional angle x. The values found for the adenine (a) and the cytosine (c) in the RNase S-@A* complex are shown.

(b). Relevant region of the (w,w’ )phosphodiester energy surface as calculated by Newton (1973). On it are marked the positions corresponding to the energy levels of (2’-S’)ApU* (Shefter et r/Z., 1969) and CpA*.

surface as calculated by Newton (1973) and has approximately the same energy level as the phosphodiester conformation observed in the crystal structure of A(2’5’)U (see Fig. 4(b)). The overall shape of CpA* is extended, the purine and pyrimidine bases are nearly perpendicular to each other ami the distance between them, as defined by the atoms C(6) on the adenine and C(2) on the cytosine (Webb et al., 1973), is 13.1 A. Many of the enzyme-dinucleoside interaotions are between polar atoms and several involve hydrogen bonds. Considered as such in this study are bonds for which the donor-acceptor distance does not exceed 3.4 A. Considerable lattitude has been allowed in the N-H--O and O-H--O angles since covalent bond angles and bond distances were not allowed to vary. The hydrogen-bonding scheme that we propose for the cytosine ring to the enzyme is as follows: atom N(3) of the base forms a hydrogen bond with the hydroxyl group (0~1) of Thr45. In this interaction the proton is provided by the protein, since N(3) of the base is unprotonated. This hydrogen bond has been previously observed in the crystallographic binding studies of UMP, CMP and UpcA to RNase S (Richards & Wyckoff, 1971; Allewell, 1968; Richards et CL, 1971; Carlson, 1976). The binding of uracil is similar to that of cytosine because the Oyl of Thr45 can also act as an hydrogen acceptor; it assumes that role when hydrogen-bonded to N(3) of uracil which unlike cytosine is protonated at pH 5.5. In the above-mentioned binding studies another hydrogen bond has been observed between the O(2) group of the pyrimidine base and the backbone nitrogen of Thr45. In the RNase S-CpA* complex however, the distance between the O(2) atom of the cytosine and the imino group of Thr45 is

Cp,p,

,A-RIBONUCLEASE

COMPLEX

867

4.0 A. indicating that this hydrogen bond is not formed and in addition that the itnino group of Thr45 is inaccessible to solvent. The cytosine rihose forms no hydrogen bonds with the enzyme ; rather, the 3’ hydroxyl and 5’ hydroxyl groups are pointing away from the active site and into the solution (see Figs 5 and 6). Our calculations indicate however, that if the Lys41 side-chain is allo~r~l to movp freely during the energy minimization process, its c nitrogen ;I ppro:\ctlc~s to wit’hin 2.6 -4 of tjhe 0( 1’) oxygen of the cytosine ribose and could form

FIG. 5. Sterro views of the model of the complex of RN&se S with @A*, where only the r&x-ant, rvgion around the active site is shown. On the dinucltv~side monophosphate only the non-hydrogen atoms are explicitly marked. On the cnzymo only the amino acid residues which hydrogen-bond to the CpA* molecule are named and numbered. The backbone atoms for the enzyme include hydrogens. The latter appear ai well in side-chains where oxygen and nitrogen groups are potential protond onors in a hydrogen bond. (a) General view of the complex with few details at the adenine binding site. (b) Detailrtl virw of the adenine binding site.

868

S. Y.

WODAK

ET

AL.

Adenine (CPA*) CCpA*l

(bl

\ Gln69 (a)

Phosphate (CPA*) Adenoslne

(CPA*)

0 (2’)

-..,c8/o \

Glnll

\

N-H----O(II)

/

‘\ ‘H\ p ...N\Ca’c \ ‘.. LP / Phel20

‘c H1sl2 CT

)( C(2’1

,I),.,. “.. *denme .

HAkp/

p CP ‘CO’

(d) H-N

c

/

(cl

hydrogen-bonding interactions between several portions of CpA* and the FIG. 6. Detailed amino acids in the active site of RN&se S. (a) The hydrogen-bonding interactions of adenine and tho amino acids Gln69, Asn71 and Glulll. (b) The hydrogen-bonding interactions of cytidine with Thr45. (0) The interactions of the phosphate group with Glnll, His12 and Phel20. (d) The interactions of His119 and the adenine ribose ring.

a hydrogen bond. For further discussion of this interaction see section (d)(i) (3),below, for a description of the movements of the side-chain of Lys41. (b) Binding

oj the phosphate group

The phosphate group of CpA* binds to the enzyme in roughly the position thought to be the binding site of the sulfate ion in the native enzyme and the phosphate groups of t,he mononucleotide complexes such as LIMP and CMP. The specific inter-

C2,p,,-44RIBONUCLE4SE

COMPLEX

nm

of the CpA* phosphate group with RNase S are not well understood. One of the phosphate oxygens forms a hydrogen bond with the imino nitrogen of Phel20: the same oxygen is also very close to the main-chain oxygen of Phe120 (see Table 2). with which itI could probably not form a hydrogen bond at physiological pH. The other phosphate oxygen is within hydrogen-bonding distance of the NED amino group of Glnll and the NED group of Hisl2. A close interaction between the phosphate of bound mononucleotides and His12 has been previously observed, however. that of the phosphate with the side-chain of Glnll was found to occur vita. a water molecule (Richards & Wyckoff, 1973). In view of the sulfate-phosphate displacement problem, a careful study of the 2 A derivative Fourier map rather than the differenpc. Fourier map, might help to assign the correct interactions. Both the native and derivative crystals used in this work were prepared in thtl presrnce of a 75% solution of ammonium sulfate. Therefore, if the sulfate and phosphate ions were t)o occupy the same position in the active site of RNase S! the difference map should be relatively flat at the assumed phosphate position. This was indeed observed in previous studies of RNase S complexes with nucleotides (Allewell. 1968) and with UpcA (Carlson, 1976). With CPA*, and using the commerciall? purchased native enzyme, however, the difference electron density shows a deep hole at the assumed phosphate-sulfate position. Given the fact t,hat the rest of the CpA* molecule is so well localized in the active site, no other position could 1~. assigned to the phosphate than that which places it in the middle of the hole (sea Fig. 7). Several possible explanations could account for t,he observed hole: (1) the incomplete overlap of the phosphate and sulfate positions in this region. (2) Thea CpA* molecule reduces the amount of sulfate bound to the enzyme even when not in the specific position assigned in this study. (3) A heavier ligand than the sulfate: actually occupies the specific site in the native enzyme. (4) The map is noisy, eitt1t.r

actions

Fro. 7. Interpretation of the difference Fourier map in t,he regions of the phosphate group and His1 19. Thick cont,ours represent negative electron density, and thin lines, posit,ive electron density. The contour levels were drawn at l/3 the height of the maximum density level.

870

S. Y. WODAK

ET

AL.

due to random error in the data or to systematic error from the phasing procedure, with either of these compounded by a low occupancy of the dinucleotide. These hypotheses could be tested especially if more complexes of 2’-5’ nucleosides with RNase S are to be studied. (c) Bindin]

of the adenine nucleoside

The bound adenine is more exposed to the solvent than the cytosine. The N(6) nitrogen of the purine base hydrogen bonds to the 0~2 oxygen group of Gln69 and to the 062 oxygen group of Asn71 (see Figs 5 and 6). The N(1) nitrogen of the adenine base is within 2.7 A of the oxygen of the Glulll side-chain, close enough to form a hydrogen bond. The presence of this interaction could indicate that the N( 1) nitrogen was protonated when the binding to the enzyme occurred. The side-chains of residues 69 and 71, but not of residue 111, appear to have moved from their original positions in order to form these hydrogen bonds with the base. Residues Gln69 and Asn71 are part of an exposed loop in the polypeptide chain that extends from residues 65 to 72 and which has not been well determined in the native Fourier map (Richards & Wyckoff, 1971). The side-chains of residues 69 and 71 seem therefore to have the necessary freedom of movement that would allow them to readjust upon the binding of the purine base. Such readjustment does not seem possible in the deep enzyme pocket in which the cytosine base binds. The hydrogen-bonding pattern of adenine to RNase S suggests that guanine at the 5’ position of the phosphodiester linkage (which has an oxygen instead of an amino group at position 6) could theoretically bind to the enzyme in a similar manner, although it might be able to form only one hydrogen bond with either Asn71 or Gln69, this time via O(6) and with the amide group acting as proton donor. Steric hindrance would most probably prevent both side-chains from interacting simultaneously with the O(6) atom. When the structure of the CpA*-RNase S complex is compared to that of UpcA and RNase S, it appears that the conformations of the 5’ portions of both complexes are very similar, while the conformations of the pyrimidine portions are different, probably reflecting the differences between the 2’ and 3’ phosphoester linkages. Both nucleosides appear to bind to the protein in an extended form with hydrophilic groups pointing into the protein to provide a complement to polar groups in the active site. (d) Structure-function

relationship

The two-step catalysis carried out by RNase is thought to be initiated when the phosphorous of the ribonucleoside is attacked by an oxygen atom (0(2’)) of the pyrimidine ribose made more nucleophilic by the enzyme. As a result, a pentacovalent intermediate is formed (Findlay et al., 1961; Witzel & Barnard, 1962; Usher et al., 1972). The formation of this complex is followed by the specific protonation of the leaving O(5’) group and the formation of the 2’-3’ cyclic phosphate which is the substrate of reaction step 2. Many closely related mechanisms for RNase activity have been proposed. While an ever-growing body of knowledge derived from experiments with chemically modified forms of the enzyme and from detailed structural studies is available, it has not yet been possible to decide in favor of any one mechanism unambiguously. For a detailed review of this subject, the reader is referred to the article by Richards &

C2,p,,A-RIBONUCLEASE

871

COMPLEX

Wyckoff (1971). It is nevertheless generally accepted that at least two of the three residues, i.e. Hisl2, His119 and Lys41, are intimately involved in the catalytic mechanism. His12 and His119 are believed to be involved in the nucleophilic attack and protonation, while Lys41 is thought to be a stabilizing positive charge in the formation of the pentacovalent intermediate. (i) Positions

of Hisl2,

His119 and Lys41 in the RNase X-CpA*

complex

(1) His-12. No movement of His12 upon the binding of CpA* can be detected. While maintaining its original position as in the native enzyme the ~2 nitrogen of the histidine side-chain is 3.2 A away from the O(I1) atom of the phosphate group of the dinucleoside, possibly forming a hydrogen bond as well as a close electrostatic intcra&ion at low pH. (2) H&119. The position of this amino acid is not well-defined in the native prot&l electron density map. In the difference map of the Rh’ase S-CPA* complex, however. a dist,inct pair of positive and negative peaks appear at the region of the His119 side-chain (see Fig. 7), indicating that a shift has occurred upon CPA* binding. The negative peak corresponds to a fair approximation to the His119 side-chain position befort, the CpA* has bound and the positive peek to the position in bhe presence of CpA* in the active site. The His1 19 position in the native enzyme is partially occupied I)y the adenine base and ribose in the RNase S-CPA* complex. The side-chain dihedral angles corresponding to the two conformations are: x1 :150”, x2 = 94” in the enzyme-CPA* complex and x1 = 270”, xZ = 94” in the proposed native conformation. In both positions the strain in the His119 residue was found to l:e similar and small. Inspection of the energy profile of His119 in the active site of RKase S as a function of x1 showed that no significant energy barrier is encountered when x1 is varied from 150” to 270” by a positive rotation of 120”. A considerable energy barrier is encountered however when the rotation is performed from 150” in t,he opposite direction. This could suggest that the movement of His119 o(ncurs readily upon substrate binding and does not represent a ma,jor contribution to t,hr activation energy barrier of that process. In the CPA*-RNase S complex the histidine Sl nitrogen has close interactions wit11 t’hree oxygens : it, is 3.0 A away from the 0( 1’) oxygen of the 5’ ribose sugar, 3.1 4 from (0)5’ and 3.4 A from O(1). The orientation of the histidine ring suggests a direct hydrogen bond to O(1’). The NED nitrogen of His119 is 3.5 Ai away from ttuh carboxyl group of Aspl21, probably forming a salt bridge, a.nd is only 2.9 A an’+> from t,he backbone oxygen of Phel20 forming a hydrogen bond with the Iatter (see Table 2). The HisllS-Asp121 salt bridge and the close interact,ion of His119 with the phosphate group have been previously observed in the binding studies of nuoleosides and nucleotides (Richards & Wyckoff, 1971). The hydrogen-bonding interact,ion \vit#h the O(1’) oxygen of the adenine ribose, however, has not been observed before. lft,he position of His119 which is observed here represents the conformation of this group in the actual enzyme-substrate complex, a readjustment’ of His119 and flexing of the dinucleoside would seem to be necessary in order to form the pentacovalent, int,ermediate complex. (3) Lys41. No movement of Lys41 as a result of the binding of CPA* was observed from examination of the difference Fourier maps. This amino acid seems too far away from the dinucleoside (the closest distance being 55 A from the O(1’) of the p-yrimidine rihose) for any direct hydrogen-bonding interaction but sufIicient,ly close

872

S. Y. WODAK

ET AL.

for electrostatic interactions with either the 2’ ribose ring or the phosphate group. However, if the side-chain of Lys41 is allowed to rotate freely maintaining the /I carbon fixed during the minimization procedure, the final position of the nitrogen of this residue comes to within 2.6 A of the O(1’) oxygen of the pyrimidine ribose, possibly forming a hydrogen bond. Because of the difference between CpA* and the naturally 3’-5’ dinucleosides, this specific interaction with the pyrimidine ribose might not occur in the actual enzyme-substrate complex, and the role of Lys41 might be confined to the subsequent sequence of events such as in stabilizing the pentacovalent intermediate or in the binding of the (2’,3’)-cyclic nucleotide monophosphate. (ii) CpA*

versus the actual substrate

From the structure of this complex we have learned that the atoms involved in the recognition of the pyrimidine base by the enzyme are the same as those involved in the recognition of the pyrimidine bases of UMP, CMP and UpcA, although the details of binding, especially for the 2’ nucleoside portion are different. This has several implications, the most important being that the binding of the pyrimidine ring to the active-site pocket can probably occur in only a limited number of ways always involving the same crucial atoms on the protein as well as on the base. As a result, most of the substrate analogs are competitive inhibitors of RNase, and studying their binding can supply direct information on what the enzyme actually recognizes on the pyrimidine base of the substrate. Similarly, the phosphate group of CpA* binds to the same region of the active site as phosphate groups of other ligands including the sulfate ion, strongly indicating that the recognition is charge specific and localized to a small region of the active site. It is not yet clear that all phosphate groups are similarly oriented relative to the enzyme. The hydrogen bond between the phosphate oxygen of CpA* and the imino group of Phel20 has not been previously reported, and should be investigated in other derivatives. From the binding studies of Z’AMP, 3’AMP, 3’CMP, B’AMP, ATP and UpcA (Richards & Wyckoff, 1971), as well as from the present study, it can be deduced that the interactions of the purine base with the enzyme are very similar. In all cases it is probable that the N(6) nitrogen of the purine base hydrogen-bonds to the carbonyl groups of Asn71 or Gln69, and the N(,) of the base forms a hydrogen bond with the side-chain of Glulll. It has also been shown that 3’AMP and S’CMP bind to RNase S simultaneously without displacing each other (Richards & Wyckoff, 1971). One can therefore conclude that preferential independent binding sites exist for purine and pyrimidine bases. This preference might lead to the 3’-pyrimidine-5’-purine versus 3’-purine-5’pyrimidine specificity when the phosphodiester linkage acts as an additional constraint on the orientation of the bases relativ-e to their respective binding sites. It should be emphasized however, that enzymatic specificity can only be derived from thermodynamic parameters such as the free energy of enzyme-substrate or reaction intermediate complex forma.tion. In order to attempt a quantitative evaluation of these parameters from structural data it is necessary to settle first the question of what are the structures of the active site and the substrate when both form the transition-state complex. This in turn could only be achieved through investigations of the interactions of side-chains in the active site of the free enzyme by spectroscopic

C2,p,,A-RIBONUCLEARE

COMPLEX

X73

methods or X-ray diffraction analysis, the latter preferably at low temperature (Albert et al., 1976). In conclusion, CpA* is recognized by the enzyme in much the same way as are other inhibitors and probably also the substrate. This in itself is sufficient for competitive inhibition. In addition to the inhibitory properties of CPA*, chemical studies have demonstrated (unpublished results) that CpA* is not turned over by the enzyme. There is no intrinsic reason why a (2’,3’)-cyclic phosphate could not be formed starting from a (2’,5’)-dinucleoside monophosphate instead of a (3’,5’)-dinucleoside. In the 2’,5’ linkage the 0(3’) ribose could be made nucleophilic in much the same way as an 0(2’) oxygen would normally. This, however, could not happen in CPA*, since the orientation of the cytosine ribose is such that the 3’OH and 5’OH groups point away from the active site of the enzyme into the solution where no enzymatic chemical group can attack the 0(3’) in order to make it more nucleophilic. This finding is cous&tent with the lack of turnover of CpA* by RNase. One should keep in mind that the details of the model and the protein interactions presented here stem from crystallographic data, model-building and energy minimization. In particular the detailed conformation of the adenine ribose and the phosphate cannot be stated to be determined crystallographically but, rather represent a reasonable solution to the problem.

4. Discussion From this work and from previous studies which we have extensively quoted in the previous sections it appears that the binding of nucleotides and nucleosides to RKase occurs through general hydrophobic interactions and through hydrogen bonds. The latter involve both main-chain and side-chain groups. Edges of two fi sheet structures are involved in contacts where the obligate donor or acceptor roles of C=O and N-H come into play. An edge of a distorted /I structure at residues 118, 119, 120 divides the surface of the protein into two rather independent domains where the two bases bind and provides a “cutting edge” where the cleavage occurs (see the illustration ou p. 52 in Atlas of Protein Structures, Richards & Wyckoff, 1973). Strong positive charge contacts with the phosphate group should not dominate the situation at neutral pH, nor necessarily play an obvious role in stabilizing the bound nucleoside. Specific motion of some side-chains is associated with complex formation but it is the flexibility of the dinucleoside that is used to facilitate the fit. As was pointed out by Wyckoff et al. (1977), several of these features are shared by complexes of the coenzyme nicotinamide adenine pyrophosphoryl dinucleotide (NAD + ) with lactate dehydrogenase, and with other dehydrogenases (Rossman et al.. 1975). In the dehydrogenases, as in the RNase S-dinucleoside complex the binding domains for the two bases are separated by the tip of a fl strand which provides backbone to ligand hydrogen bonds among others. The nicotinamide and adenine bases are nearly perpendicular and the C(6) adenine-C(2) nicotinamide distance is about 14 A. The details of the enzyme-nucleic acid interactions appear, however, to be different from those we report here. This could be expected solely on the basis of the marked differences in structure between the dinucleoside monophosphate and the coenzyme NAD + . The coenzyme structure involves two 5’ base-phosphate linkages, it contains two instead of one phosphate group as well as a nicotinamide ring rather than a usual

S. Y. WODAK

874

ET AL.

base. For example, in the LDHaset-NAD+-pyruvate complex the adenjne portion of the coenzyme interacts with the protein, almost exclusively, via non-polar groups, whereas in the RNase S-CPA* complex the bases make quite a few hydrogen bonds with the protein. The adenine and nicotinamide ribose rings form hydrogen bonds with LDHase which involve the 2’ and 3’ hydroxyl groups while the hydroxyl groups in the 5’ moiety of the bound CpA* point into the solution. On the other hand the interactions of the coenzyme phosphate groups with the protein seem, as in the present study, not to be dominated by strong electrostatic attraction. Although charged side-chains are nearby (Lys58, ArglOl in LDHase, and Lys41 in RNase S) close non-bonded interactions of charged atoms with the phospha#te oxygens are not always observed. Similarly, in the complex of flavin mononucleotide (FMN) with flavodoxin from Clostridium MP (Ludwig et d., 1974), the phosphoryl ion is bound in a neutral environment with no net positive charge nearby. The absence of strong charge-charge interactions with the bound phosphate ion is puzzling. One could envisage that the charge distribution of a whole region of the active site rather than one specific close interaction could be important in “lurjng” the ligand to the correct binding area. Such an effect, however, would operate only at short distances due to the large dielectric constant of water. The role of electrostatic interactions in the mechanism of substrate binding obviously requires further investigation. We thank Y. Lapidot for the CpA* material and J. Sussman for having suggested the binding experiment. We are particularly indebted to Helen Schwartz for her help in establishing that CpA* inhibits RNase S. We are grateful to C. Levinthal for encouragament and helpful suggestions, to J. Greer for comments on a previous version of this paper, and to T. Steitz, R. Fletterick and other members of the protein crystallography laboratory at Yale University for valuable help at all stages of this work. Last but not least we thank R. Abba, C. Tountas and R. Bornhold at Columbia University for programming the Protein Manipulation Package. This work was supported by National Institutes of Health grant GM-10025, National Institutes of Health Facility grant RR-00442, and the Columbia University Computer Center. This material has been submitted in partial fulfilment of the requirement for the degree of Doctor of Philosophy in the Faculty of Pure Sciences, Columbia University, 1974. REFERENCES Albert, T., Petsko, G. A. & Tsernoglou, D. (1976). Nature (London), 263, 297-301. Allwell, N. M. (1968). Ph.D. thesis, Yale University. Allwell, N. M., Mitsui, Y. & Wyckoff, H. W. (1973). J. Biol. Chem. 248, 5291-5298. Arnott, S. & Hukins, D. W. L. (1969). Nature (London), 224, 886-888. Atlas of Protein Structures (1973). Vol. 1. Oxford University Press. Brown, D. M. & Todd, A. R. (1955). In The Nucleic Acids (Chargaff, E. & Davidson, G. N., eds), p. 409, Academic Press, New York. Carlisle, C. H., Palmer, R. A., Mazumdar, S. K., Gorinsky, B. A. & Yeates, D. G. R. (1974). Nature (London), 213, 8622865. Carlaon, W. (1976). Ph.D. thesis, Yale University. Davidson, W. C. (1968). Comp. J. 10, 4066415. Diamond, R. (1971). Acta Cryatallogr. sect. A, 27, 436-452. Donohue, J. & Trueblood, K. N. (1960). J. Mol. Biol. 2, 3633371. Fletcher, R. & Powell, M. J. P. (1963). Comp. J. 6, 163-168. Findlay, D., Herries, D. G., Math@ A. P., Rabin, B. R. & Ross, C. A. (1961). Nature (London), 190, 781-784. Gibson, K. D. & Scheraga, H. A. (1967). Proc. Nat. Acad. Sci., U.S.A. 58, 1317-1323. t Abbreviation

used: LDHase,

lactacte

dehydrogenase.

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8 75

Henderson, R. & Moffat, J. K. (1971). Acta Crystallogr. sect. B, 27, 1414-1420. Honig, B., Ray, A. & Levinthal, C. (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 1974.-1978. IUPAC-IUB Commission On Biochemical Nomenclature (1970). Biochemistry, 9, 34713479. Kartha, G., Bello, .J. & Harker, D. (1967). Nature (London), 213, 862-865. Knrtha, G., Bollo, J. & Harker, D. (1968). In Structural Chemistry and Jfolecular Biolog,q (Rich, A. 85 Davidson, N., eds), pp. 29-37, W. H. Freeman and Co., New York. Knt,z. L. & Levinthal, C. (1972). Annu. Rev. Biophys. Bioen,g. 1, 465-504. Kim, S. H., Berman, H., Seeman, N. C. & Newton, M. (I 973). Acta Crystallogr. sect. H, 29,

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I,evinthal, C., Wodak, 8. J., Kahn, P. & Dadivanian, A. K. (1!175). I’roc. Sat. dead. ,‘+i., r’.S.A. 72, 1330-1334. Limpkin, D., Talbert, P. T. & Cohn, M. (1954). J. Amer. Chem. Sot. 76, 2871-2879. I,udwig, M. L., Burnett, R. M., Darling, S. R., Jordan, S. It., Kendall, D. S. & Smitll, TV. 11;. (1974). 4th Steenbock Symp., pp. 407-426, Madison, Wisconsin. Martirr. P. D., Petsko, G. A. & Tsernoglou, D. (1976). J. Mol. Biol. 108, 265-268. McGuire, R. F., Momany, F. A. & Scheraga, H. A. (1972). J. Phys. Chem. 76, 375-393. Newton, M. (1!173). J. Amer. Chem. Sot. 95, 256-258. North, A. C. T., Phillips, D. C. & Mathews. S. S. (1968). Acta Crystallogr. sect. B, 23,351-369. Powers, T. B. (1976). Ph.D. thesis, Yale University. Reekth. G. N., ,Jr (1971a). The CRYM Sorting and Merging Subprogram for the IBMj36(/. The Rockefeller University, New York. Reeke, G. N., Jr (197lb). GRNFOURI, 9 Crystallographic Fourier Summation. I’rograw~ for the IBM/360, The Rockefeller University, New York. Itenugopalakrishnan, V., Lakshiminarayanan, A. V. & Saisekharan, V. (1971). Hiq~olymers, 7, 115991167. Richards, F. M. (1968). J. Mol. Biol. 37, 225-230. Richards, P. M. 85 Vithayatil, P. J. (1959). J. Biol. Chem. 234, 145991465. Richards, F’. M. & Wyckoff, H. W. (1971). The Enzymes, 4, 647-806. Richards, P. M. & Wyckoff, H. W. (1973). Atlas of Protein Structures (Phillips, D. (‘. &z Richards, F. M., eds), vol. 1, Oxford University Press. Micha,rds. I”. M., Wyckoff, H. W. & Alwell, N. M. (1970). The Neurosciences Second Study I’rogram (Schmitt, F. O., ed.), pp. 901-925, The Rockefeller University Press. Richards, F. M., Wyckoff, H. W., Carlson, W. D., Allwell, N. M., Lee, B. K. & Mitsui, Y. (1971). Cold Spring Harbor Symp. Quant. Biol. 36, 35-43. Rossman. M. G., Liljas, A., Branden, C. I. & Banazak, L. ,J. (1975). The En,zymes. 11, pp. 61l102. Sliefter, E., Barlow, M., Sparks, R. A. & Trueblood, K. N. (1969). -4cta Crystallogr. sect. t!., 25, 895 901 Sundaralingam. M. (1969). Biopolymers, 7, 821-860. ITslicr, D. A., Evely, S., Erenrich, A. & Eckstein, F. (1972). hoc. LVat. ,4cad. Sci.. Iv.b’.d. 69, 115118. \Vebb. L. E.. Hill, E. ,J. & Banazak, L. J. (1973). Biochemistry, 12, 5101 -5109. \\‘itztl, H. & Barnard, E. A. (1962). Biochem. Biophys. Res. Commun. 7, 295- 299. \l:yckoff, H. W., Doscher, M., Tsernoglou, D., Ingami, T.. Johnson, L. N., Hardman. K. D., Allwell, N. M., Kelly, D. M. & Richards, F. M. (1967). J. Mol. Riol. 27, 563 578. \Vyckoff, H. W., Tsernoglou, D., Hanson, A. W., Knox, ,J. R., Lee. B. K. & Richards. F. M. (1970). .I. Biol. Chem. 245, 305-328. \l-yckoff, H. W., Carlson, W. & Wodak, S. J. (1977). In h’ucelic Acid -Protein Recognition (k’ogel, H. .J., rd.), pp. 569-580, Academic Press, New York,