Computer-generated Models of Blood Coagulation Factor Xa, Factor IXa, and Thrombin Based upon Structural Homology with Other Serine Proteases*

Transcription

1 THE JOURNAL OF BIOLOGKAL CHEMISTRY Vol. 257, No. 7, Issue of April 10, pp Printed in U.S.A. Computer-generated Models of Blood Coagulation Factor Xa, Factor IXa, and Thrombin Based upon Structural Homology with Other Serine Proteases* (Received for publication, July 22, 1981, and in revised form, September 29, 1981) Bruce Furie"76, David H. Bined, Richard J. Feldmann', David J. Robison", John P. BurnieraJ, and Barbara C. Furie"" From the "Division of Hematology-Oncology, Department of Medicine and Department of Biochemistry and Pharmacology, Tufis-New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02111, 'Center for Blood Research, Boston, Massachusetts 02115, and "Division of Computer Research and Technology, National Institutes of Health. Bethesda, Maryland Computer-generated molecular models of the trypsin-like domains of blood coagulation Factor IXa, Factor Xa, and thrombin have been prepared. These hypothetical models are based upon the sequence homology of the blood coagulation enzymes with the pancreatic serine proteases and the known three-dimensional structure of the pancreatic serine proteases. The internal structures and active sites of these enzymes are highly conserved. The high degree of substrate specificity which characterizes the blood coagulation enzymes appears to be defined not entirely by the active site, but by the unique molecular surface surrounding the active site of each enzyme. Several regions which demonstrate high sequence variability among these enzymes likely participate in forming the putative extended substrate binding sites. The blood clotting enzymes, Factor TXa, Factor Xa, and thrombin, bear marked sequence homology with chymotrypsin and trypsin (3-5). Like the digestive proteases, these enzymes are serine proteases with trypsin-like specificity for arginyl bonds. These serine proteases are synthesized as zymogens, activated to enzymes by limited prot,eolysis, and inactivated by protein protease inhibitors (for reviews, see Refs. 6 and 7). However, Factor IXa, Factor Xa, and thrombin are larger than trypsin and have domains which are not analogous to domains in other serine proteases. Factor X, Factor IX, and prothrombin contain y-carboxyglutamic acid (8, 9), an amino acid which confers unique metal binding properties on these proteins (10, 11). Furthermore, calcium, protein cofactors, and membrane surfaces are required for rapid conversion of these proenzymes to their active state (7). A prominent characteristic of these enzymes is their high degree of specificity for a limited number of plasma protein substrates. Since these enzymes are structurally and mechanistically homologous to trypsin and chymotrypsin, the functional differences must relate to differences in the active sites or putative extended substrate recognition sites. The nature of these differences is not known. To date, the blood coagulation proteins have not been crystallized in forms suitable for x-ray diffraction studies. In the absence of such studies, we have developed three-dimensional computer models of the trypsin-like domains of bovine Factor IXa, Factor Xa, and thrombin based upon the known tertiary structure of bovine chymotrypsin and trypsin and the sequence homology between the clotting proteins and the digestive proteases. In this communication we present these models as first approximations of the structures of the heavy chain of Factor IXa, the heavy chain of Factor Xa, and the B chain of thrombin. Six regions in the primary structures of these proteins have been identified which show minimal conservation of amino acid sequence when compared to chymotrypsin and trypsin. These regions, located on the surface of the protein models, are likely to confer, in part, the characteristic substrate specificity of each of these enzymes. Furthermore, these hypothetical models suggest that the limited substrate specificity which characterizes these enzymes is defined not only within the active site, but in an extended substrate binding site that surrounds the active site. * This work was supported by Grants HL 21543, HL 18834, and AM from the National Institutes of Health. Portions of this work have been previously presented at a Symposium on Contributions to Hemostasis, Detroit, MI, May 1980 (1) and the International Congress on Thrombosis and Hemostasis, Toronto, Canada July 1981 (2). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 EXPERIMENTAL PROCEDURES The protein models were constructed and displayed on the comsolely to indicate this fact. puter macromolecular surface graphics system at the Division of Recipient of a grant from Merck, Sharp and Dohme. Established Investigator of the American Heart Association and their Massachusetts affiliate. A limited number of stereo slide sets of Fig. 3 will be Computer Research and Technology, National Institutes of Health. We have previously described this facility, which includes a DECSystern 10 computer, a DEC PDP 11/70 computer, and an Evans and made available to investigators with sufficient need. Address corre- Sutherland Picture System 2 (12). In brief, protein structures are spondence and reprint requests to Dr. Bruce Furie, Tufts-New Eng- presented on the vector display as an array of points (depicting land Medical Center, I71 Harrison Ave., Boston, MA atoms) connected by lines to represent the covalent connectivity. The "Recipient of a grant from the Burroughs Wellcome Co. Estab- position of each atom except hydrogen is defined by the atomic lished Investigator of the American Heart Association and their coordinates which have been determined by x-ray diffraction tech- Massachusetts affdiate. niques. Hydrogens are added to the structure using known average 'Recipient of Institutional (1 T32 HL07437) and Individual (1 F32 bond angles and bond lengths. This display is translated into a space- HL06235) National Research Service Awards from the National In- filling molecular model on a cathode ray tube. The displayed molecstitutes of Health, ular structure is a two-dimensional projection of a three-dimensional 'Recipient of a Research Career Development Award from the array of points. Each point, representing an atom, is the center of a National Institutes of Health. shaded, colored sphere. Spheres can be color-coded to delineate 3875

2 3876 Models of Blood Coagulation Proteins atoms, residues, domains, or chemicophysical features of the molecular surface. Some aspects of the molecular modeling were performed manually. The displayed model was translated and rotated to optimize viewing of specific molecular details. A subroutine allowed rotation of the + and.i. torsion angles of four adjacent residues on either side of a given residue, thus allowing alteration of the structure of the peptide backbone. Additional amino acid residues (insertions) were incorporated into the peptide backbone or amino acid residues in the structure (deletions) were eliminated and the backbone reconnected using this program under direct user control. The amino acid sequences of bovine Factor IXa (3), bovine Factor Xap (heavy chain) (4), bovine thrombin (5), bovine trypsin (13), and bovine a-chymotrypsin (14) have been taken from published data. A correction in the thrombin B chain, based upon the nucleotide sequence coding for thrombin, has been incorporated into this analysis (15). The atomic coordinates for bovine chymotrypsin and bovine trypsin used were updated versions of Tulinsky et al. (16) and Krieger et al. (17). The sequence homologies of analogous regions in the serine proteases were compared by inspection. On this basis, conserved regions were identified in which 50% or greater sequence homologies were preserved. The remaining sequences were considered part of variable region domains. RESULTS AND DISCUSSION Sequence Comparison Analysis-The amino acid sequences of Factor Xa, Factor IXa, and thrombin were aligned with the sequences of chymotrypsin and trypsin. Highest priority was accorded to the preservation of the alignment of regions with identical amino acid sequences and homologous disulfide bonds. Insertions and deletions were introduced in the blood clotting proteins relative to the sequence of trypsin and chymotrypsin where stretches of the sequence were out of register. To preserve the integrity of the P-barrels, insertions and deletions were not incorporated into regions which, in trypsin or chymotrypsin, define the,c3 sheet structure. Despite these rules, the precise placement of insertions and deletions was often ambiguous. Inspection of the three-dimensional structure of the chymotrypsin peptide backbone aided in the identification of regions of the peptide backbone that could accommodate these changes. The alignments of thrombin, Factor Xa, and Factor IXa are shown in Fig. 1. Relative to chymotrypsin, thrombin has 9 insertions and 2 deletions. The largest insert is of 10 residues between residues 60 and 61. Two 5-residue inserts are located between residues 146 and 147 and between residues 185 and 186. The remaining insertions are 3 residues or less. A total of 29 additional amino acid residues have been inserted into the chymotrypsin backbone. Two 1-residue deletions have also been noted. The Factor Xa model contains 5 insertions of 1 or 2 residues each and 4 deletions (Fig. 1). These include a total of 8 inserted residues and 6 deleted residues relative to chymotrypsin. Five insertions of 1 or 2 amino acids and 3 deletions of a single residue are proposed for Factor IXa (Fig. 1). These include a total of 8 inserted residues and 3 deleted residues in Factor IXa relative to chymotrypsin. The aligned sequences of chymotrypsin, thrombin, Factor Xa, Factor IXa, and trypsin (Fig. 1) were compared and analyzed. Seven regions of the sequences were identified that are generally well preserved in the five proteins. These regions are termed CR, for conserved regions. CR1, stretching from residues 16 to 33, contains the NH2-terminal amino acid which, when exposed after zymogen activation, is observed in close proximity to Asp 194 in trypsin and chymotrypsin. CR2 (residues 41 to 58) includes His 57 of the catalytic triad in the active site. CR3 (residues 102 to 124) includes Asp 102 of the catalytic triad. CR4 (residues 139 to 145) and CR5 (residues 155 to 185) include, in part, residues which participate in P- barrel structures. CR6 (residues 189 to 200) includes the active site Ser 195 of the serine proteases. CR7 (residues 211 to 239) contains a substrate binding domain, including Trp 215. As shown in Table I, about 50% or more of the residues in CR1- CR7 are identical in the blood coagulation proteins when compared to chymotrypsin or trypsin. These conserved residues are indicated by the solid circles in Fig. 1. Although conservative substitutions are not specifically denoted, these regions also contain many of these substitutions. Six variable regions, VR, were identified by sequence com- parison analysis. VR1 (residues 34 to 40) is a short stretch with few conserved residues and short deletions and insertions relative to chymotrypsin. VR2 (residues 59 to 101) contains 3 insertions (including the major 10-residue loop) and 1 deletion in thrombin, 2 insertions and 1 deletion in Factor Xa, and 2 insertions and 1 deletion in Factor IXa. Although the character of certain residues is preserved, e.g. residues 65 to 67,77, 82,95, these regions contain sequences unique to each enzyme. Similarly, VR3, VR4, VR5, and VR6 contain sequences which are highly variable. Insertions and deletions characterize these regions. As shown in Fig. 1, these regions show little conservation of structure except in approximate number of amino acids. Although these five proteases overall show marked sequence homology, these variable regions may contribute to the features of each protease that define their diverse physiologic properties. Generation of Protein Models Using Interactive Computer Graphics Techniques-Ample evidence has been put forth to indicate that the serine proteases, including trypsin, chymotrypsin, elastase, Factor Xa, Factor IXa, and thrombin, share marked sequence homology (3-7). X-ray crystallographic studies of trypsin, chymotrypsin, and elastase have revealed that these proteins also share homology of the three-dimensional structure of the peptide backbone. Using the known crystallographic coordinates for these structures, we have shown that the backbones of trypsin and chymotrypsin are nearly superimposable when the structures are oriented in a plane defined by the Ca of Trp 215, Ser 195, and Val 17. This analysis has been performed quantitatively by Greer, giving emphasis to the conformational homology of this class of proteases (18). Furthermore, Remington and Matthews have shown that proteins within the serine protease class that have modest sequence homology and multiple insertions andeletions relative to each other share very similar secondary structures (19). On this basis, we have constructed models of thrombin, Factor IXa, and Factor Xa based upon the homologous sequences of these proteins and the structures of the polypeptide backbones of chymotrypsin and trypsin. A working hypothesis employed in the construction of these models has been that of the conservation of the secondary structural elements of trypsin or chymotrypsin in the corresponding regions of the clotting enzymes. In support of this, Salemme has found that /? sheets of similar plan determine similar tertiary conformations (20). More specifically, /3 struc- tures of rhombic form tend to form cylindrical structures known as fl-barrels. We have employed the Chou and Fasman analysis (21) to compare the secondary structural elements of these serine proteases. The a or P potential, an index which was derived empirically from analysis of many known protein structures, was averaged over four adjacent residues. The hydrophobicity constants of Nozaki and Tanford (22) were averaged over 5 adjacent residues, as described by Rose (23). The results of these analysis (Fig. 2) show striking similarities between these different proteins as well as several interesting differences. The results predict considerable p-structural elements in the clotting enzymes in domains which correspond to regions of trypsin and chymotrypsin that are known to participate as strands in the P-barrels. The major differences

3 Models of Blood Coagulation Proteins 3877,

4 3878 Models of Blood Coagulation Proteins TABLE I Conserved and variable regions of the serineproteases The chymotrypsin numbering system is employed Regions Variable VRl VR2 VR3 VR4 VR5 VR6 Conserved CRI CR2 CH3 CR4 CR5 CH6 CR I Ser 195 His 57 Asp 102 Lys SEQUENCE NUMBER FIG. 2. Comparison of the predicted secondary structure of bovine trypsin and Factor Xa. The /3 potential of the sequence of these enzymes,less the insertedresidues,are compared. Trypsin, shaded; Factor Xa, -. The location of certain amino acid residues in the linear sequence is indicated. occurred in regions which were identified independently by sequence comparison analysis as regions of variability. The conclusions that we have drawn from these analyses of the predicted secondary structure and the sequence homology of the primary structure strongly suggest that these proteases share homology of their three-dimensional structure. Preliminary studies in which structural models of the blood clotting enzymes were prepared from both the chymotrypsin and trypsin backbone indicated that use of the chymotrypsin polypeptide backbone was advantageous. Thi structural model minimized the number and size of insertions and deletions necessary to develop the models of the blood coagulation proteins. However, in contrast to trypsin, thrombin, Factor Xa, and Factor IXa, a-chymotrypsin is composed of two polypeptide chains linked by disulfide bonds. This protein lacks residues 147 and 148. For this reason, some of the favorable features of the trypsin peptide backbone were extracted and incorporated into a hybrid structure primarily based upon the chymotrypsin backbone. The segment in trypsin was spliced into the hybrid structure to yield a single continuous polypeptide chain. angles of the adjacent residues were minimally altered. Residue 218 of chymotrypsin was deleted to make the position of Cys 220 trypsin-like in its spatial orientation. The resulting hybrid backbone structure represents the combined features of chymotrypsin and trypsin, with maximum homology with the blood coagulation proteins preserved. A computer program RESSUB was developed to facilitate the replacement of the side chains of the protein of known three-dimensional structure with the side chains of proteins of known sequence. According to Fig. 1, the nonhomologous amino acid side chains of the modeled protein were substituted at each appropriate residue of the peptide backbone. The angles describing C-Ca-Cp, Cn-Cp-Cy, and so forth, in the original side chains were preserved. When a substituted side chain was longer than the original (e.g. Glu for Gly) a standard but arbitrary side chain formation was incorporated into the model. Amino acid residues not present in the hybrid, the deletions, were eliminated from the model. The resulting NH2- terminal and COOH-terminal amino acids were spliced manually through manipulation of the projected structure on the vector display. Minimal changes in angles of the residues adjacent to the modeled peptide bond were necessary. The inserted residues relative to the chymotrypsin-trypsin hybrid structure were more difficult to place. RESSUB cleaved the peptide bond into which the inserted residues were to be placed. The inserted peptide backbone, initially in an arbitrary conformation, was joined to the NHs-terminal and COOH-terminal amino acid at the cleavage site. Using the vector display of the two-dimensional graphics projection of the three-dimensional structure of the protein model, we manually altered torsion angles of the inserted peptide backbone and the backbone adjacent (within 4 residues) to the inserted peptide. Efforts were made to minimally perturb the backbone structure of the protein. The inserted peptide backbones were placed in plausible positions. However, the structures chosen were not unique, but fall within the constraints of the covalent connectivity, close packing of the atoms, and elimination of van der Waals overlap. Insertions of 1 or 2 residues, characteristic of Factor Xa and Factor IXa, proceeded easily. The placement and structure of the longer insertions of thrombin must be considered crude approximations of the true structure of the protein. In the final steps, van der Waal overlap of amino acid side chains in the protein was eliminated or minimized by systematic rotation of the Ca-C/, Cp-Cy, etc. bonds of every amino acid. This routine minimized contacts among neighboring amino acids. In the absence of further constraints, however, side chains are often extended into the surrounding solution of the protein. Since the current studies are directed at un- derstanding the structural and functional roles of large domains on the protein surface, we have not refined the side chain conformations. Hypothetical Models of Factor Xa, Factor IXa, and Thrombin-The molecular surface projections of the active site-containing aspect of thrombin, Factor Xa, Factor IXa, and trypsin models are shown in Fig. 3. The benzamidine binding site is located in the center and the catalytic triad (His 57, Asp 102, Ser 195) just below the center (Fig. 3, Row 1, Column B). In Fig. 3 (Column A) we have employed the functional coloring code to emphasize the chemical and phys- The functional color code includes: peptide backbone carbonyl oxygen pink, carbon (gray), and amide nitrogen (light blue); all other atoms are black except as follows: red, 0,1 and Or2 of Glu. 081 and Oaz of Asp; blue, Nt of Lys and Nql and Nq2 of Arg; light blue, NB and NN of His, Ne2 of Gln NS2 of Asn, N,I of Trp, N, of Arg; pink, O,I of Gln

5 Models of Blood Coagulation Proteins 3879 Lc 1 I I L ia L r -1, FIG. 3. Computer graphics models of thrombin, Factor Xa, and Factor M a. Frontal views of the active site. Column 2, Row 1: specific residues of the trypsin-active site are indicated Ser195 (red), His 57 (yellow), Asp 102 (green), Gln 192 (black), Try 215 (turquoise), Asp 189 (blue). Column I shows trypsin and theblood coagulation enzymes depicted in the functional coloring code. This coloring algorithm, which connotes the chemical, physical, and topographical properties of the molecular surface, has been previously described in detail (12). Column 2 shows the same projections, but with a coloring algorithm that compares each protein to trypsin. Identical amino acids (pale blue), substituted amino acids (yellow), inserted amino acids (red), and amino acids adjacent to deleted residues (blue). Column 3 shows the same projections with the variable regions indicated. VR1 (white), V R 2 (red), VR3 (green), VR4 (blue), VR5 (yellow), and VR6 (black). The constant regions are displayed in pale blue. Row 1, trypsin; row 2, thrombin; row 3, Factor Xa; row 4, Factor IXa. ical differences in the surfaces of trypsin and the blood clotting enzymes (12). For example, trypsin has few negative charges (red) on the front surface, but many negative charges are observed on this surface on the three blood coagulation enand Orl of Asn; white, 0 6 of Ser, of Thr, O,,of Try, N,? or N,wof His can be presented in blue depending upon the pk of the histidine and the assumed ph of the environment. Hydrogen atoms assume the same color as the atomto which they are bound. zymes. As a general rule, the charge distribution, topography, and hydrophobic veins on each surface is highly individualized. This is likely to be a reflection of the unique substrate specificity that characterizes each of these enzymes. The individual characteristics of the front projections are also true Of Other parts Of the molecular surface (not shown). For example, thrombin, Factor Xa and Factor 1% have in cornmon a lower molecular weight chain which is disulfide-linked

6 3880 Models Coagulation Proteins of Blood Trypsin TABLE I1 Amino acid substitutions in the conserved regions which may effect substrate binding Clotting I. proteases Region Role in trypsin-ligand complexes Thrombin Factor Xa Factor IXa Phe 41 CR2 Leu Phe Leu 99 VR2 Leu Tyr Asn 143 CR4 Asn Arg Ser 190 Ala CR6 Ala Gln 192 Gln CR6 Glu Ser 217 CR7 G~Y Glu GlY 219 GlY CR7 GlY 221 Ala CR7 ASP Ala Phe Hydrogen bonding to amide N of substrate P'2 region Tyr Hydrophobic bonding potential to substrate P2 site Lys Salt bridge of Ile 16 to Asp 194 Ser Hydrogen bonding of 0 to amidino N in benzamidine Gln Hydrogen bonding to carbonyl 0 of substrate at P2 site Glu At top entrance to the substrate binding pocket; may affect substrate recognition Glu Hydrogen bonding of carbonyl 0 to amidino N of benzamidine Ala Participates with Asp in forming hydrogen bonding to the amidino nitrogen of benzamidine to the heavy chain at Cys 122. The molecular weights of the thrombin, Factor Xa, and Factor IXa models (front view) light chain in thrombin, Factor Xa, and Factor IXa are 4,600, have been color-coded to denote the location of the variable 17,000 and 18,000, respectively. In chymotrypsin Cys 122 is regions, VR1 through VR6, of the serine proteases. The seven linked to an octapeptide, residue 1 to 8, derived from the 16- amino acid polypeptide which is cleaved in chymotrypsinogen conserved regions, shown in pale blue, form part of the surface and most of the internal structure. Considering that the conduring activation. Based upon identification of Cys 122 on the stant regions include 137 residues of the 229 residues (not rear surface of the models projected in Fig. 3, we can infer that the light chain domains of the blood clotting enzymes are centered on these rear surfaces. including deletions and insertions), or 60% of the amino acid residues, it would appear that the structural core of the serine proteases are defined by these sequence regions. Comparison of the surfaces of the trypsin-like blood clotting Substrate Binding Domains-The variable regions likely enzymes with the surface of trypsin revealed regions of simi- play a major role in the definition of the diverse binding larity and dissimilarity. In Fig. 3 (Column B), the same properties of these enzymes. The locations of these variable projections of the computer models of thrombin, Factor Xa, regions, VR1-VR6, were identified in each of the blood coagand Factor IXa are displayed. The coloring algorithm em- ulation enzymes. Three variable regions, VR1, VR2 and VR4, ployed codes for comparative features of these proteins. Atoms surround the conserved active site. VR2 (red) is particularly of residues which are identical with those of trypsin are prominent and substantial in terms of surface area. It includes presented in pale blue. Atoms of residues which differ from those in trypsin are presented in yellow. The atoms of inserted amino acids in the blood clotting enzymes are shown in red. The atoms of residues of amino acids on either side of deleted residues are shown in blue. A common feature characterizes all of the three models: the cores of these proteins appear highly conserved. In contrast, much of the surface below the active site and to the right side of the active site. All of the clotting enzymes have insertions in this domain in close proximity to the active site. In thrombin, the VR2 region also contains the carbohydrate covalently linked to Asn 60G.2 This part of the surface thus represents a major difference between the three proteases. Lys 69 and Arg 78 in VR2 are the sites of proteolytic cleavage which results the surface structures are defined by amino acids that vary in the conversion of a-thrombin to P-thrombin. This form, from those of trypsin. This emphasizes that our molecular lacking residues 70 to 78, cannot convert fibrinogen to fibrin. modeling approach leads to prediction of a hypothetical mo- However, P-thrombin retains full activity with respect to lecular surface structure of proteins, and not the molecular structure in toto. One area of the surface stands out in the three enzymes because, unlike other aspects of the surfaces, it is preserved in the serine proteases. This is the region about and including the active site: the catalytic triad (His 57, Asp synthetic ester hydrolysis. This site likely represents an area of the thrombin surface which is involved in protein substrate recognition. This interpretation is consistent with and supported by the spin label experiments of Berliner et al. (24). To the right side of the active site is the VR1 domain (white). 102, Ser 195), the benzamidine binding site (including Asp Above and to the right side is the VR4 domain (blue). We 189), and the extended substrate binding site (including Trp 215). The conserved active site is surrounded by a highly substituted surface. In thrombin this surface includes 5 insertions envision that these surfaces might define recognition domains for the binding of large protein substrates to the clotting enzymes. By comparison of the location of the VR1, VR2, and VR4 and 1 deletion. Factor Xa and Factor IXa both contain 4 domains and the amino acid residues on the surface which insertions and 2 deletions on the front surface. Since these have undergone substitution in the generation of the models, proteases are highly specific for certain protein substrates and it is clear that some amino acid residues in the conserved since their active sites are nearly identical, it would follow regions also define the surface characteristics about the active that the substrate recognition site must include an extensive surface surrounding the active site which is complementary to the protein substrates. site. Although conserved regions were identified as regions in which greater than 50% of the amino acid residues are conserved, up to 50% of the amino acids in these regions may be We have examined the front surface of the models to altered. Some of these altered residues may play a key role in determine which of the variable regions of the sequence define ' Amino acid residues in insertions are lettered sequentially. Each the surface in the vicinity of the active site. These regions residue is identified by the residue number of the amino acid NHLmay then be assigned as likely domains responsible for the terminal to the insertion and the letter of inserted residue (eg. 60A, definition of substrate specificity. In Fig. 3 (Column C), the 60B, 60C, etc.).

7 Models Coagulation Proteins of Blood 3881 defining substrate recognition. Conserved regions in or near the variable external surface which must define their diverse the active site include CR2, CR3, CR6, and CR7. Janin and Chothia (25) have studied the structures of the trypsin-panproperties and biological function as enzymes. One area of the surface, the immediate region about the creatic trypsin inhibitor and trypsin-soybean trypsin inhibitor active site, is conserved in all of the trypsin-like serine protease complexes in order to gain insight into the nature of the models. This is the surface responsible for efficient catalysis substrate binding regions on both sides of the cleavage site. of peptide bond hydrolysis and recognition of arginyl and lysyl Only a small proportion of inhibitor and protease are in residues. Experimental data using substituted benzamidines contact in the complex: 14 out of 58 residues in the inhibitor have emphasized the similarity of the interaction of these and 24 out of224 residues in trypsin. The contacts in the relatively small ligands with the active sites of the serine complex are characterized by a large number of hydrogen proteases studied (27). These data also support the chemical bonds and van der Waals contacts a tightly in packed protein- similarity of the active sites in these enzymes. A question protein complex (26). In Table 11, we have summarized this arises as to why this hypothetical surface, which is nearly information and have further indicated what these residues identical with trypsin, does not confer on the blood coagulaare in each of the clotting proteases. While there is a high degree of conservation at positions close to the cleavage (Pltion enzymes the enzymatic properties trypsin, these enzymes bind benzamidine of trypsin? Unlike with much lower P l) site, there are some notable exceptions. These include (i) the change from a serine to alanine position at 190 in thrombin and Factor Xa, which eliminates the possibility of hydrogen affinity. It is likely that lysyl and arginyl residues in polypeptides, suitable ligands for trypsin, are not bound tightly by the blood coagulation enzymes. A large extended substrate bindbond formation between the serine hydroxyl of the protein ing site on these enzymes may serve to recognize a limited and the amidino nitrogen of benzamidine; and (ii) the change number of protein substrates of these enzymes to promote from glycine to glutamate at position 219 in Factor IXa which interaction with a suitably high affinity. We believe that VR1, may alter the size and charge of the binding pocket. The VR2, and perhaps VR4 define this recognition site. Thus, changes at the P2 site may affect substrate recognition. For substrate specificity is not defined entirely by the active site example, the glutamine to glutamic acid change at residue 192 in thrombin facilitates possible electrostatic interaction with the substrate rather than the hydrogen bonding that is observed in trypsin between the carbonyl oxygen of glutamine and the peptide backbone of the substrate in the P2 region. (as is the case for enzymes with lower molecular weight substrates), but by an extended substrate recognition and binding site which surrounds the active site. COMMENTS Although we cannot assume that the binding of pancreatic With the advent of sophisticated electronic graphics systrypsin inhibitor to trypsin necessarily serves as an ideal tems interfaced with high speed computers and the increase model for the interaction of protein substrates (Factor X, in the primary structural data on proteins facilitated by auprothrombin, and fibrinogen) with the clotting enzymes, this tomated sequence techniques, it has become highly desirable analysis does suggest the potential for the partial definition of and facile to develop models of proteins of known sequence substrate specificity by residues in the conserved regions. which are structurally homologous to proteins of known threedimensional structure (28). These models may be considered CONCLUSIONS From this analysis we can conclude that the architecture as first approximations of the three-dimensional structures. They do not replace or substitute for models based upon x- and interior framework is likely to be similar for the blood ray diffraction methods. Yet only a very small percentage of coagulation proteins and the proteases of known tertiary proteins of known sequence have been crystallized to yield structure-trypsin, chymotrypsin, and elastase. In contrast, the surfaces of these protein models are composed in part of detailed information about the tertiary structure. In the absence of such studies, computer-generated models offer conamino acid residues from six variable regions of the serine siderable insight into the structure-function relationship of a protease sequences. These regions define molecular surfaces that may be important to the physiologic function of this class of proteins. Macromolecular assembly and protein substrate recognition is an important component of the complex cascade pathways of blood coagulation (6, 7). Although we cannot be altogether certain whether the trypsin-like domains or the class of proteins. As with all protein models, these hypothetical structures can facilitate the design of experiments which test the structural hypothesis. They furthermore yield insights into both the unifying features that characterize a family of proteins and the features which define their diverse properties and mechanisms of action. nonhomologous domains, or surfaces defined by both, are responsible for the interaction of these enzymes with other Acknowledgments-We thank Dr. Michael Potter (National Canproteins or membranes, some of the molecular surface recog- cer Institute) for many helpful discussions and Eileen OBrien for nition must be conferred by structures on or near the active site. For example, thrombin cleaves fibrinogen, Factor VIII, Factor V, Factor XIII, and a protein on platelet membranes. It binds to and activates platelets. is inhibited It by antithrompreparation of the manuscript. We also thank Mindy M. Tai for the computer program used in the secondary structure analysis. REFERENCES bin 111, among other plasma protease inhibitors, and also 1. Bing, D. H., Laura, R., Robison, D. J., Furie, B., Furie, B. C., and binds heparin. Recognition of these macromolecules is a fun- Feldmann, R. J. (1981) Ann. N. Y. Acad. Sci. 370, damental property of the thrombin surface. In contrast, Factor 2. Furie, B., Bing, D. H., Furie, B. C., Robison, D. J., Burnier, J. P., and Feldmann, R. J. (1981) Thromb. Haemostasis 46, 14 Xa cleaves prothrombin, Factor VII, Factor IX, and a peptide 3. Katayama, K., Ericsson, L. H., Enfield, D. L., Walsh, K. A,, bond on Factor X and Factor Xa which does not alter biolog- Neurath, H., Davie, E. W., and Titani, K. (1979) Proc. Natl. ical function. Factor Xa binds specifically to a receptor on Acad. Sci. U. S. A. 76, platelet membranes which involves, at least in part, Factor V. 4. Titani, K., Fujikawa, K., Enfield, D. L., Ericsson, L. H., Walsh, It is also inhibited by antithrombin 111. Factor IXa cleaves K. A., and Neurath, H. (1975) Proc. Natl. Acad. Sci. U. S. A. Factor X, a reaction accelerated by Factor VIII, calcium, and 72, platelet membranes. It too is inhibited by antithrombin Magnusson, S., Peterson, T. E., Sottrup-Jensen, L., and Claeys, H. (1974) in Proteases and Biological Control (Reich, E., Although these proteins share the same organizational frame- Rifkin, D. B., and Shaw, E., ed) pp , Cold Spring work in their interior and the same mechanism of action, it is Harbor Laboratory, Cold Spring Harbor, NY

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