©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Modeling of the Substrate Specificity of Prohormone Convertases SPC2 and SPC3 (*)

Gregory Lipkind (1), Qiuming Gong (1), Donald F. Steiner (1) (2)

From the (1) Department of Biochemistry and Molecular Biology and the (2) Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
Structures of the Active Sites of Subtilisins and SPCs
Conformational Models and Methods of Calculation
Investigation of the Primary Substrate Specificity of SPC2 and SPC3
Secondary Specificity of SPC2 and SPC3
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In this paper we describe the results of molecular modeling of the structures of the active sites of two subtilisin-like prohormone convertases (SPCs), SPC2 (PC2) and SPC3 (PC1/PC3). These enzymes are members of a recently discovered family of cellular proteases involved in the processing of precursor proteins. Although these proteases all possess catalytic domains similar to the bacterial subtilisins no tertiary structural data from x-ray analysis are yet available. We have shown that despite the high structural homology of the subtilisins and the SPCs, the structure of the loop which lies immediately below the active sites differs due to the presence of a cis-peptide bond (Tyr-Pro) in this loop in the subtilisins and its absence in the SPCs. Accordingly, we have proposed a new alignment for the amino acid sequences of the SPCs in this region. Both SPC2 and SPC3 participate in the processing of prohormones at dibasic cleavage sites, typically Lys-Arg or Arg-Arg. To investigate the structural basis of the substrate specificity of these SPCs, we have carried out molecular mechanic calculations of the optimal arrangement and interactions of peptide substrates containing several residues of arginine or lysine, i.e. Arg, Ala-Ala-Ala-Arg, Arg-Ala-Ala-Arg, Arg-Ala-Arg-Arg, Arg-Ala-Lys-Arg, in the putative active sites. Such subtilisin-based modeling has allowed us to identify those negatively charged residues, Asp and Glu, in the S1, S2, and S4 subsites, which can directly interact with basic residues in the substrates via formation of salt bridges and thereby contribute to the substrate selectivity of the SPCs.


INTRODUCTION

In neuroendocrine cells many peptide hormones and neuropeptides are synthesized as part of larger, less active or inactive precursors. An essential step in the formation of bioactive peptides is the selective endoproteolytic cleavage of their precursors (1, 2, 3, 4, 5) , as was first demonstrated in the processing of proinsulin to insulin (6) . Within the last few years a novel family of cellular endoproteinases involved in precursor processing has been discovered which includes the yeast mating pheromone processing enzyme kexin (7, 8) , the mammalian endoproteinases furin (SPC1)()(9) , PACE4 (SPC4) (10) , and the prohormone convertases PC2 (SPC2) (11, 12) , PC1/PC3 (SPC3) (13, 14) , PC4 (SPC5) (15) , and PC5/PC6 (SPC6) (16) .

Surveys of the cleavage sites of a large variety of precursors that are processed within the secretory pathway have led to the conclusion that most of them possess complex multibasic cleavage sites of the general type Arg-X-Lys/Arg-Arg (R-X-K/R-R) (17, 18) . Most prohormones on the other hand are cleaved at paired basic residue sites, primarily Lys-Arg or Arg-Arg. However, further analysis of sites cleaved in mouse AtT-20 cells, which contain SPC3 as their major endogenous protease, has revealed that, in addition to the foregoing sites, others consisting of Arg-X-X-Arg (R-X-X-R) are also cleaved (19) . Therefore, a basic residue at position -4 relative to the scissible bond (P4) may play a very important role in enhancing substrate site selectivity of the SPCs generally, as is illustrated especially in the processing carried out by furin (20) . Thus, both furin and SPC3, and possibly also SPC2, catalyze the processing of protein and peptide precursors containing Arg-X-Lys/Arg-Arg or Arg-X-X-Arg cleavage sites in the constitutive and/or regulated exocytic pathways.

When kexin was cloned and sequenced (7) , it was found to contain a catalytic domain that is homologous to that of the bacterial subtilisins. Amino acid sequence alignments of SPC2, SPC3, furin, and kexin showed a very high degree of similarity in their catalytic domains (4, 21) . The most significant difference between the subtilisins and the catalytic domains of the SPCs is the large increase in the number of negatively charged residues (Glu and Asp), relative to the subtilisins, which may contribute to the great selectivity of this family of enzymes for substrates containing paired basic residues (5, 22, 23) .

Although x-ray investigations of SPCs have not yet been carried out, molecular modeling of the catalytic domain of furin has indicated that it is closely related in its three-dimensional structure to the subtilisins (21, 22, 23, 24) . This circumstance opens the way for molecular modeling and protein engineering of the SPCs on the basis of their homology with subtilisins.


Structures of the Active Sites of Subtilisins and SPCs

The three-dimensional structures of four subtilases have been established, subtilisin BPN` (Novo) (25, 26, 27) , subtilisin Carlsberg (27, 28), thermitase (29) , and proteinase K (30) . The overall conformation of these enzymes is very similar (21) , especially in their active site regions (27) . In molecular modeling the active sites of SPC2 and SPC3, we have used the x-ray data on subtilisins BPN` and Carlsberg (27) . The substrate-binding site of the subtilisins was identified by analysis of their crystalline complexes with inhibitors or inactive analogs of substrates (26, 27) . The active site grooves of the subtilisins can bind at least 6 amino acid residues (P4-P2`)() of peptide inhibitors and/or substrates.

Residues forming the active sites of subtilisin BPN` and Carlsberg are indicated in . This information is taken from Ref. 27, describing contacts of less than 4 Å between subtilisins and the peptide inhibitors eglin-c or chymotrypsin inhibitor 2. The presumptive residues at structurally equivalent positions in the catalytic domains of SPC3 and SPC2 are also given in in accordance with the amino acid sequence alignment of known subtilases as proposed by Siezen et al.(21) . In accord with our modeling results, we have included 3 additional amino acid residues because their side chains potentially interact with residues P1 and P4 of substrates: 165 in subsite S1, and 131 and 135 in subsite S4 of SPC3 and SPC2.

However, we have noted a significant difference between the primary structures of the subtilisins and PCs that appears to be important in the structural organization of these proteins. In subtilases with known x-ray structures, a cis-peptide bond always occurs between residues 167-168 and in all cases residue 168 is proline, as is usually the case following this kind of bond (32) . Moreover, in dipeptide fragments of the type Tyr-Pro (Tyr-Pro in the subtilisins), cis-peptide bonds predominate (32) . In the case of the subtilisins, this cis-peptide bond forms a sharp type VI -turn (33, .2) in the inner loop 165-171 which serves as a floor for the active sites.

In contrast to the subtilisins, all the SPCs contain Ala or Thr instead of Pro at positions corresponding to 168 of the subtilisins (21, 24) . This suggests that a trans-, rather than cis-, peptide bond occurs in this position. In most proteins cis-peptide bonds occur exceptionally rarely preceding residues other than Pro (33) , e.g. the loop formed by the sequence, Thr-Gln-Ser-Pro-Ser-Ser (residues 5-10), of the Fab domain of immunoglobulin MCPC 603 contains a cis-peptide bond between Ser and Pro, whereas in the analogous loop, Thr-Gln-Thr-Thr-Ser-Ser, of Ig F19 all bonds are trans (files 1MCP and 1F19 of the Brookhaven Protein Data Base). Therefore, the alignment of amino acid residues for the loop 165-171 given by Siezen et al.(21) in the SPCs may be incorrect. Moreover, in their alignment the small Gly or Ala residues at position 169 in the subtilisins are replaced by Asp (in SPC3, ) or Asn (in kexin and furin), which is unrealistic because the methyl group of the side chain of Ala makes close van der Waals contacts with the backbone and the side chains of residues 152 and 153, as well as with the side chain of residue 165 (Asp in SPC2 and SPC3, ).

To appropriately align the amino acid sequences of the subtilisins and SPCs, we have taken into account the characteristic interactions of the side chains of the residues Tyr and Tyr at ends of the aforementioned loop. This packing has an energetically favored perpendicular arrangement of the aromatic rings (34) and is likely to be conserved in the structures of both the subtilisins and SPCs. In the alignment of Siezen et al.(21) , residue 167 is tyrosine in all enzymes; however, the downstream residue (Tyr) in the sequences of the SPCs has been aligned with residue 172 (Pro or Asp) of the subtilisins. Such an alignment is unlikely according to the matrix of mutual substitutions of amino acid residues in proteins (35) . For this reason it is difficult to accept the latest alignment given by Siezen et al.(24) where Tyr and Tyr of the subtilisins and thermitase are substituted by Gly and Ser in the SPCs. Such substitutions would be expected to lead to a loss of the energy of nonbonded interactions between residues of Tyr and Tyr which for thermitase comprises -7 kcal/mol. It is important that all known SPCs contain these 2 highly conserved aromatic residues in this loop (24, ).

In order to preserve the Tyr-Tyr interaction in the SPCs, we propose a new alignment for sequences 165-171 of the subtilisins and the corresponding residues in SPC3 and SPC2 (). In this alignment a small residue (Ser) occurs in position 325 (169) () at the bottom of the S1 binding cavity, and the bulky side chain of Pro (168) of the subtilisins is substituted by the side chains of residues Asp, Ser, or Asn, which are located outside the S1 groove in the SPCs. As a consequence, the primary structures of the SPCs contain an inserted residue of Ala or Thr between residues (167) and (168) in the subtilisins. We propose that this additional residue in this loop in the SPCs compensates for the loss of the cis peptide bond of the subtilisins and promotes the formation of a new central -turn, involving residues 320-327 of SPC3, which consists entirely of trans-peptide bonds ().()

To conserve structural correspondence between the two loops,(165-171) and 320-327, in the preliminary stage of molecular modeling we introduced range constraints on the arrangements of the main chains of these two peptide strands between the C atoms of residues(165-167) and(168-171), removed the cis-peptide bond, and introduced a new residue, either Ala or Thr, between the C atoms of residues (167) and (168) . This residue could be accommodated if it adopted the appropriate conformation of a classical -turn, which corresponds on a Ramachandran plot to the lower right square H (33; .2). This -turn forms a sharp reverse turn in the mid-region of the 320-327(165-171) loop of the SPCs which is stabilized by the hydrogen bond CO (Tyr) HN (Asp) and consists entirely of trans-peptide bonds (Fig. 1).


Figure 1: Backbone structures of the loop 165-171 in subtilisin (a) and the loop 320-327 (165-171) in SPCs (b).



Upon energy optimization of this initial structure (see ``Conformations Models and Methods of Calculation''), we found a new conformation for the loop 320-327(165-171) in SPC3, which conserved effective interactions of the aromatic side chains of residues 322 (167) and 327 (171) . The new version of this loop (Fig. 1) has been introduced into the structures of subtilisins BPN` and Carlsberg by means of superposition of equivalent -carbon atoms (except for C of the inserted residue) of the new and old loops with a root-mean-square deviation of 0.35 Å. All the following calculations of enzyme-substrate interactions have been performed with SPC active site models having this new loop structure.


Conformational Models and Methods of Calculation

Any theoretical study of enzyme-substrate interactions should begin with considerations of factors which govern the primary specificity. It is most practical to undertake this initially at the stage of the formation of covalent complexes between the catalytic side chains of the enzyme and the atoms of the scissible bond of the substrate. Investigation of nonbonded Michaelis complexes would make this problem significantly more complex since the introduction of six additional variables for the orientation of the nonbonded substrate as a whole relative to the enzyme would be necessary.

Subtilisin, like other serine proteases, cleaves peptide bonds in two steps (36) . The first step is the formation of a covalent bond between the C` atom of the scissible peptide bond and the hydroxyl oxygen O of the side chain of the catalytic Ser residue. This acyl-enzyme intermediate proceeds through a negatively charged tetrahedral intermediate where the nitrogen atom of the peptide bond is protonated by His. The second step of the reaction, deacylation, includes hydrolysis of the acyl-enzyme by the attack of a water molecule, which is activated also by His, to regenerate the free enzyme and release the product. This step also proceeds through a negatively charged tetrahedral intermediate. The side chain of Asn and the NH group of Ser take part in the stabilization of the tetrahedral intermediate in the catalytic pathway of the subtilases (36, 41). The only exception at this position in the SPCs is Asp in SPC2 (5) . However, SPC2 is optimally active at acidic pH 5.5, suggesting that this Asp could interact with the tetrahedral intermediate in its protonated form (11) .

Therefore, the acyl-enzyme intermediate and the corresponding tetrahedral adduct formed in the deacylation step with a single P1 residue (Arg) as the substrate component are the simplest models for calculating enzyme-substrate interactions and investigating the nature of the primary specificity of SPCs. Moreover, comparison of the structures of the acyl-enzymes and the tetrahedral intermediates allows us to investigate the stereochemistry of their mutual transformations. Models of substrate components (consisting of a single Arg residue) for both intermediate states of the enzymes SPC2 and SPC3 are shown in Fig. 2 .


Figure 2: Conformational parameters of the substrate component in the complexes of SPC3 or SPC2 with Arg (P1) in the stage of formation of the acyl-enzyme (a) and the tetrahedral intermediate (b).



The conformational possibilities for substrate components are determined by the dihedral angles of rotation around the C-N and C-C` bonds of the main chain, (C-N) and (C-C`), and around the side chain bonds, angles (C-C), (C-C), (C-C) and (C-N), of the Arg residue (Fig. 2). In addition, the dihedral angles of rotation around the side chain bonds of Ser(221) [( (C-C), (C-O)) and around the complex or simple ester bond ( (O-C`) between this Ser residue and Arg (P1)] were calculated. The determination and description of the angles of rotation in the peptide chain corresponds to standard nomenclature (33, 35). The angles of rotation equal zero when the corresponding bonds in the backbone and in the side chain have a cis orientation. For the values of bond lengths and valence angles, the averaged parameters for peptide and ester bonds were used (35) . Zero approximation for the valence angles at the C` atom of the scissible bond of the tetrahedral adduct was 109.5°.

In the case of more complex substrate components that include several Arg residues (e.g. Arg-Ala-Ala-Arg), we have taken into account the same conformational parameters for the side chain of P4 Arg as for the P1 Arg and also have calculated the angles of rotation around the C-N and C-C` bonds of the main chain; i.e. and for residue P1, and for P2, and for P3, and and for P4.

Molecular modeling was carried out using the Insight and Discover graphical environments (Biosym Technologies, Inc., San Diego). The molecular mechanics energetic calculations utilized the force field cvff (consistent valence force field), which was specifically derived to apply to structures of peptides and proteins. In this approximation the formation of hydrogen bonds is modeled as an electrostatic interaction. Electrostatic interactions were calculated using a dielectric constant = 4. This value corresponds to the microscopic dielectric constant of polypeptides (39) and usually is used for conformational calculations in polar media (37) . N- and C- ends of peptide fragments of active sites were considered in neutral form as N-methyl and N-acetyl amides, correspondingly. For minimization procedures the steepest descends and conjugate gradients have been used. Atomic coordinates of the enzymes were taken from the Brookhaven Protein Data Base for subtilisin BPN` and subtilisin Carlsberg (PDB codes 2 SNI and 2 SEC (27) ). We have not had any problems with substitutions of some amino acid residues in the active site of subtilisin by the new residues present in SPC3 or SPC2 (Tables I and II); the initial conformations of the side chains of corresponding residues was conserved, with the exception of the bulky side chain of Trp(126) for which additional packing calculations were performed. In the case of the new loop 320-327 (165-171) the side chain at position 324 (168) (Asp in SPC3) forms a hydrogen bond with the side chain of residue Trp(126) .

The procedure for minimization of the potential energy of enzyme-substrate interactions consisted of two steps. First the arrangement of the substrate component was optimized within the field of the rigid binding cavity of the putative active sites of the enzymes SPC3 and SPC2, taking into account simultaneous variations in all the above described dihedral angles of rotation , , , , , (and also , , ) for the P1 Arg residue (Fig. 2). Then the potential energy of the entire enzyme-substrate complex was minimized, allowing possible changes in the positions of amino acid residues within the active sites.


Investigation of the Primary Substrate Specificity of SPC2 and SPC3

With few exceptions the SPCs possess unique primary specificity toward Arg residues (19) . To account for this our investigation of enzyme-substrate interactions began with an analysis of the structural correspondence of the side chain of Arg (P1) to the S1 pocket of the active sites of SPC2 and SPC3.

Minimization of the potential energies of interaction of substrate components (including P1 Arg) (Fig. 2) with active sites of SPC2 and SPC3 was performed on the basis of zero approximation for angles of rotation (C-C), (C-C`), and (C-C), found in complexes of subtilisin with different inhibitors. In complexes, atom O of the side chain of residue Ser(221) is turned by 120° relative to its original position ( 180°) in the free enzymes. Thus, in the case of the complex of subtilisin Carlsberg with eglin-c the value of angle = -81.6 (27) . For the residue Leu (P1) in the same complex (C-C) = -66.2°.

Among the three possible values for the angle of rotation around bond C-C () of the side chain of the P1 residue, -60, 180 and 60°, corresponding to minima of the rotational potential, only in the case of -60° can this side chain be arranged in the S1 subsite, while the rotamer 60° leads to significant hindrances with residues on the bottom of the active site and the 180 promotes its location in the external region of the enzyme. By following similar mutual considerations only the values in proximity to 120° and -60° are correspondingly admissible for (C-O) and (C-C`). The angle (O-C`) was taken equal to 180°, which corresponds to the trans configuration of the complex ester group, as is usual for acyl-enzymes (38). Initial values for the remaining angles of the side chain of Arg, , , and , corresponded to the trans orientation of the C-C bonds (180°), since this side chain has a tendency to accept extended conformations in active sites of enzymes, as for example, in trypsin (39) .

As mentioned above, minimization of the potential energy of complexes has been carried out in two steps in the field of rigid and flexible active sites. As a result of the second step of minimization, displacements of the positions of the C backbone atoms did not exceed 0.3 Å relative to the initial rigid model of the active sites. However, such small changes were enough to remove non-bonded repulsions between the guanidinium group of the side chain of Arg (P1) and the atoms of the walls of the S1 subsite, primarily with 3 Gly residues (269 (127) , 308 (154) , and 321 (166) ). A similar effect was observed in an x-ray study of the adduct of 2-phenylethaneboronic acid with subtilisin BPN` (40) . An expansion of the binding crevice of subtilisin by perhaps as much as 0.5 Å was interpreted as an effect induced by binding of the aromatic ring of the inhibitor.

The optimal arrangements found by energy minimization for the side chain of Arg in the S1 subsite in the tetrahedral intermediates formed by SPC3 and SPC2 are shown in Fig. 3 , a and b. Angles of rotation around bonds of both substrate components are -70.1°, 100.4°, -172.1°, -50.9°, -81.1°, 175.5°, 178.8°, 170.4° (in the acyl-enzyme) and -68.8°, 101.5°, -167.8°, -66.5°, -69.9°, 175.1°, 179.9°, -170.1° (in the tetrahedral intermediate). These calculations show that the conformation of the substrate component of the tetrahedral adduct at the deacylation step is practically coincident with its conformation in the acyl-enzyme. It is also valid for the arrangement of the guanidinium group of the side chain of Arg in both intermediate states.


Figure 3: Spatial models of the tetrahedral adducts SPC3 with Arg (P1) (a); Arg (P1) (for SPC2 with Asp at position 310 (155)) (b); Ala-Ala-Ala-Arg (P1-P4) (c); Arg-Ala-Ala-Arg (d); Arg-Ala-Arg-Arg (e). The substrates and important residues of enzymes are shown in a space-filling image.



The size and shape of the S1 pocket of enzymes SPC3 and SPC2 exactly corresponds to the extended conformation of the Arg side chain. The walls of the binding cavity are restricted by 3 residues of Gly, 269 (127), 308 (154) , and 321 (166) , and the guanidinium group forms close van der Waals contacts primarily with hydrogen atoms of C-H bonds of these residues (Fig. 3, a and b). Due to such dense packing the energy of nonbonded interactions of the side chain of Arg (P1) with these three glycines is -4.5 kcal/mol. Thus, in this case we have an example of high conformity between the structures of the substrate and the active site S1 of the enzyme. Moreover, the P1 Arg side chain is located parallel to the peptide backbone of the extended strand formed by residues 267-270(125-128), and the energy of nonbonded and weak polar interactions with this strand comprises -10 kcal/mol. Therefore, this calculation allows us to conclude that the nonbonded van der Waals attraction of the side chain of Arg with the three walls formed by peptide segments of the S1 subsite comprises a significant energetic contribution to the binding of the P1 Arg residue in SPC substrates.

However, Asp(165) probably plays the most significant role in determining the high primary specificity of the SPCs for P1 Arg residues. In the subtilisins the bulky hydrophobic side chain of residue 165 (Val or Ile) is surrounded by Ala, Leu, Ala, and by the salt bridge formed by Lys and Glu, whereas in the SPCs all of these residues are replaced by small polar residues of Ser (Thr in the case of Leu), and therefore, the side chain of Asp(165) can easily change its conformation. Such changes in the environment of residue 320 (165) in the SPCs shows that it is an internal rather than a surface residue of the protein. Binding of substrates containing Arg residues induces conformational transformations in the side chain of Asp(165) from a state with an angle (C-C) = 60° in native subtilisin to a new state with (C-C) = 180° when this side chain forms the bottom of the S1 binding cavity and interacts directly with the guanidinium group of P1 Arg. In the most optimal orientation, the distance between the carboxyl oxygen of Asp(165) and one of the protons of N-H groups of the guanidinium group is 2 Å, which is suitable for a hydrogen bond (35) . The energy of electrostatic interaction between Arg (P1) and Asp(165) comprises -7.1 kcal/mol, whereas the energy of nonbonded interactions is -1.2 kcal/mol. The hydrogen bond between the backbone groups in SPC3 (NH (Gly)O=C (Ser)) is also weakened due to direct interaction of the side chain of Asp(165) with the NH bond of Gly(166) .

Moreover, the high degree of structural correspondence of the S1 pocket to the side chain of Arg, especially its guanidinium group, allows us to explain the preferential cleavage by SPCs of peptide bonds formed by Arg residues in comparison with Lys in P1 positions (15, 19) , i.e. the shorter length of the side chain of Lys does not allow formation of the hydrogen bond with the side chain of Asp(165) (the distance N(Lys)O (Asp) is 4.5 Å). Mutagenesis of the corresponding Asp residue in furin to Asn (Asp(165) in our alignment) reduced its activity dramatically to below detectable levels (23) . Thus, this aspartic acid residue (320 in SPC3) clearly participates in the recognition of scissible peptide bonds. In sum then, the triad of glycine residues (269 (127) , 308 (154) , and 321 (166) ), in the S1 pocket of SPC2 and SPC3 provides suitable structural complementarity for the P1 Arg side chain of substrates, while the primary specificity of the SPCs for Arg residues is determined by the side chain of Asp(165) , which is located deeply in the bottom of the S1 pocket.

The foregoing conclusions do not agree with the amino acid alignment recently proposed (24) for furin in which the conserved Gly residue of the subtilases is aligned with Asp in the processing endoproteases (Asp in SPC3). The presence of the additional C atom at position 166 hinders the binding of substrates having long bulky side chains at position P1, i.e. Tyr (41) . Moreover, the side chain at position 166 is external, does not form hydrogen bonds with Arg (P1), and is likely to interact with the aqueous medium. Indeed, double mutation of subtilisin BPN` (GlyAsp and AsnAsp) produces a subtilisin variant having greater specificity for dibasic pairs where Lys occupies the P1 position, i.e. Lys Lys and Arg Lys (42) . However, the SPCs exhibit a strong preference for sites containing P1 Arg residues and discriminate significantly against Lys at this position. An exception is the cleavage of some Lys-Lys bonds by SPC2, which may result from an alternative mechanism for binding of the side chain of P1 Lys residues by Asp(155) that is unique to this enzyme.()

It is well established that Asn plays an important role in catalysis by the subtilisins. Its side chain is believed to provide one of the hydrogen bonding groups for stabilizing the negatively charged tetrahedral intermediate in the transition state (36, 43) . According to our calculations of the optimal conformation of the substrate component in the acyl-enzyme, the carbonyl of the complex ester group forms two hydrogen bonds with amide groups of the main chain of Ser(221) and the side chain of Asn(155) in SPC3, and the corresponding OH distances equal 2.4 Å. In the case of the tetrahedral intermediate (Fig. 3a) the negatively charged oxygen of the scissible bond also forms the same hydrogen bonds, but they now become significantly shorter, the OH distances in both segments C-OH-N equal 1.8 Å, an optimal value for hydrogen bonds of this type (35) . A similar hydrogen bond can be formed with the protonated side chain of Asp(155) in the case of PC2 (5) (Fig. 3b). The tendency for formation of strong hydrogen bonds appears to be maximal in the tetrahedral intermediate. Evaluation of the energetic stabilization of the tetrahedral intermediate by the additional electrostatic interactions with residues Asn(155) and Ser(221) relative to the acyl-enzyme gives a calculated value of -3 kcal/mol. The experimental value for the energy of stabilization of the transition state in subtilisin catalysis is -3.7 kcal/mol (44) .


Secondary Specificity of SPC2 and SPC3

For the correct alignment of peptide bonds undergoing cleavage by serine proteinases, substrates must also satisfy additional secondary specificity requirements. In subtilisin-like enzymes the main chain of residues P1-P4 of the substrates forms a -strand structure with the surface crevice (loci S2-S4) of the active sites, residues 100-103 and 125-128 (36) . Secondary specificity requirements appear to be significantly more stringent for the SPCs than the subtilisins in that Lys and Arg residues at P2 and P4 play an essential role in determining substrate specificity (15, 20) .

Before consideration of the binding of real substrates containing Arg or Lys at positions P2 and P4 it is necessary to determine the optimal packing of the main chain of these residues. For this aim we considered the binding of a tetrapeptide containing Arg (P1) and 3 alanines at P2-P4 under the conditions of forming the tetrahedral intermediate. The orientation of the side chain of the P1 Arg residue was rigidly fixed as found in the previous step of calculation (Fig. 3a) and then the angles of rotation, and , around bonds C-N and C-C`, respectively, were optimized for each of the main chain residues P1-P4. As an initial approximation and were chosen to satisfy the formation of hydrogen bonds within the limits of 2.8-3.5 Å between the substrate component and the peptide backbone of segments 243-246(100-103) and 267-270(125-128) of the enzymes.

The angles of rotation . . . found as a result of minimization of the potential energy of the interactions of Ala-Ala-Ala-Arg with SPC3 are -75.5°, -63.1°, 107.1°, -136.9°, 148.1°, -82.4° and 151.0°. The network of hydrogen bonds formed by this peptide with segments 243-246 (100-103) and 267-270(125-128) of the enzyme are practically the same as in the complex of subtilisin with eglin (27) . However, the hydrogen bond with the carbonyl group of Gly(127) is less satisfactory because the adjacent Pro(128) in the case of SPC2 or SPC3 hinders the formation of hydrogen bonds by this residue.

In the molecular model of the tetrahedral adduct of SPC3 with Ala-Ala-Ala-Arg (Fig. 3c), it can be seen that the P4 Ala is located exactly on top of another potential binding pocket of SPC3, the S4 locus. In the subtilisins this region is filled by bulky hydrophobic residues (Tyr, Ile, Leu, and Pro) which enhance the binding of hydrophobic P4 residues (45) . However, in the SPCs the corresponding residues at positions 247 (104) and 250 (107) are Asp and Glu, respectively (). Moreover, such changes in the S4 region allow residue 284 (135) , which is Ala or Thr in the SPCs instead of the bulky residues (Leu or Met) of the subtilisins, to form a new floor for the S4 cavity. The distance between the C atom of the P4 Ala of the substrate Ala-Ala-Ala-Arg and the C atom of the new residue 284 (135) (Ala) corresponds closely to the length of the Arg side chain in extended conformation. It is likely that the substitution of Gly in the subtilisins by Pro in SPCs () provides further orientation for the backbone of the side chain of residue P4.

The deduced packing of the main chain of residues P2-P4 in the crevice S2-S4 of the active sites provides a basis for consideration of the binding of a real substrate of SPC3 having Arg at position P4, i.e. Arg-Ala-Ala-Arg. Optimization of the arrangement of the side chain of Arg (P4) in the binding cavity S4 was carried out after fixation of the main chain of residues P1-P4 in the previous step of calculation and simultaneous changes in the conformations of the side chains both of Arg (P4) and residues Asp(104) , Glu(107) , Asp(131) , and Trp(126) . Optimal values for the angles of rotation around bonds of the side chain of the P4 Arg are -53.6°, -159.6°, 161.1°, -169.9°. Only the rotamer with the value of (C-C) -60° was available while other angles , , and corresponded to the extended conformation (180°) of the side chain. As in the case of the P1 Arg, the side chain of the P4 Arg conformed well with the surface of the S4 binding cavity. The guanidinium group of the P4 Arg made van der Waals contacts with the side chains of residues Asp(104) , Glu (107), Ala(135) , and Trp(126) , and the energy of nonbonded dispersion interactions between the side chain of Arg (P4) and residues of the binding site S4 comprised -11 kcal/mol. A spatial model of the binding of Arg-Ala-Ala-Arg by SPC3 (loci S1-S4) is shown in Fig. 3d.

All three negatively charged residues forming the walls of the S4 locus, Asp(104) , Glu(107) , and Asp(131) , can form strong hydrogen bonds or salt bridges with the guanidinium group of Arg (P4). This is possible when the side chain of Asp(104) adopts a conformation with (C-C) 180°, of Glu(107) , -60°, and of Asp(131) , -160° (Fig. 3d). It is evident that electrostatic interactions with these side chains (calculated value -19.5 kcal/mol) participate significantly in the energy of binding of the P4 Arg residue. However, Asp(104) is a surface residue of the SPCs, and thus its side chain can easily change its conformational state, while the side chains of residues Glu(107) and Asp(131) are located deeply within the bottom of the S4 locus. Binding of the P4 Arg residue of a substrate freezes their conformations. Indeed, substitution of Asp(104) by Asn in furin had little effect on the activity of this enzyme (23) . The side chain of Asp(104) is thus not likely to interact with the P4 Arg side chain. On the other hand, site-directed mutation of the 2 neighboring Asp residues (272 (130) and 273 (131) ) () of furin (Asp-Asp Ser-Gly) led to inhibition of the processing of precursors at sites containing a P4 Arg residue (Arg-X-X-Arg) (23) . However, in our model the Asp(130) side chain is directed away from the S4 subsite. Thus, the substrate specificity of the SPCs relative to Arg (P4) is determined mainly by internal (but not external) carboxyl groups within the spatial structures of these proteins.

To summarize, our molecular modeling indicates that the structure of the S4 loci of the SPCs corresponds well to the size and shape of the side chain of Arg and provides significant electrostatic stabilization of binding for this residue. It is possible that the presence of Asn in position 274 (131) of SPC2 instead of Asp (as in SPC3) explains the reduced sensitivity of PC2 to the nature of the residue in the P4 position (5) .

The conformational possibilities for Lys and Arg residues at the P2 position of substrates have been investigated by simple substitution of the P2 Ala by Arg or Lys in the model peptide Arg-Ala-Ala-Arg in order to assess its optimal arrangement (Fig. 3e). As a rule, in the subtilisins the S2 loci (mainly comprised of Asp, His, Leu, and Asn) bind small nonpolar residues (Thr in the case of eglin c). Moreover, it is impossible to direct the relatively long side chains of Lys or Arg into the S2 pocket of the SPCs due to strong, nonbonded repulsions with the residues lining the inner wall of this pocket (for example Asp(33) ; see ). Therefore the P2 Lys and Arg side chains can be located only on the external surface of the active site where they may interact with the side chains of His (64) and Asn(62) . As before, the side chain of the P2 Arg adopts an extended conformation ( 180°) with an optimal value for the (C-C) angle of 88.5. In the orientation we have deduced for the P2 Arg, the conserved Asp(33) of the S2 subsites of the SPCs does not interact directly with this residue; however, it can play an important orienting role by fixing the conformation of the side chain of Asn(62) such that its amide group forms hydrogen bonds with the side chains of P2 Lys or Arg, thereby promoting the binding of these basic residues. A stereo view of a model of the binding of Arg-Ala-Arg-Arg is shown in Fig. 4.


Figure 4: Stereo view of the proposed arrangement of the substrate Arg-Ala-Arg-Arg (heavy line) in the active site of SPC3 (in accordance with Fig. 3e).



Participation of residue 206 (62) in processing follows from data for kexin in which the presence of Asp instead of Asn at this position significantly increases the preference for substrates containing P2 Lys residues, while the nature of the P4 residue is irrelevant (46) . It is likely that the interaction of the side chain of Asp (62) with the side chains of P2 Lys or Arg compensates for the loss of effective S4 binding in kexin. In contrast, it has been shown that sites of the type Arg-X-X-Arg are effectively processed by both SPC3 (15, 19) and furin (20) . Thus, the processing by these SPCs requires cleavage sites containing primarily a P1 Arg residue and a second basic residue at either P2 or P4 such that the loss of effective binding in the S2 subsite is compensated by binding in the S4 subsite and vice versa.

In summary, from the considerations we have discussed here we can conclude that the structures of the active sites of the SPCs, SPC2 and SPC3, which were modeled on the basis of their assumed homology with the tertiary structures of the subtilisins, satisfy the conditions for formation of dense energetic packing with substrates of the type Arg-X-Lys/Arg-Arg in the acyl-enzyme and tetrahedral intermediate states. Each of the active site subsites (S1-S4) contain deeply located residues of Asp or Glu (primarily Asp(165) in the S1 subsite, Asp(33) in the S2 subsite, and Glu(107) and Asp (131) in the S4 subsite), and these determine the high specificity of these enzymes toward substrates having basic amino acid residues at positions P1, P2, and P4. It appears that all the SPCs share this fundamentally very similar specificity for P1, P2, and P4 basic residues but their selectivity for individual substrates is dictated in part by additional features (possibly residues at P5, P6, P1`, and P2`), as well as by the more acidic pH optima of the prohormone convertases, SPC2 and SPC3.

  
Table: Alignment of amino acid residues in active site regions of subtilisins BPM` and Carlsberg and SPCs (21)

Residue numbering corresponds to SPC3 (13) and subtilisin BPN (27) (in parentheses). Numbering for this alignment of the SPCs and kexin is given in Ref. 13.


  
Table: New proposal for alignment of amino acid residues in SPCs (sequences 319-329 of SPC3 and 164-175 in subtilisin BPN`)



FOOTNOTES

*
This work was supported by United States Public Health Service Grants DK 13914 and DK 20595 and in part by the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: The Howard Hughes Medical Institute, 5841 S. Maryland Ave., Chicago, IL 60637. Tel.: 312-702-1334; Fax: 312-702-4292.

See Ref. 5 for an explanation of this terminology.

According to the nomenclature of Schechter and Berger (31), the scissible peptide bond is located between the P1 and P1` substrate residues. Amino acid residues of the substrate are numbered P2, P3, P4 , etc. toward the N terminus. The complementary subsites of the binding region of the enzyme are correspondingly numbered S1`, Sl, S2, S3, S4 . . .

The residue numbers for the subtilisins (BPN` and Carlsberg) are given in parentheses after those for SPC3 hereinafter.

In order to verify our hypothesis, we have prepared the mutant ProThr of subtilisin BPN` (Q. Gong, unpublished data). Western blot analysis of the culture medium of the mutant revealed the absence of mature processed subtilisin BPN`. Therefore, this single mutation may interfere with the formation of a cis-peptide bond and disrupt the folding pathway for this subtilisin. This result provides evidence that Pro plays an important role in the self-organization of the subtilisins and that the tertiary structures of loops 165-171 in the subtilisins and 320-327 in SPCs differ significantly. Moreover, in accordance with our proposal the mutant GlyAsp also was not active, indicating that the alignment of the position 169 (324) as given in Ref. 21 is not acceptable.

G. Lipkind, unpublished results.


ACKNOWLEDGEMENTS

We thank Florence Rozenfeld for her expert assistance in the preparation of this manuscript.


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