Structural Basis for Reduced Staphylocoagulase-mediated Bovine Prothrombin Activation*

Staphylocoagulase (SC) is a protein secreted by the human pathogen, Staphylococcus aureus, that activates human prothrombin (ProT) by inducing a conformational change. SC-bound ProT efficiently clots fibrinogen, thus bypassing the physiological blood coagulation pathway. The crystal structure of a fully active SC fragment, SC-(1-325), bound to human prethrombin 2 showed that the SC-(1-325) N terminus inserts into the Ile16 pocket of prethrombin 2, thereby inducing expression of a functional catalytic site in the cognate zymogen without peptide bond cleavage. As shown here, SC-(1-325) binds to bovine and human ProT with similar affinity but activates the bovine zymogen only very poorly. By contrast to the ∼2-fold difference in chromogenic substrate kinetic constants between human thrombin and the SC-(1-325)·human (pro)thrombin complexes, SC-(1-325)·bovine ProT shows a 3,500-fold lower kcat/Km compared with free bovine thrombin, because of a 47-fold increase in Km and a 67-fold decrease in kcat. The SC-(1-325)·bovine ProT complex is ∼5,800-fold less active compared with its human counterpart. Comparison of human and bovine fibrinogen as substrates of human and bovine thrombin and the SC-(1-325)·(pro)thrombin complexes indicates that the species specificity of SC-(1-325) cofactor activity is determined primarily by differences in conformational activation of bound ProT. These results suggest that the catalytic site in the SC-(1-325)·bovine ProT complex is incompletely formed. The current crystal structure of SC-(1-325)·bovine thrombin reveals that SC would dock similarly to the bovine proenzyme, whereas the bovine (pro)thrombin-characteristic residues Arg144 and Arg145 would likely interfere with insertion of the SC N terminus, thus explaining the greatly reduced activation of bovine ProT.

Pathogenic bacteria exploit and subvert several host processes and signaling pathways (1). For example, some secreted or cell wall-bound bacterial proteins can efficiently activate trypsin-like serine proteinase zymogens circulating in the blood plasma of the host. From a mecha-nistic point of view, these bacterial activators can be grouped into proteolytic and nonproteolytic ones (2). The former class comprises proteinases capable of cleaving host zymogens at their physiologic Arg 15 -(Ile/Val) 16 activation sites (using the chymotrypsinogen numbering for the catalytic domain residues of serine proteinases). This cleavage liberates a new N terminus with a typical (Ile/Val) 16 -(Val/Ile) 17 sequence, which inserts into the preformed "Ile 16 pocket" of the zymogen and engages in a strong salt bridge with the Asp 194 carboxylate. The corresponding rotation of the Asp 194 side chain induces formation of a functional active site (for a recent review on zymogen activation mechanisms see Ref. 3). The plasminogen (Pg) 4 activator from Yersinia pestis (i.e. Pla proteinase), and related membrane-bound omptins belong to this class of bacterial activators (4,5).
The second group of activators is formed by several nonenzymatic proteins, which upon binding induce functional active sites in their cognate serine proteinase zymogens. Proteolysis of the Arg 15 -(Ile/Val) 16 bond is not needed for activation but can occur as an epiphenomenon of the activation mechanism (6,7). Notably, these bacterial activators can modify the specificity of the bound host proteinase toward macromolecular substrates by providing novel docking sites for substrate recognition (8). The mechanism of nonproteolytic, cofactor-induced activation has been intensively investigated for streptokinase (SK), a Pg activator secreted by ␤-hemolytic streptococci (9,10), as well as for staphylocoagulase (SC), a prothrombin (ProT) activator from Staphylococcus aureus (11)(12)(13). Another Pg activator from S. aureus, staphylokinase, is structurally related to SK (8,14) but requires active plasmin (Pm) to form an activator complex of free Pg molecules (15,16).
The N-terminal sequences of SK and SC ( SK Ile 1 -Ala-Gly and SC Ile 1 -Val-Thr, respectively; superscripts identify cofactor residues) mimic those of mature trypsin-like catalytic domains. These conformational zymogen activators are thought to be necessary to spread and maintain infections by their respective Gram-positive pathogens. Species-specific zymogen activation by SK and SC is a well documented and poorly understood caveat to their cofactor function. SK is highly specific for conformational activation of human Pg among other species, but the catalytic SK⅐Pg/Pm complexes proteolytically activate Pg from a broad range of species (17). The resistance of murine Pg to activation by SK has impeded the development of versatile animal models of human streptococcal infection. As elegantly shown recently in a transgenic mouse model expressing only human Pg, tissue invasion by the human pathogen Streptococcus pyogenes is critically dependent on human Pg, SK, and the bacterial Pg/Pm-binding M-like protein (i.e. PAM) to generate Pm that degrades fibrin and the extracellular matrix (18). Given the conservation in sequence and fold exhibited by members of the chymotrypsinogen family of serine proteinases across species, the high specificity of SC for activation of human ProT is similarly quite remarkable. Here we present for the first time peptide substrate and Fbg clotting studies that establish limits on the ability of SC-(1-325) to activate bovine ProT compared with human ProT and the effect of SC-(1-325) binding on bovine thrombin activity. The results suggest that compared with SC-(1-325)⅐human ProT, the catalytic site in SC-(1-325)⅐bovine ProT is incompletely formed. Surprisingly, equilibrium binding results indicate that SC-(1-325) binds bovine ProT with a 1 to 1 stoichiometry and only ϳ12-fold weaker than to the human zymogen. We also present the crystal structure of SC-(1-325) bound to bovine ␣-thrombin, along with a more detailed description and comparison of the atomic interactions in the SC-(1-325)⅐human (pre)thrombin and SC-(1-325)⅐bovine thrombin complexes to define structural differences responsible for the species specificity of ProT activation by SC.
Chromogenic Substrate Kinetics and Fbg Clotting Activity-Hydrolysis of the thrombin substrate, H-D-Phe-Pip-Arg-pNA by bovine ␣-thrombin and the SC-(1-325)⅐bovine (pro)thrombin complexes was followed by monitoring the absorbance increase at 405 nm in 50 mM Hepes, 110 mM NaCl, 5 mM CaCl 2 , 1 mg/ml polyethylene glycol 8000, pH 7.4 buffer containing 1 mg/ml bovine serum albumin and 0.1 mg/ml soybean trypsin inhibitor, at 25°C. Stoichiometric factors (n) were determined by fitting of the quadratic binding equation to initial rates of hydrolysis of 100 M H-D-Phe-Pip-Arg-pNA by bovine ␣-thrombin (1 nM) or bovine ProT (50 nM) as a function of the total SC-(1-325) concentration. The kinetic parameters, k cat , K m , and K i for the product, when appropriate, were determined by simultaneous fits of the integrated Michaelis-Menten equation including competitive product inhibition to full reaction progress curves determined at H-D-Phe-Pip-Arg-pNA concentrations of 15, 31, and 62 M with 1 nM bovine thrombin in presence or absence of saturating SC-(1-325). For SC-(1-325)⅐bovine ProT cleavage of H-D-Phe-Pip-Arg-pNA, kinetic parameters were determined by fitting the Michaelis-Menten equation to initial rates collected over a substrate concentration range of 0 -4 mM. In all of the assays, SC-(1-325) and bovine (pro)thrombin were preincubated for at least 20 min, and the reactions were initiated by the addition of sub-strate. Fbg clotting activity relative to a human thrombin standard was measured with a fibrometer from the slopes of plots of clotting time against the reciprocal of the enzyme concentration essentially as described (19,23).
Competitive  (20). Direct titration of [OG]FPR-ProT with SC-(1-325) in the picomolar range was performed by incubation of individual reactions for Ն16 h in the dark to assure that equilibrium had been reached. These long incubations were not necessary for titrations at probe concentrations in the nanomolar range. Fluorescence measurements of individual binding reactions and analysis of competitive binding were performed as described in the companion paper (20). The error estimates represent Ϯ 2 S.D.
Crystallization and Structure Solution-Bovine ProT was purified from fresh ox blood and activated using Oxyuranus scutellatus venom (Latoxan). Thrombin was inhibited with D-Phe-Pro-Arg-chloromethyl ketone (Bachem) and mixed with an equimolar amount of purified SC-(1-325). The complex was purified by anion exchange chromatography on a TSK gel DEAE-5PW column (Tosoh Bioscience) and concentrated using Centricon YM 30 filters (Millipore). Crystals grew out of solutions containing 0.1 M imidazole, pH 7.0 -7.5, 0.2 M ammonium formate, 12% polyethylene glycol 4,000. These crystals diffracted only to medium resolution but allowed collection of a complete data set "in house" from a single crystal mounted on a capillary, using a MAR345 image plate system (Marresearch) installed on a RIGAKU rotating anode generator (Rigaku Corp.). The data were processed with MOSFLM (www.mrc-lmb.cam.ac.uk/harry/mosflm/) and scaled and reduced using programs supported by CCP4 (www.ccp4.ac.uk/ main.html). The structure was solved by molecular replacement using the coordinates of the corresponding human complex as search model and refined using data to 3.5 Å resolution with programs of the CNS suite (xplor.csb.yale.edu/cns_solve/). Strong noncrystallographic restraints were applied throughout refinement. The atomic coordinates of the SC-(1-325)⅐bovine ␣-thrombin complex have been deposited in the Protein Data Bank under accession code 2A1D.

Activation of Bovine ProT by SC-(1-325) and Its Effect on Bovine
␣-Thrombin Activity-There have been conflicting reports regarding the species specificity of SCs from different isolates. SC from strain Tager 104 was reported to protect human thrombin, but not bovine thrombin, from antithrombin-mediated inhibition (24). More recently, Raus and Love (25) reported qualitative differences in the clotting activities of SCs isolated from Staphylococcus intermedius and S. aureus from different animal sources when their complexes with human or bovine ProT were probed against human, bovine, or equine Fbg. No affinity constants have been determined for ProT from various species, however, so it remains to be clarified whether these differences result from impaired ProT activation because of loss of affinity or loss of activity or because the SC⅐ProT activator complex is unable to cleave substrate Fbg.
We compared the cofactor activities of SC-(1-325) against human ProT and its bovine homolog. Despite the high similarities both at the sequence and structural level of human and bovine thrombin (81% overall and 87% catalytic domain sequence identity, respectively; ϳ0.45 Å root mean square deviation of all defined ␣-carbon atoms in the catalytic domains (26) (Table 1). Kinetic constants determined for the SC-(1-325)⅐bovine ProT complex by initial rates indicated a 3,500-fold lower k cat /K m compared with free ␣-thrombin. In addition to the 47-fold increase in K m , indicating significantly weaker substrate binding, k cat for hydrolysis of H-D-Phe-Pip-Arg-pNA by the SC-(1-325)⅐bovine ProT complex was 1.4 Ϯ 0.1 s Ϫ1 (Table 1), reduced 67-fold compared with ␣-thrombin. The results demonstrated that SC-(1-325) activates bovine ProT but that the activity of the complex (k cat /K m ) toward H-D-Phe-Pip-Arg-pNA compared with the SC-(1-325)⅐bovine thrombin complex is 740fold lower, whereas the SC-(1-325) complex with human ProT is ϳ2-fold more active than SC-(1-325)⅐human thrombin measured with the same substrate (20).
Fibrinogen Clotting Activities of Human and Bovine Thrombin and SC-(1-325)⅐(Pro)thrombin Complexes-Fbg clotting assays were performed to assess the role of the low catalytic activity of the SC-(1-325)⅐bovine ProT complex in bovine and human Fbg substrate recognition (Table 2). Compared with human thrombin, the SC-(1-325) complexes with human (pro)thrombin had indistinguishable activity toward human Fbg and also exhibited a similar, 3-4-fold lower activity with bovine Fbg. Bovine thrombin and SC-(1-325)⅐bovine thrombin complex had essentially the same activities of 67 and 96%, respectively, as human thrombin toward human Fbg and lower activities of ϳ30% toward bovine Fbg. By contrast, the SC-(1-325)⅐bovine ProT complex exhibited only 0.07% of the clotting activity of human thrombin toward human Fbg. Moreover, the clotting activity of the bovine complex with bovine Fbg was undetectable at concentrations up to 46 M. These results indicated a small 3-4-fold dependence of the clotting activity on the species of Fbg for human and bovine thrombin and the corresponding SC-(1-325)⅐thrombin complexes. In these results, the 5-fold lower

Effect of SC-(1-325) binding to bovine ProT and ␣ -thrombin on the hydrolysis of H-D-Phe-Pip-Arg-pNA
Michaelis-Menten kinetic parameters determined for hydrolysis of H-D-Phe-Pip-Arg-pNA by the indicated enzyme species are listed. The parameters were obtained by nonlinear least squares fitting of the Michaelis-Menten equation to the initial rates for the SC-(1-325)⅐bovine ProT complex or fitting of the integrated equation to progress curves for bovine ␣ -thrombin in the presence and absence of saturating concentrations of SC-(1-325). The experiments were performed, and the data were analyzed as described under "Experimental Procedures."   (Table 1) was not reflected in the clotting activity (Table 2). Remarkably, however, the 3,470-fold lower k cat /K m for chromogenic substrate hydrolysis by the SC-(1-325)⅐bovine ProT complex was correlated with a 1,400-fold lower clotting activity toward human Fbg and an undetectably lower activity toward bovine Fbg. These results indicated that the greatly reduced Fbg clotting activity of the SC-(1-325)⅐bovine ProT complex results from its low catalytic activity and suggested a minor role for species-specific recognition of substrate Fbg.  (Fig. 2). These results indicated that SC-(1-325) binds very tightly to bovine ProT and suggested that high affinity binding may not be the sole determinant of effective zymogen activation by SC- (1-325).
The Two Helical Domains of SC-(1-325) Form a Continuous Structure-We have previously presented crystal structures of SC-(1-325) bound to human ␣-thrombin and to its immediate precursor, Pre 2  ) and the C-terminal domain D2 (helices ␣ 1 D2 to ␣ 6 D2 ) and is represented as a green ribbon. The anion-binding exosite I, the active site (Ser 195 ), and the 148 loop (Trp 148 ) of bovine ␣-thrombin are labeled. The N terminus of SC (defined from SC Ser 7 onwards) is placed close to the Ile 16 activation pocket of thrombin but is disordered in the complexes with bovine and human thrombin and extends away from the enzyme surface. (12). We have now solved the structure of the SC-(1-325) complex with bovine thrombin to low but still satisfactory resolution. A summary of crystallographic parameters and refinement statistics for this crystal structure is given in Table 3. All of the stereochemical parameters were better than average in structures solved at this resolution, as assessed with PROCHECK (www.biochem.ucl.ac.uk/ϳroman/procheck/pro-check.html). Here we present a detailed analysis of the three crystal structures, as a basis for understanding the species specificity of SC and the mechanism of Fbg processing by SC⅐(pro)thrombin complexes.
Each boomerang-shaped SC-(1-325) molecule consists of two ␣-helical domains, which stick together at an angle of about 110°. The N-terminal domain D1 (residues SC Ile 1 to SC Gln 142 ) essentially comprises three ␣-helices (␣ 1 D1 to ␣ 3 D1 ), which range in length from 25 to 41 residues (Fig. 3). The three ␣-helices are slightly wound around each other to form a left-handed ␣-helical coiled-coil. The C-terminal SC domain D2 comprises residues SC Thr 150 to SC Gly 281 and also contains a three-helix bundle formed by helices ␣ 1 D2 to ␣ 3 D2 . The two major helix bundles are topologically similar (Fig. 4A) but are only distantly related at the sequence level, pointing to an early gene duplication event. The centers of the helix bundles are occupied by a number of medium-sized hydrophobic side chains, which are mainly leucine residues in D1 but more varied in D2. These side chains pack together in a "knobs-inholes" manner, creating a quite hydrophobic core free of buried water molecules or polar groups. In addition, D2 is C-terminally extended by three shorter ␣-helices. Together, 75% of all defined residues of SC-(1-325) are found in segments with helical conformation in agreement with results obtained by circular dichroism (not shown). D1 and D2 contact each other along an interface centered on the side chain of the strictly conserved linker residue SC Leu 146 , burying a surface area of about 1,150 Å 2 from bulk solvent (Fig. 4B). The SC Leu 146 side chain is enwrapped by the aliphatic parts of polar/charged residues donated by the C-terminal ends of helices ␣ 1 D1 and ␣ 3 D1 (e.g. the strictly conserved SC Tyr 55 ) and the N-terminal parts of ␣ 1 D2 and ␣ 3 D2 . Several of these side chains engage in a network of hydrogen bonds and salt bridges, most notably the superficial interdomain salt bridge, SC Glu 54 . . . SC Arg 209 . Together, this interface is well packed and rigid, so  325). A, the two major helix bundles ␣ 1 D1 -␣ 3

D1
(yellow) and ␣ 1 D2 -␣ 3 D2 (orange), which are shown superimposed here, are structurally related. Side chains of all topologically equivalent residues conserved in both domains are shown with all their non-hydrogen atoms and labeled. B, close-up of the D1-D2 interdomain interface, with important residues shown with their full side chains. Notice the multiple contacts between polar/charged side chains (e.g. SC Glu 54 -SC Arg 209 ), which separate the strictly conserved SC Leu 146 side chain from bulk solvent. The three loops are shown against the solid SC surface as seen in SC⅐human Pre 2, which is colored according to its electrostatic surface potential (red, negative; blue, positive). Notice in particular the insertion of the Trp 148 indole ring in the SC groove lined by hydrophobic residues of helices ␣ 1 D1 and ␣ 2 D1 and the displacements of the following Thr 149 -Gln 151 segments in SC-(1-325) complexes with human Pre 2 and bovine thrombin compared with free human FPR-thrombin (partially hidden behind the SC surface). The small surface tube extending toward the top represents the inserted N-terminal SC segment.
that the two SC domains would presumably maintain their relative orientation in solution also in the free cofactor.
The C Terminus of SC-(1-325) Is Intrinsically Disordered-The C-terminal SC-(1-325) segments from SC Glu 282 to SC Leu 325 are not defined by appropriate electron density in any of the six equivalent SC-(1-325) moieties of the human (pre)thrombin and bovine thrombin complexes, suggesting enhanced flexibility. Three-state secondary structure prediction methods (cubic.bioc.columbia.edu/ predictprotein/), which correctly identify all major ␣-helices within the defined SC-(1-325) fragment, indicate with high probability that this molecular region is almost entirely solvent-exposed and devoid of regular secondary structure. An inherent disorder of this C-terminal peptide is also in agreement with VL-XT predictor analyses (www.pondr. com/), according to which most of the SC region following SC Gln 300 is intrinsically disordered. Around the defined C termini of both SC-(1-325) molecules, the crystals contain large solvent-filled cavities, which would seem to accommodate this disordered SC Glu 282 -SC Leu 325 segment without steric hindrance.
The D1-Thrombin Interface Differs in the Bovine and Human Thrombin Complexes-Upon SC-(1-325)⅐thrombin complex formation, ϳ3,400 Å 2 of accessible surface is removed from contact with the bulk solvent (Fig. 3). Domain 1 contacts the cognate thrombin molecule primarily at the 148 loop that borders the "south" rim of the thrombin active site (with respect to the standard orientation) (27). Of note, this loop moves considerably compared with its position in human FPRthrombin (27), Gly 149D being displaced to a maximum of about 11.5 Å (Fig. 5). SC-(1-325) helices ␣ 1 D1 and ␣ 2 D1 as well as the ␣ 2 D1 -␣ 3 D1 connector engage in particularly extensive contacts with the indole moiety of Trp 148 . In addition to these hydrophobic interactions, a few polar/charged contacts are observed at this SC-(1-325)⅐thrombin interface. For example, the thrombin-internal Glu 146 . . . Arg 221 salt bridge becomes embedded in a hydrophobic pocket formed by the strictly or well conserved residues SC Leu 74 , SC Lys 78 , and SC Phe 105 , with the Arg 221 guanidyl group frontally opposing the SC Asp 106 carboxylate.
Notably, human and bovine thrombin diverge considerably in the 148 loop sequence, with mostly nonconservative substitutions Leu 144 3 Arg, Lys 145 3 Arg, Ala 149A 3 Thr, Asn 149B 3 Ser, Gly 149D 3 Ala, Glu 149E 3 Lys, and Gly 150 3 Val. These variations in turn provoke changes at the SC-thrombin interface. For example, the 149 -150 segments adopt different conformations in the two thrombin complexes, but in neither case seem to make particularly specific interactions with the SC moiety (Fig. 5).
The exchanges of Leu 144 and in particular of Lys 145 by Arg residues in the bovine species have more dramatic consequences. In SC⅐human (pre)thrombin, the distal ammonium group of Lys 145 , framed by SC Leu 67 , SC Gln 71 , and the Glu 146 -Thr 147 main chain segment, is fixed through favorable hydrogen bonds with SC Gln 71 O⑀1, SC Asp 70 O␦1, and Glu 146 O. By contrast, steric clashes of the bulkier Arg 145 side chain of bovine (pro)thrombin would prohibit it to be similarly buried at the interface with SC. Indeed, Arg 145 extends away from the thrombin moiety in the SC⅐bovine thrombin complex (shown in yellow in Fig. 6).
To insert into the Ile 16 pocket (marked by the Asp 194 carboxylate group at its bottom; Fig. 6) and to induce functional active and substrate binding sites, the N-terminal SC segment would have to circumvent or displace the neighboring, charged side chains of the bovine-specific (pro)thrombin residues Arg 144 and Arg 145 . The N-terminal SC Ile 1 -SC Tyr 6 hexapeptide, which is flexibly disordered in both human and bovine thrombin complexes with SC-(1-325), is highlighted green in Fig. 6. Given the limited flexibility at the SC-(pre)thrombin interface, the exposed side chains of Arg 144 and Arg 145 would clash with the approaching N-terminal segment of the bound cofactor. In addition, they are likely to repel the incoming free N terminus of SC Ile 1 electrostatically. As a consequence, the positively charged "gate" to the Ile 16 pocket formed by these basic residues would efficiently hamper or even impair the correct insertion of the N terminus into the activation pocket. Thus, the energetically unfavorable approach and insertion of the N-terminal SC residues may explain the impaired activator activity of the bacterial cofactor against bovine ProT.

Comparison of Exosite I Interactions in the SC-(1-325)⅐Bovine and Human Thrombin Complexes-
The positively charged anion-binding exosite I is extremely similar in both human and bovine (pre)thrombin. It is a slightly notched surface depression "east" of the thrombin active site centered on the 70 -80 loop and bordered by the 37 loop (Fig. 3) (28,29). Its central Arg 73 , Arg 67 , Arg 75 , and Arg 77A side chains are surrounded by other positively charged residues such as Arg 35 , Lys 36 , Lys 81 , Lys 109 , and Lys 110 , only interspersed by a few hydrophobic side chains such as the phenolic group of Tyr 76 . The vast majority of residues that form exosite I are strictly conserved in human and bovine thrombin. The edge residue Asn 78 (in the human enzyme) represents an exception to this pattern of conservation, because it is replaced by a lysine in bovine thrombin. This side chain, however, is not involved in direct contacts with SC. Consequently, SC interactions with exosite I are virtually identical in the human and bovine (pro)thrombin complexes, in Notice the location of bovine thrombin-specific Arg 144 and Arg 145 (yellow). The stick model in front represents the N-terminal segment of SC (green, disordered residues SC Ile 1 to SC Tyr 6 ) in complex with bovine thrombin. To insert its N-terminal residues into the Ile 16 pocket of a bound bovine Pre 2 molecule, the cofactor main chain would have to change its direction at SC Lys 8 to circumvent the Arg 145 side chain. Because of collisions of the SC N terminus with the Arg 144 and Arg 145 side chains, its activator capability toward the bovine zymogen would be impaired or at least considerably weakened, as observed. agreement with the similar SC affinities for both thrombin species (see above).

DISCUSSION
Although SC-(1-325) activates bovine ProT, the SC-(1-325)⅐bovine ProT complex is about 5,800-fold less active than its human counterpart, consistent with early reports that S. aureus isolates activated human ProT better than bovine ProT (25). Our studies demonstrated a low intrinsic activity of the SC-(1-325)⅐bovine ProT complex toward a tripeptide-pNA substrate, which may explain early reports of no activation of bovine ProT by SC (30).
The role of substrate Fbg and (pro)thrombin species specificity in the clotting activities of the SC-(1-325)⅐(pro)thrombin complexes was evaluated by comparing the activities of human and bovine thrombin and the corresponding SC-(1-325)⅐(pro)thrombin complexes toward both human and bovine Fbg. The results demonstrate a relatively small, 3-4fold preference of both human and bovine thrombin as well as SC-(1-325)⅐thrombin complexes for human Fbg. Although cofactor complexes with human thrombin and its precursor have similar activity, the bovine SC-(1-325)⅐ProT complex shows a drastically reduced activity toward both human and bovine Fbg. The results support the conclusion that the low catalytic activity of SC-(1-325)⅐bovine ProT is mainly responsible for its low Fbg clotting activity. The species specificity of SC-(1-325) cofactor activity in ProT activation and Fbg clotting is evidently primarily due to species-specific differences in conformational activation of ProT in the SC-(1-325)⅐ProT complexes.
In view of these results, the similar affinity of SC-(1-325) for bovine and human ProT was unanticipated. Indeed, analysis of the competitive binding results indicated an approximate 12-fold higher affinity of SC-(1-325) for human [OG]FPR-ProT compared with bovine ProT, but this was indistinguishable from SC-(1-325) binding to native human ProT (K D 72 Ϯ 36 pM) (20). Because of the significant experimental error involved in determining affinities of very tight interactions, such small differences may not be meaningful. The results of our previous studies of human ProT activation with N-terminal and domain mutants of SC-(1-325) indicated that binding affinity was correlated with activation of the human ProT zymogen (12). In the case of bovine ProT, however, the low activity of the SC-(1-325)⅐bovine ProT complex was not associated with a significantly lower affinity of the activator for the zymogen, indicating that tight binding alone is not enough for full activation. The results suggest at least one of the following: (a) N-terminal insertion is much less favorable in the SC-(1-325) complex with bovine ProT as compared with the human zymogen, such that the former is only partially activated; (b) the SC N terminus inserts into the Ile 16 pocket of bovine ProT but adopts a nonoptimal orientation for full ProT activation; or (c) zymogen activation by SC can occur at a low level independently of N-terminal insertion.
The structure of the SC-(1-325)⅐bovine thrombin complex supports the idea that SC N-terminal insertion is hindered in the cofactor complex with bovine ProT, mainly because of the presence of two bovine (pro)thrombin-specific residues in the 148 loop, Arg 144 and Arg 145 . These bulky and positively charged residues appear to repel or partially deflect the N-terminal segment of SC-(1-325), thus favoring either conformations of the complex with an exposed cofactor N terminus and/or a less than optimal positioning of the SC Ile 1 ␣-amino group in the ProT Ile 16 pocket. Lastly, we confirmed our previous observation that a variant lacking the N-terminal SC Ile 1 -SC Val 2 dipeptide, SC-(3-325), retains ϳ2% activator activity of SC-(1-325) toward human ProT (results not shown), indicating that a low level of conformational activation without N-terminal insertion cannot be ruled out completely.
Our crystal structures of SC-(1-325) bound to human and bovine thrombin, together with that of the SC-(1-325)⅐human Pre 2 complex, allow for a dissection of molecular events during ProT binding and activation. First, we notice that in these three complexes the interactions of SC-(1-325) domains D2 and D1 with exosite I and the 148 loop, respectively, are almost identical. Exosite I is in a precursor, low affinity state in ProT (proexosite I) (31), whereas the more solvent-exposed, longer 148 loop is commonly disordered in thrombin crystal structures. Therefore, considering the excellent electrostatic complementarity of the two surfaces, it appears reasonable to assume that docking of the more C-terminal D2 to proexosite I represents the initial binding event. This strong electrostatic interaction stabilizes proexosite I in its active, thrombin-like conformation. Further, because D1 and D2 are rigidly connected, the N-terminal domain D1 would be appropriately positioned to dock the tip of the 148 loop into a shallow hydrophobic groove. The entropic penalty of stabilizing this long flexible loop is compensated for by a multitude of van der Waals' and electrostatic interactions, most notably with the indole moiety of the thrombin-specific Trp 148 .
Finally, positioning of D1 locates the N-terminal activation hexapeptide SC Ile 1 -SC Tyr 6 at the correct distance to insert into the Ile 16 pocket of the zymogen, triggering the Asp 194 side chain rotation linked to functional active site generation in a manner similar to the endogenous Ile 16 N terminus formed upon proteolytic activation. The latter step is essential for cofactor-induced activation, because deletion of the SC Ile 1 -SC Val 2 dipeptide reduces SC activator activity ϳ98% (12).
We reported previously that both Met-SC-(1-325) and the truncated form, SC-(2-325) generate complexes of only slightly different maximal activity with human ProT, whereas their affinities for the zymogen are 60-and 6-fold lower than SC-(1-325), respectively (12). These unanticipated findings imply that presentation of a free N terminus that can reach into the Ile 16 pocket is more critical for activation than both the exact nature of the N-terminal residue and the conformation of the preceding segment. The substantially higher affinity for activation of ProT by the truncated variant is explained by the close physicochemical similarity of its N-terminal SC Val 2 to the natural SC Ile 1 residue, as compared with the nonbranched SC Met 0 of Met-SC-(1-325) and its ability to extend into the activation pocket.
Contrasting with these observations, SK-mediated Pg activation more strictly requires the N-terminal SK Ile 1 residue (32). The crystal structure of SK bound to the Pm catalytic domain reveals that the first residue defined by electron density, SK Ser 12 , is located ϳ21 Å away from the endogenous Ile 16 residue (10). Considering that the similarity of catalytic domains of thrombin and Pm implies conserved interactions of the N-terminal cofactor residue with the Ile 16 pocket, we predict that the N-terminal SK segment approaches Pg in a more extended conformation compared with the SC-(1-325)⅐Pre 2 complex and that the impaired activator activity of the SK Ile 1 -deleted variant results from the inability of SK Ala 2 to reach the zymogen Ile 16 pocket.
A previously unappreciated effect of fixing the (pro)thrombin 148 loop conformation by cofactor binding is the deflection of residues that might interfere with insertion of the activation peptide into the Ile 16 pocket. Unexpectedly, bovine thrombin, although engaging in similar overall interactions with SC-(1-325), differs from its human counterpart in the location of several side chains of the 148 loop, most notably the pair of consecutive basic residues, Arg 144 and Arg 145 . These bulky side chains would seem to interfere directly with an incoming SC Ile 1 -SC Tyr 6 segment, both through direct steric hindrance and by generating a positively charged region that repels the N terminus of SC. This finding adds a new example to the reported phenomenon of coevolution of host and bacterial proteins, as recently reported for the SK-Pg system (33).