Fibrinogen Substrate Recognition by Staphylocoagulase·(Pro)thrombin Complexes<sup><a href="#FN4" rid="FN4" class=" fn">*</a></sup>

Peter Panizzi

Rainer Friedrich

Pablo Fuentes-Prior

Klaus Richter

Paul Bock

Wolfram Bode

^{Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232}

^{Proteinase Research Group, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany}

^{Cardiovascular Research Center, Institut Català de Ciències Cardiovasculars-Consejo Superior de Investigaciones Cientificas, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain}

^{Department of Biotechnology, Technical University Munich, D-85747 Garching, Germany}

^{To whom correspondence should be addressed: Dept. of Pathology, Vanderbilt University School of Medicine, C3321A Medical Center North, Nashville, TN 37232-2561. Tel.: 615-343-9863; Fax: 615-322-1855; E-mail:}ude.tlibrednav@kco4uap

^{These authors contributed equally to this work.}

^{Supported in part by National Institutes of Health Training Grant HL07751.}

Abstract

Thrombin generation and fibrinogen (Fbg) clotting are the ultimate proteolytic reactions in the blood coagulation pathway. Staphylocoagulase (SC), a protein secreted by the human pathogen Staphylococcus aureus, activates prothrombin (ProT) without proteolysis. The SC·(pro)thrombin complex recognizes Fbg as a specific substrate, converting it directly into fibrin. The crystal structure of a fully active SC fragment containing residues 1–325 (SC-(1–325)) bound to human prethrombin 2 showed previously that SC inserts its Ile^-Val^{N terminus into the Ile}^{pocket of prethrombin 2, inducing a functional active site in the cognate zymogen conformationally. Exosite I of}α-thrombin, the Fbg recognition site, and proexosite I on ProT are blocked by domain 2 of SC-(1–325). In the present studies, active site-labeled fluorescent ProT analogs were used to quantitate Fbg binding to the SC-(1–325)·ProT complex. Fbg binding and cleavage are mediated by expression of a new Fbg-binding exosite on the SC-(1–325)·ProT complex, resulting in formation of an (SC-(1–325)·ProT)₂·Fbg pentameric complex with a dissociation constant of 8–34 nM. In both crystal structures, the SC-(1–325)·(pre)thrombin complexes form dimers, with both pro-teinases/zymogens facing each other over a large U-shaped cleft, through which the Fbg substrate could thread. On this basis, a molecular model of the pentameric (SC-(1–325)·thrombin)₂·Fbg encounter complex was generated, which explains the coagulant properties and efficient Fbg conversion. The results provide new insight into the mechanism that mediates high affinity Fbg binding and cleavage as a substrate of SC·(pro)thrombin complexes, a process that is central to the molecular pathology of S. aureus endocarditis.

Abstract

Staphylococcus aureus is a pathogen that causes diseases in humans ranging in severity from superficial skin infections to life-threatening conditions such as endocarditis and septic shock (1). Coagulase-positive S. aureus secretes staphylocoagulase (SC),⁴ which conformationally activates the central coagulation zymogen, prothrombin (ProT) and mediates cleavage of fibrinogen (Fbg) to fibrin (Fbn). Fbn generated in these reactions is thought to aid the bacteria in evading host immune cell defense mechanisms in acute bacterial endocarditis (2).

We recently solved the crystal structure of the immediate human thrombin zymogen precursor, prethrombin 2 (Pre 2), bound to an SC fragment, SC-(1–325), which possesses full ProT activator and Fbg clotting activity (3, 4). The structure demonstrates that SC activates ProT conformationally by a mechanism known as “molecular sexuality” (5). Serine proteinase zymogens are activated normally by cleavage at Arg^-(Ile/Val)^{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)}^-(Val/Ile)^{sequence, which inserts into the “Ile}^{pocket” of the zymogen and forms a strong salt bridge with the Asp}^{carboxylate (}5, 6). Formation of this critical salt bridge triggers folding of the “activation domain” of the zymogen, resulting in formation of the substrate-binding site and oxyanion hole (7). As postulated in the molecular sexuality hypothesis, the N-terminal SC-(1–325) dipeptide, ^Ile^-^Val^{, occupies the Ile}^{pocket of the cognate Pre 2, similar to the endogenous}^Ile^-Val^{N terminus in mature}α-thrombin (8). The SC-(1–325)·Pre 2 and thrombin structures raised questions concerning the nature of Fbg substrate recognition and cleavage by the SC-(1–325)·(pro)thrombin complexes because domain 2 (D2) of SC-(1–325) blocks exosite I, the Fbg recognition site.

Fbg is a large glycoprotein (M_r = ~340,000), formed by three pairs of Aα-, Bβ-, and γ-chains covalently linked to form a “dimer of trimers,” where A and B designate the fibrinopeptides released by thrombin cleavage. The elongated molecule folds into three separate domains, a central fragment E that contains the N termini of all six chains and two flanking fragments D formed mainly by the C termini of the Bβ- and γ-chains. These globular domains are connected by long triple-helical structures (9–11). SC·(pro)thrombin complexes, which efficiently convert human Fbg to the self-polymerizing Fbn, are not targeted by circulating thrombin inhibitors (12, 13). Thus, SC action bypasses the physiological blood coagulation pathway.

Fbg interactions with specific residues located in thrombin exosite I are required for fibrinopeptide removal and consequent Fbn generation (14, 15). For example, the reversal-of-charge mutant Arg^{→ Glu is severely compromised as a Fbg activator (}16). Further, variants in which alanine replaces residues Lys^{, His}^{or Tyr}^{are practically devoid of Fbg clotting ability, and mutants Arg}^{→ Ala and Arg}^{→ Ala possess Fbg clotting activities below 25% of wild-type thrombin (}17). Finally, occupancy of exosite I by the physiological regulator of the blood coagulation cascade, thrombomodulin (18, 19), or by a thrombin-specific inhibitor, triabin (20), impairs Fbg processing by competing with substrate binding.

Because the major Fbg recognition surface on α-thrombin is blocked in the SC-bound complexes, the mechanism of Fbg clotting by cofactor-bound (pro)thrombin differs from that of the free enzyme. Investigation of the underlying mechanism was the goal of the present studies. Quantitation of interactions between Fbg and the SC-(1–325)·ProT complexes supports the conclusion that SC-(1–325) mediates specific Fbg binding and cleavage by expression of a new Fbg-binding exosite absent in the individual proteins. Molecular modeling of the complex formed between Fbg fragment E and the (SC-(1–325)·(pre)thrombin)₂ heterotetramer found in the crystals, together with results of equilibrium binding studies employing active site-labeled fluorescent ProT analogs described in the preceding paper (4), support the formation of a pentameric (SC-(1–325)·ProT)₂·Fbg complex, underpinning a novel cofactor-mediated mechanism of Fbg substrate recognition.

Footnotes

^{This work was supported by National Institutes of Health Grants HL038779 and HL071544 (to P. E. B.) and by the SPINE Project QLG2-CT-2002-00988 of the European Union and the Fonds der Chemischen Industrie (to W. B.).}

^{The abbreviations used are: SC, staphylocoagulase; SC-(1–325), SC fragment, residues 1–325; Met-SC(1–325), SC-(1–325) containing an additional Met residue at the N-terminus; D1, domain 1 of SC, residues 1–146; D2, domain 2 of SC, residues 147–281; Fbg, fibrinogen; Fbn, fibrin; ANS, 2-((4′-iodoacetamido)anilino)naphthalene-6-sulfonic acid; ACR, 6-acryloyl-2-dimethylaminonaphthalene; BAD, 6-bromoacetyl-2-dimethyl-aminonaphthalene; AF350, the sulfonated coumarin derivative called AlexaFluor 350 C}₅ maleimide; BD, BODIPY^{507/545 IA or}N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl)iodoacetamide; TMR, tetramethylrhodamine-5-io-doacetamide dihydroiodide; 4′F, 4′-(iodoacetamido)fluorescein; 5F, 5-(iodoacet-amido)fluorescein; OG, Oregon Green 488 iodoacetamide or 5-(and 6)-(iodo-acetamido)-2′,7′-difluorofluorescein; ProT, prothrombin; Pre 2, prethrombin 2; Pre 1, prethrombin 1; SK, streptokinase; SAK, staphylokinase; Pg, plasminogen; Pm, plasmin; DAN, dansylaziridine.

^{Fluorescent analogs of ProT are represented as: [probe abbreviation]connecting thio-ester peptide chloromethyl ketone-ProT.}

^{Schechter-Berger (}51) notation referring to the residues of a substrate (from the N-terminal end) as …-P4-P3-P2-P1-P1′-P2′-…, with the scissile bond at P1-P1′, which interact with complementary specificity sites …-S4-S3-S2-S1-S1′-S2′-… on the proteinase.

^{P. Panizzi, H. K. Kroh, and P. E. Bock, unpublished observations.}

Footnotes

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