X-ray Structure of Active Site-inhibited Clotting Factor Xa

Brandstetter, Hans; Kühne, A; Bode, Wolfram; Huber, Robert; Saal, Wolfgang von der; Wirthensohn, Klaus; Engh, Richard A.

doi:10.1074/jbc.271.47.29988

Cited by 275 publications

(167 citation statements)

References 33 publications

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“…Contrary to thrombin, factor X a contains three basic amino acids at positions 222-224 (Arg-222, Lys-223, and Lys-224) that can interact with the acidic residue at P 3 (3). Both Glu and Asp are found in the activating sequences of prothrombin, together with the well conserved Gly residue at the P 2 site (52). Surprisingly, the effect of the P13D mutation in BPTI was not as positive as expected.…”

Section: Fig 3 Inhibition Curves Of Human Factor Xiia By Wild Type mentioning

confidence: 87%

Inhibition of Six Serine Proteinases of the Human Coagulation System by Mutants of Bovine Pancreatic Trypsin Inhibitor

Grzesiak¹,

Krokoszynska²,

Krowarsch³

et al. 2000

Journal of Biological Chemistry

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A series of 12 bovine pancreatic trypsin inhibitor variants mutated in the P 4 and P 3 positions of the canonical binding loop containing additional K15R and M52L mutations were used to probe the role of single amino acid substitutions on binding to bovine trypsin and to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X a and XII a , thrombin, and protein C. The mutants were expressed in Escherichia coli as fusion proteins with the LE1413 hydrophobic polypeptide and purified from inclusion bodies; these steps were followed by CNBr cleavage and oxidative refolding. The mutants inhibited the blood-clotting proteinases with association constants in the range of 10 3 -10 10 M ؊1 . Inhibition of plasma kallikrein, factors X a and XII a , thrombin, and protein C could be improved by up to 2 orders of magnitude by the K15R substitution. The highest increase in the association constant for P 3 mutant was measured for factor XII a ; P13S substitution increased the K a value 58-fold. Several other substitutions at P 3 resulted in about 10-fold increase for factor X a , thrombin, and protein C. The cumulative P 3 and P 1 effects on K a values for the strongest mutant compared with the wild type bovine pancreatic trypsin inhibitor were in the range of 2.2-(plasmin) to 4,000-fold (factors XII a and X a ). The substitutions at the P 4 site always caused negative effects (a decrease in the range from over 1,000-to 1.3-fold) on binding to all studied enzymes, including trypsin. Thermal stability studies showed a very large decrease of the denaturation temperature (about 22°C) for all P 4 mutants, suggesting that substitution of the wild type Gly-12 residue leads to a change in the binding loop conformation manifesting itself in non-optimal binding to the proteinase active site.Blood coagulation is a series of proteolytic events resulting in clot formation. Equally important are the processes of anticoagulation and fibrinolysis that are also mediated by proteolytic enzymes. Research during the past decade has resulted in the determination of spatial structures for many of these enzymes, including thrombin, factors VII a , IX a , and X a , protein C, tissue plasminogen activator, and plasmin (1-6). The specificity of a particular enzyme toward its cognate sequence results from well defined subsites on the enzyme surface recognizing not only the scissile peptide bond but often also more extended regions. These structural studies nicely explain earlier data on sequence-specific cleavage of natural substrates and provide a framework for drug design efforts.Because the coagulation/fibrinolysis processes are of vital importance, they are precisely controlled. Protein inhibitors are one of the most essential regulating factors. Interestingly, the scaffold of Kunitz-type inhibitors is used both to control the natural coagulation process in human blood and to prevent blood clotting in a blood-sucking organism, the soft tick. In the former case, tissue factor pathway inhibitor (TFPI) 1 compo...

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Section: Fig 3 Inhibition Curves Of Human Factor Xiia By Wild Type mentioning

confidence: 87%

Inhibition of Six Serine Proteinases of the Human Coagulation System by Mutants of Bovine Pancreatic Trypsin Inhibitor

Grzesiak¹,

Krokoszynska²,

Krowarsch³

et al. 2000

Journal of Biological Chemistry

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“…erence for glycine residues in P2 (34). A plot of the transition state stabilization energy differences for all substrates revealed a considerably broadened substrate selectivity of rfXYa compared with rtrypsin and rfXa (Fig.…”

Section: Design Of the Hybrid Enzymementioning

confidence: 99%

New enzyme lineages by subdomain shuffling

Hopfner

Kopetzki

Kresse

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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Protein functions have evolved in part via domain recombination events. Such events, for example, recombine structurally independent functional domains and shuff le targeting, regulatory, and͞or catalytic functions. Domain recombination, however, can generate new functions, as implied by the observation of catalytic sites at interfaces of distinct folding domains. If useful to an evolving organism, such initially rudimentary functions would likely acquire greater efficiency and diversity, whereas the initially distinct folding domains would likely develop into single functional domains. This represents the probable evolution of the S1 serine protease family, whose two homologous ␤-barrel subdomains assemble to form the binding sites and the catalytic machinery. Among S1 family members, the contact interface and catalytic residues are highly conserved whereas surrounding surfaces are highly variable. This observation suggests a new strategy to engineer viable proteins with novel properties, by swapping folding subdomains chosen from among protein family members. Such hybrid proteins would retain properties conserved throughout the family, including folding stability as single domain proteins, while providing new surfaces amenable to directed evolution or engineering of specific new properties. We show here that recombining the Nterminal subdomain from coagulation factor X with the C-terminal subdomain from trypsin creates a potent enzyme (fXYa) with novel properties, in particular a broad substrate specificity. As shown by the 2.15-Å crystal structure, plasticity at the hydrophobic subdomain interface maintains activity, while surface loops are displaced compared with the parent subdomains. fXYa thus represents a new serine proteinase lineage with hybrid fX, trypsin, and novel properties.Consideration of hierarchical features of protein architecture is particularly important in the design of novel enzymatic functions (1, 2). The simplest protein design problems involve the recruitment of existing evolved structural and functional elements (domains or modules) in straightforward recombination of associated properties, for example the combination of separate targeting and effector domains in immunotoxin design (3) or the shuffling of cofactor binding domains of coagulation factors (4). Many applications would benefit from the recombination of properties within a single functional domain; however, recombination of hierarchical units smaller than functional domains (referred to here as subdomains) generally risks loss of function or folding stability. A cautious attempt in this direction is the recombination of subdomains selected from homologous proteins that themselves have diversified through evolution but that retain relatively conserved catalytic machinery and subdomain interactions. The evolution of protein function via domain duplication and͞or recombination has generated numerous functional domains that comprise two such subdomains (5-9). This strategy allows one to swap specific subdomain linked func...

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“…the crucial role of the thrombin in the pathophysiological mechanisms of anticoagulant and antiaggregation processes has been clearly demonstrated (13) in numbers of studies published in the last ten years (Fig. 1 thrombin is a serine protease included in the blood coagulation cascade which in the same time also plays a key role in the process of thrombin-induced platelet aggregation.…”

Section: Introductionmentioning

confidence: 99%

“…this is affected via different substances with defence function called antithrombocytes. They include inhibitors of thrombin, factor Ха, Factor IXa, factors participating in the extrinsic and intrinsic pathways of the coagulation cascade (13).…”

Section: Introductionmentioning

confidence: 99%