Interaction between Yersinia pestis YopM protein and human alpha-thrombin.
YopM, a 41.5 kDa virulence protein of Yersinia pestis is believed to have an anti-inflammatory role in bubonic plague. It has been shown previously that YopM binds human alpha-thrombin but not prothrombin and inhibits thrombin-induced platelet aggregation in vitro. In the present studies we carried out crosslinking reactions between purified YopM and alpha-thrombin or its blocked form FPR-alpha-thrombin in the presence of various competitors to identify where on thrombin YopM binds. We found that thrombin cleaves YopM at the C-terminus, indicating that this part of YopM must interact with thrombin's catalytic site. Hirudin, a 65 amino acid natural thrombin inhibitor, prevents both the YopM degradation and the formation of a ca. 75 kDa crosslinking complex between YopM and alpha-thrombin. A similar effect is observed when hirugen, a short peptide corresponding to hirudin's C-terminus (amino acids 58-64), is used as a synthetic thrombin inhibitor. A 15 bp long specific oligonucleotide known to block alpha-thrombin successfully competes with YopM for the thrombin-binding site, whereas a control, scrambled sequence aptamer does not. As these competitors block a fibrinogen binding site (also called anion binding exosite I), our crosslinking data indicate that YopM binds not only to the active site of alpha-thrombin but also to the abeI.
Skrzypek
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Department of Microbiology and Immunology Chandler Medical Center, University of Kentucky, Lexington 40536-0084, USA.
Straley
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SC
D000595:Amino Acid Sequence; D000991:Antithrombins / Q000378:metabolism* / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D005340:Fibrinogen / Q000378:metabolism; D006629:Hirudins / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D013917:Thrombin / Q000378:metabolism*; D015010:Yersinia pestis / Q000378:metabolism*
D000991:Antithrombins; D001425:Bacterial Outer Membrane Proteins; D006629:Hirudins; D010446:Peptide Fragments; C062510:hirugen; C060772:yopM protein, Yersinia; D005340:Fibrinogen; D013917:Thrombin