Thrombomodulin Changes the Molecular Surface of Interaction and the Rate of Complex Formation between Thrombin and Protein C

Xu, Hong; Bush, Leslie A.; Pineda, Agustin O.; Caccia, Sonia; Di, Enrico

doi:10.1074/jbc.m412869200

Cited by 53 publications

(73 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Comparable effects are observed for the cleavage of fibrinogen, PAR1 and protein C in the absence of thrombomodulin, confirming a molecular origin of the perturbation within the active site of the enzyme. Binding of thrombomodulin partially relieves the perturbation, as documented for other mutants of the active site region [18]. However, the gain in relative anticoagulant potency, measured as the ratio between protein C activation in the presence of thrombomodulin relative to fibrinogen cleavage, is modest compared to wild type and makes the R4A mutant a poor competitor of other well-established protein C activators [19,20].…”

Section: Resultsmentioning

confidence: 90%

Role of the A chain in thrombin function

Papaconstantinou

Bah

2008

Cell. Mol. Life Sci.

View full text Add to dashboard Cite

The A chain of thrombin is covalently linked to the catalytic B chain but is separate from any known epitope for substrate recognition. In this study we present the results of the Ala replacement of 12 charged residues controlling the stability of the A chain and its interaction with the B chain. Residues Arg4 and Glu8 play a significant role in substrate recognition, even though they are located >20 Å away from residues of the catalytic triad, the primary specificity pocket and the Na + site. The R4A mutation causes significant perturbation of Na + binding, fibrinogen clotting and PAR1 cleavage, but modest reduction of protein C activation in the presence of thrombomodulin. These findings challenge our current paradigm of thrombin structure-function relations focused exclusively on the properties of the catalytic B chain, and explain why certain naturally occurring mutations of the A chain cause serious bleeding.

show abstract

Section: Resultsmentioning

confidence: 90%

Role of the A chain in thrombin function

Papaconstantinou

Bah

2008

Cell. Mol. Life Sci.

View full text Add to dashboard Cite

show abstract

“…Dominant involvement of the primary specificity pocket is confirmed by perturbation of fibrinogen and PAR cleavage that is affected to the same extent as FPR and FPK. Protein C activation is compromised to lesser extent, presumably because this substrate engages thrombin with minimal interactions in the presence of thrombomodulin (31). Also noteworthy is the large effect on antithrombin binding, which suggests perturbation of the 186 and 220 loops in addition to the primary specificity pocket.…”

Section: Discussionmentioning

confidence: 96%

Important Role of the Cys-191–Cys-220 Disulfide Bond in Thrombin Function and Allostery

Bush-Pelc

Marino

Chen

et al. 2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Little is known on the role of disulfide bonds in the catalytic domain of serine proteases. The Cys-191-Cys-220 disulfide bond is located between the 190 strand leading to the oxyanion hole and the 220-loop that contributes to the architecture of the primary specificity pocket and the Na ؉ binding site in allosteric proteases. Removal of this bond in thrombin produces an ϳ100-fold loss of activity toward several chromogenic and natural substrates carrying Arg or Lys at P1. Na ؉ activation is compromised, and no fluorescence change can be detected in response to Na ؉ binding. A 1.54-Å resolution structure of the C191A/ C220A mutant in the free form reveals a conformation similar to the Na ؉ -free slow form of wild type. The lack of disulfide bond exposes the side chain of Asp-189 to solvent, flips the backbone O atom of Gly-219, and generates disorder in portions of the 186 and 220 loops defining the Na ؉ site. This conformation, featuring perturbation of the Na ؉ site but with the active site accessible to substrate, offers a possible representation of the recently identified E* form of thrombin. Disorder in the 186 and 220 loops and the flip of Gly-219 are corrected by the active site inhibitor H-D-Phe-Pro-Arg-CH 2 Cl, as revealed by the 1.8-Å resolution structure of the complex. We conclude that the Cys-191-Cys-220 disulfide bond confers stability to the primary specificity pocket by shielding Asp-189 from the solvent and orients the backbone O atom of Gly-219 for optimal substrate binding. In addition, the disulfide bond stabilizes the 186 and 220 loops that are critical for Na ؉ binding and activation.Thrombin is a Na ϩ -activated, allosteric serine protease involved in blood clotting (1, 2) and is composed of two polypeptide chains of 36 (A chain) and 259 (B chain) residues covalently linked through the Cys-1-Cys-122 disulfide bond (3, 4). The shorter A chain runs in the back of the B chain that has the typical fold of serine proteases, with two six-stranded ␤-barrels of similar structure that pack together asymmetrically to accommodate at their interface the residues of the catalytic triad His-57, . The B chain is stabilized by three disulfide bonds, Cys-42-Cys-58, Cys-168 -Cys-182, and Cys-191-Cys-220, that connect contiguous strands shaping the Na ϩ binding site, the primary specificity pocket, and the active site. These bonds are highly conserved among serine proteases, but the Cys-191-Cys-220 disulfide bond is absent in cathepsins and granzymes. Previous studies using reduction with dithiothreitol have addressed the role of disulfide bonds in the mechanism of thrombin unfolding (6). The role of disulfide bonds in thrombin function and allostery, however, remains unexplored. Indeed, remarkably little is known on the role of conserved disulfide bonds for serine proteases in general (7), and no structural information on relevant mutants lacking one or more such bonds is currently available.Previous mutagenesis studies on trypsin have addressed the role of the Cys-191-Cys-220 bond and offered conflicting r...

show abstract

“…Cleavage of H-D-PhePro-Arg-p-nitroanilide, fibrinogen, and PAR1 and activation of protein C were determined as reported elsewhere (9,(21)(22)(23) under experimental conditions of 5 mM Tris, 0.1% polyethylene glycol, 145 mM NaCl, pH 7.4, at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of Thrombin in a Self-inhibited Conformation

Pineda

Chen

Bah

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The activating effect of Na ؉ on thrombin is allosteric and depends on the conformational transition from a low activity Na ؉ -free (slow) form to a high activity Na ؉ -bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na ؉ have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na ؉ site and the active site regions. Here, we report a 1.87 Å resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow 3 fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10 Å to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na ؉ -binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na ؉ and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215.

show abstract

Thrombomodulin Changes the Molecular Surface of Interaction and the Rate of Complex Formation between Thrombin and Protein C

Cited by 53 publications

References 42 publications

Role of the A chain in thrombin function

Role of the A chain in thrombin function

Important Role of the Cys-191–Cys-220 Disulfide Bond in Thrombin Function and Allostery

Crystal Structure of Thrombin in a Self-inhibited Conformation

Contact Info

Product

Resources

About