Molecular mapping of thrombin‐receptor interactions

Ayala, Youhna M.; Cantwell, Angelene M.; Rose, Thierry; Bush, Leslie A.; Arosio, Daniele; Di, Enrico

doi:10.1002/prot.1130

Cited by 101 publications

(167 citation statements)

References 33 publications

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“…Trp-56 interacts with Ile-82 of thrombin at the boundary of the binding epitope. The contacts documented by the crystal structure are in agreement with the results of Ala scanning mutagenesis of thrombin (40). In particular, mutagenesis studies have pointed out the peculiar contribution of Arg-67 to PAR1 recognition.…”

Section: Resultssupporting

confidence: 83%

Structural identification of the pathway of long-range communication in an allosteric enzyme

Gandhi

Chen

Mathews

et al. 2008

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

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103

142

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Allostery is a common mechanism of regulation of enzyme activity and specificity, and its signatures are readily identified from functional studies. For many allosteric systems, structural evidence exists of long-range communication among protein domains, but rarely has this communication been traced to a detailed pathway. The thrombin mutant D102N is stabilized in a self-inhibited conformation where access to the active site is occluded by a collapse of the entire 215-219 ␤-strand. Binding of a fragment of the protease activated receptor PAR1 to exosite I, 30-Å away from the active site region, causes a large conformational change that corrects the position of the 215-219 ␤-strand and restores access to the active site. The crystal structure of the thrombin-PAR1 complex, solved at 2.2-Å resolution, reveals the details of this long-range allosteric communication in terms of a network of polar interactions.protease activated receptor ͉ thrombin ͉ x-ray crystallography E ver since its original formulation (1, 2), the allosteric concept of enzyme regulation has captivated the interest of structural biologists. The idea that events occurring at a given site of the protein can be transmitted long-range to affect affinity or catalytic efficiency at a distant site offers an elegant explanation for linkage and cooperativity (3) but poses a challenge to the crystallographer who seeks to identify the communication pathway underlying the functional effects. Perutz (4) was the first to offer a molecular explanation for the allosteric mechanism of hemoglobin cooperativity. For multimeric proteins like hemoglobin, conformational transitions tend to affect the quaternary structure and signatures of allostery have been detected crystallographically (5-9). Allostery is not limited to multimeric assemblies and, in fact, many monomeric proteins feature conformational plasticity that translate into allosteric behavior at equilibrium and steady state (6, 10, 11). For such systems, the structural signatures of long-range communication tend to manifest themselves as small changes in tertiary structure or Hbonding connectivity (12, 13) and lack the amplification seen in large quaternary structural reorganization. Identification of the pathway of communication underlying an allosteric mechanism remains a difficult task in general and continues to receive utmost attention (11, 13). Alternative approaches have been proposed to identify such pathways based on the statistical analysis of evolutionary records of protein sequences (14) or anisotropic thermal diffusion (15). Although promising and insightful, such approaches must ultimately find validation by structural investigation.Among Na ϩ -activated allosteric enzymes (6), the serine protease thrombin has received much attention in view of its critical roles in hemostasis and thrombosis (12,16,17). Thrombin features considerable structural plasticity and exists predominantly in two forms at equilibrium, the Na ϩ -free slow form E (Ϸ40% of the molecules in vivo) and the Na ϩ -bound fast f...

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Section: Resultssupporting

confidence: 83%

Structural identification of the pathway of long-range communication in an allosteric enzyme

Gandhi

Chen

Mathews

et al. 2008

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

Self Cite

103

142

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“…PAR-1 has been identified as the primary target receptor for thrombin on platelets and endothelial cells (24). The cleavage of PAR-1 by thrombin elicits potent prothrombotic and proinflammatory responses (24,25). Because thrombin is responsible for the activation of protein C and it also cleaves PAR-1 with a catalytic efficiency that is 3-4 orders of magnitude higher than that of APC, whether APC in the presence of thrombin produces physiologically significant protective signaling events by PAR-1 cleavage is controversial (14).…”

Section: Discussionmentioning

confidence: 99%

“…Thrombin utilizes the basic residues of exosite-1 to interact with the hirudin-like sequence on PAR-1 (24,25) or the acidic residues of epidermal growth factor-like domains on TM (26,27) to function in either the procoagulant/proinflammatory or the anticoagulant/antiinflammatory pathways, respectively (24,28). Noting the high affinity of thrombin for TM (Ͻ1 nM) (29), physiological concentrations of thrombin may primarily interact with TM in lipid rafts, thereby blocking its interaction with PAR-1 and activating EPCR-bound protein C that is located next to PAR-1 in the same lipid raft and/or PAR-1 recruited from another nearby raft through a regulated mechanism.…”

Section: Discussionmentioning

confidence: 99%

Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells

Bae

Yang

Rezaie

2007

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

176

212

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Ever-increasing evidence in the literature suggests that the antiinflammatory and cytoprotective properties of activated protein C (APC) are mediated through its endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on endothelial cells. However, recent results monitoring the cleavage rate of PAR-1 on human umbilical vein endothelial cells, transfected with an alkaline phosphatase-PAR-1 fusion reporter construct, have indicated that the catalytic activity of thrombin toward PAR-1 is several orders of magnitude higher than that of APC. Because thrombin is required for generation of APC, and because it also functions in the proinflammatory pathways through the activation of PAR-1, it has been difficult to understand how APC can elicit protective cellular responses through the activation of PAR-1 when thrombin is present. In this study we provide a plausible answer to this question by demonstrating that the critical receptors required for both protein C activation (thrombomodulin and EPCR) and APC cellular signaling (EPCR and PAR-1) pathways are colocalized in the membrane lipid rafts in endothelial cells. We further show that the APC cleavage of PAR-1 on cells transfected with a PAR-1 cleavage reporter construct is not sensitive to the cofactor function of EPCR. Thus, the colocalization of EPCR and PAR-1 in lipid rafts is a key requirement for the cellular signaling activity of APC. Thrombomodulin colocalization with these receptors on the same membrane microdomain can also recruit thrombin to activate the EPCR-bound protein C, thereby eliciting PAR-1 signaling events that are involved in the APC protective pathways. endothelial protein C receptor ͉ protease-activated receptor 1 ͉ thrombin ͉ thrombomodulin

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“…The interaction with fibrinogen leading to release of fibrinopeptide A (FpA) and B (FpB), cleavage of the protease activated receptor 1 (PAR1) and activation of protein C with and without thrombomodulin were studied as reported [11,14,15] under experimental conditions of 5 mM Tris, 0.1% PEG8000, 145 mM NaCl, pH 7.4 at 37°C .…”

Section: Methodsmentioning

confidence: 99%

Role of the A chain in thrombin function

Papaconstantinou

Bah

2008

Cell. Mol. Life Sci.

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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.

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Molecular mapping of thrombin‐receptor interactions

Cited by 101 publications

References 33 publications

Structural identification of the pathway of long-range communication in an allosteric enzyme

Structural identification of the pathway of long-range communication in an allosteric enzyme

Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells

Role of the A chain in thrombin function

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