Thrombin-bound structure of an EGF subdomain from human thrombomodulin determined by transferred nuclear Overhauser effects

Srinivasan, Jayashree; Hu, Song-Qing; Hrabal, Richard; Zhu, Yongjun; Komives, Elizabeth A.; Ni, Feng

doi:10.1021/bi00250a007

Cited by 28 publications

(40 citation statements)

References 33 publications

(63 reference statements)

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“…The fact that the designed peptide was a better thrombin inhibitor strongly suggests that entropy is lost upon binding. This result is consistent with the recent transferred NOE experiments showing that the peptide does indeed become structured upon binding to thrombin (Srinivasan et al, 1994). These NMR experiments also show that the "tail" interacts with the loop in the bound structure, confirming the data presented here that indicate that the "tail" must be attached to the loop.…”

Section: Design Of a Tm-based Thrombin Inhibitorsupporting

confidence: 92%

“…First, it is known from recent NMR studies that even the designed peptide with the constrained loop, TM52-1+5, (Ki = 26.5 pM) is completely unstructured in solution. In contrast, the thrombinbound form of the peptide has a well-defined structure (Srinivasan et al, 1994). Thus, a likely explanation for the difference in binding is that the rest of the EGF-like domain imparts structure to the loop and hence decreases the entropy loss required for thrombin binding.…”

Section: The Peptides Bind More Weakly Than Larger Tm Fragmentsmentioning

confidence: 99%

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Thrombin inhibition by cyclic peptides from thrombomodulin

et al. 1995

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Peptides corresponding to the loop regions of the fourth, fifth, and sixth epidermal growth factor (EGF)-like domains of thrombomodulin (TM) have been synthesized and assayed for thrombin inhibition, as indicated by both inhibition of thrombin-mediated fibrinogen clotting and inhibition of the association of thrombin with TM that results in protein C activation. Peptides from the fifth EGF-like domain showed significant inhibition of fibrinogen clotting and protein C activation, whereas peptides from the fourth and sixth EGF-like domains were weak inhibitors in both assays.Two structural features were important for inhibitory potency of the peptides from the fifth EGF-like domain: cyclization by a disulfide bond and attachment of the "tail" amino acids C-terminal to the disulfide loop. Linear control peptides did not significantly inhibit clotting or protein C activation. The C-terminal loop alone, the "tail" peptide, or a mixture of the two were at least IO-fold less potent inhibitors of clotting or protein C activation. A more constrained peptide analog was designed by deletion of an isoleucine within the CS-C6 disulfide loop, TMS2-1+5c. This analog was a better inhibitor in both assay systems, having a K , for protein C activation of 26 pM.

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Section: Design Of a Tm-based Thrombin Inhibitorsupporting

confidence: 92%

Section: The Peptides Bind More Weakly Than Larger Tm Fragmentsmentioning

confidence: 99%

Thrombin inhibition by cyclic peptides from thrombomodulin

et al. 1995

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“…In addition, the Arg-Gly-Asp triplet in thrombin appears to be involved in thrombomodulin interactions (50). Alanine-scanning mutagenesis has shown which residues of the EGF modules are critical for these interactions (51), and x-ray crystallographic and NMR studies have determined the three-dimensional structure of thrombin with a subdomain from the EGF region of thrombomodulin bound (52,53).…”

Section: Discussionmentioning

confidence: 99%

The Shape of Thrombomodulin and Interactions with Thrombin as Determined by Electron Microscopy

Weisel

Nagaswami

Young

et al. 1996

Journal of Biological Chemistry

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Studies have been carried out to investigate aspects of the structure of thrombomodulin, an endothelial cell glycoprotein that binds thrombin and accelerates both the thrombin-dependent activation of protein C and the inhibition of antithrombin III. We have determined the shape of Solulin TM , a soluble recombinant form of human thrombomodulin missing the transmembrane and cytoplasmic domains, by electron microscopy of preparations rotary-shadowed with tungsten. Solulin appears to be an elongated molecule about 20 nm long that has a large nodule at one end and a smaller nodule near the other end from which extends a thin strand. About half of the molecules form bipolar dimers apparently via interactions between these thin strands. Electron microscopy of complexes formed between Solulin and human ␣-thrombin revealed that a single thrombin molecule appears to bind to the smaller nodule of Solulin, suggesting that this region contains the epidermal growth factor-like domains 5 and 6. Epidermal growth factor-like domains 1-4 comprise the connector between the small and large nodule, which is the lectin-like domain; the thin strand at the other end of the molecule is the carbohydrate-rich region. With chondroitin sulfatecontaining soluble thrombomodulin produced from either human melanoma cells Bowes or Chinese hamster ovary cells, a higher percentage of molecules bound thrombin and, in some cases, two thrombin molecules were attached to one soluble thrombomodulin in approximately the same region. These structural studies provide insight into the structure of thrombomodulin and its interactions with thrombin as well as aspects of the mechanisms of its actions.Human thrombomodulin is a single chain glycoprotein made up of 557 amino acids, as well as N-and O-linked carbohydrate, that acts as a co-factor in the activation of protein C by thrombin and a modulator for the specificity of thrombin (1-6). From analysis of the amino acid sequence, thrombomodulin is made up of five regions, a lectin-like domain, a group of six epidermal growth factor-like modules, a serine-, threonine-, and O-glycosylation-rich region, a transmembrane sequence, and a cytoplasmic domain. There is, however, little information on the spatial arrangement of these domains.In its actions as an anticoagulant, thrombomodulin has several functions: the binding of thrombin, resulting in the modulation of its substrate specificity; a co-factor in the activation of protein C; and a co-factor in the inhibition of thrombin by antithrombin III (1, 2, 4 -6). The binding of thrombomodulin to thrombin effectively destroys the specificity of this enzyme for catalyzing the conversion of fibrinogen to fibrin, the activation of factors V, VIII, and XIII, and the activation of platelets. At the same time, in the presence of calcium ions, thrombomodulin acts as a co-factor for the activation of protein C, which, together with protein

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“…The factor X EGFII has a four-amino acid insertion in this same loop, and it has been reported to have a structure that is similar to EGF. The fifth EGF-like domain of TM has a three-amino acid insertion, but the C-terminal loop of this domain also adopts a classic EGFlike tri-stranded antiparallel @-sheet structure when bound to thrombin (Srinivasan et al, 1994). Thus, despite the differences in lengths of the loops, the structures of all the EGF-like domains determined so far are similar.…”

Section: P Meininger Et Atmentioning

confidence: 87%

“…The fifth domain also appears to tolerate different disulfide-bonding patterns and the isomer with the highest thrombin-binding affinity is non-EGF-like . Thus, it is interesting to speculate on the roles 50 nism (Srinivasan et al, 1994 (Ye et al, 1991), but the fact that no cofactor activity is observed from mixtures of the two domains suggests that the fourth domain cannot bind if it is not contion that could be offered by the structure of the TM fragment containing both the fourth and fifth EGF-like domains, which is in progress. resulting in preliminary structure calculations utilizing 1 1.8 NOEbased distance constraints per residue, or 12.6 NOE-based distance constraints per residue if the N-terminal five residues and of each of these essential EGF-like domains in the thrombin-TM NOEs involving these residues were ignored.…”

Section: Comparison Of the Fourth Andfifth Domains Of Tmmentioning

confidence: 99%

Synthesis, activity, and preliminary structure of the fourth EGF-like domain of thrombomodulin

1995

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The fourth EGF-like domain of thrombomodulin (TM4), residues E346-F389 in the TM sequence, has been synthesized. Refolding of the synthetic product under redox conditions gave a single major product. The disulfide bonding pattern of the folded, oxidized domain was (1-3, 2-4, 5-6), which is the same as that found in EGF protein. TM4 was tested for TM anticoagulant activity because deletion and substitution mutagenesis experiments have shown that the fourth EGF-like domain of TM is essential for TM cofactor activity. TM4 showed no TM-like activity in two assay systems, both for inhibition of fibrin clot formation, and for cofactor activity in thrombin activation of protein C.A preliminary structure of TM4 was determined by 2D 'H NMR from 519 NOE-derived distance constraints.Distance geometry calculations yielded a single convergent structure. The structure resembles the structure of EGF and other known EGF-like domains but has some key differences. The central two-stranded &sheet is conserved despite the differences in the number of amino acids in the loops. The C-terminal loop formed by the disulfide bond between C372 and C386 in TM4 is five amino acids longer than the analogous loop between C33 and C42 of EGF protein. This loop appears to have a different fold in TM4 than in EGF protein. The loop forms the two outside strands of a broken, irregular tri-stranded (3-sheet, and amino acids H384-F389 lie between the two strands forming the middle strand of the sheet. Thus, although the C-terminus of EGF protein forms one of the outside strands of a tri-stranded antiparallel sheet, the C-terminus of TM4 forms the inside strand of an irregular tri-stranded parallel-antiparallel sheet. The residues D349, E357, and E374, which were shown to be critical for cofactor activity by alanine scanning mutagenesis, all lie in a patch near the C-terminal loop, and are solvent accessible. The other critical residues, Y358 and F376, are largely buried and appear to play essential structural rather than functional roles.

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Thrombin-bound structure of an EGF subdomain from human thrombomodulin determined by transferred nuclear Overhauser effects

Cited by 28 publications

References 33 publications

Thrombin inhibition by cyclic peptides from thrombomodulin

Thrombin inhibition by cyclic peptides from thrombomodulin

The Shape of Thrombomodulin and Interactions with Thrombin as Determined by Electron Microscopy

Synthesis, activity, and preliminary structure of the fourth EGF-like domain of thrombomodulin

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