Modeling Zymogen Protein C

Perera, Lalith; Foley, C. K.; Darden, Thomas A.; Stafford, Darrel W.; Mather, Timothy; Esmon, Charles T.; Pedersen, Lee G.

doi:10.1016/s0006-3495(00)76530-1

Cited by 30 publications

(32 citation statements)

References 80 publications

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“…The two prominent peaks around residues 285-296 (cn142-154) and 334-343 (cn186-194) are characteristic features of the transition from zymogen to active form. Although in the previously modeled zymogen/activated pairs for FIX 58 and protein C 48 we observed the same signature of activation, the C α deviations peak around residues 285-296 (cn142-154) for both those systems were found to have the same magnitude as the experimental X-ray crystal structures. In fact, there is a significant influence from the N-terminal region of the SP domain on the structure of this loop, due to significant structural changes associated with the N-terminus upon activation.…”

Section: Comparison With Other Zymogen/activation Form Pairssupporting

confidence: 62%

“…All crystallographic water and calcium ion coordinates assigned in the X-ray crystal structure were preserved in the initial structure. A method described in our previous work 48 was implemented to initially define the location of the N-terminus of the SP domain for the zymogen. The chymotrypsin (pdb entry, 2cga)/chymotrypsinogen (pdb entry, 4cha) X-ray crystal structures were used as references.…”

Section: Model Constructionmentioning

confidence: 99%

“…This structure was similar to Banner et al's 33 structure, but some difference was observed near the active site due to differences in inhibitors. One of the TF-free FVIIa (the pdb entry, 1qfk, of Pike et al 37 ) did not contain residues 1-44, and the X-ray intensities were disordered for residues [45][46][47][48][145][146][147][148][149][150][151][152], and 315-316. Also, the light chain residues, Lys-62, Glu-77, Lys-85, and the SP domain residues Lys-199 and Arg-202 were poorly resolved beyond the β-carbon atom, and hence, final coordinates were missing from the coordinate file for these atoms beyond β-carbon.…”

Section: Introductionmentioning

confidence: 99%

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Predicted solution structure of zymogen human coagulation FVII

Perera¹,

Darden²,

Pedersen³

2001

J Comput Chem

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A model solution structure for the complete tissue factor-free calcium ion-bound human zymogen FVII (residues 1-406) (FVII) has been constructed to study possible conformational changes associated with the activation process and tissue factor (TF) binding. The initial structure for the present model was constructed using the X-ray crystallographic structure of human coagulation FVIIa/TF complex bound with calcium ions (Banner et al., Nature 1996, 380, 41-46). This model was subsequently subjected to lengthy molecular dynamics simulations. The Amber force field in conjunction with the PME electrostatic summation method was employed. The estimated TF free solution structure was then compared with the currently available X-ray crystal structures of FVIIa (with or without TF, variable inhibitor bound) to estimate the restructuring of FVII due to TF binding and activation. The solution structure of the zymogen FVII in the absence of TF is predicted to be an extended domain structure similar to that of the TF-bound X-ray crystal structure. An additional extension of the serine protease (SP) domain of the zymogen above a reference lipid surface by approximately 7 A was in agreement with experiment. Significant Gla-EGF1 and EGF1-EGF2 interdomain motions in the zymogen were observed. Carbohydrate dimers attached to Ser-52 and Ser-60 did not cause restructuring in this domain. Minimal restructuring of the SP domain is found upon inference of the zymogen from the activated form. The catalytic triad residues maintain the H-bonded network while Lys-341 occupies the S1 specific site in the zymogen.

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Section: Comparison With Other Zymogen/activation Form Pairssupporting

confidence: 62%

Section: Model Constructionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Predicted solution structure of zymogen human coagulation FVII

Perera¹,

Darden²,

Pedersen³

2001

J Comput Chem

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“…Most of the residues in this region are either hydrophobic or acidic in nature and are surface exposed on a model of the complete protein C Gla domain with Ca 2ϩ bound (Fig. 7A) (38,39). Consequently, several of these residues form part of a putative protein-protein binding site predicted using molecular modeling based on surface hydrophobicity (40).…”

Section: Discussionmentioning

confidence: 99%

Multifunctional Specificity of the Protein C/Activated Protein C Gla Domain

Preston

Ajzner

Razzari

et al. 2006

Journal of Biological Chemistry

103

164

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Activated protein C (APC) has potent anticoagulant and antiinflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/ D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains.

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“…Ca 2ϩ influences the conformation of the activation peptide of protein C and perhaps makes it more complementary to the active site of thrombin. There are several lines of evidence in support of this hypothesis: 1) the molecular modeling data based on the crystal structures of the protease domain of activated protein C and chymotrypsinogen have predicted electrostatic interactions between the charged residues of the activation peptide and those of the 70-loop (27,28), 2) the mutagenesis of residues surrounding the scissile bond is associated with changes in the affinity of mutant zymogens for binding Ca 2ϩ (18,26), and 3) it is known that the GD-PC zymogen but not activated GD-PC undergoes a 5 Ϯ 1% quenching in the intrinsic protein fluorescence upon binding Ca 2ϩ (15). These results together with kinetic data have supported the hypothesis that the conformations of the activation peptide and the Ca 2ϩ -binding 70-loop of protein C are allosterically linked.…”

Section: Fig 3 Camentioning

confidence: 99%

The Conformation of the Activation Peptide of Protein C Is Influenced by Ca2+ and Na+ Binding

Yang

Prasad

et al. 2004

Journal of Biological Chemistry

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Previous studies have suggested that the conformation of the activation peptide of protein C is influenced by the binding of Ca 2؉ . To provide direct evidence for the linkage between Ca 2؉ binding and the conformation of the activation peptide, we have constructed a protein C mutant in the ␥-carboxyglutamic acid-domainless form in which the P1 Arg 169 of the activation peptide is replaced with the fluorescence reporter Trp. Upon binding of Ca 2؉ , the intrinsic fluorescence of the mutant decreases ϳ30%, as opposed to only 5% for the wild-type, indicating that Trp 169 is directly influenced by the divalent cation. The K d of Ca 2؉ binding for the mutant protein C was impaired ϳ4-fold compared with wild-type. Interestingly, the conformation of the activation peptide was also found to be sensitive to the binding of Na ؉ , and the affinity for Na ؉ binding increased ϳ5-fold in the presence of Ca 2؉ . These findings suggest that Ca 2؉ changes the conformation of the activation peptide of protein C and that protein C is also capable of binding Na ؉ , although with a weaker affinity compared with the mature protease. The mutant protein C can no longer be activated by thrombin but remarkably it can be activated efficiently by chymotrypsin and by the thrombin mutant D189S. Activation of the mutant protein C by chymotrypsin proceeds at a rate comparable to the activation of wild-type protein C by the thrombin-thrombomodulin complex.Protein C is a multidomain vitamin K-dependent serine protease zymogen in plasma, which, upon activation by the complex of thrombin and thrombomodulin (TM), 1 down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis (1-3). Activated protein C has a light and a heavy chain held together by a single disulfide bond, with the N-terminal light chain containing the non-catalytic ␥-carboxyglutamic acid domain and two epidermal growth factor-like domains (4, 5). The catalytic domain of activated protein C is located in the C-terminal heavy chain of the molecule (6) and contains functionally critical and conformationally linked binding sites for both Na ϩ and Ca 2ϩ (7,8). The Ca 2ϩ binding site is located in the 70-loop (chymotrypsin numbering (9)) by analogy with trypsin (10) and optimizes the catalytic activity of the protease (11,12). Ca 2ϩ binding to the 70-loop is also required for the rapid activation of protein C by the thrombin-TM complex, whereas such binding inhibits the conversion to activated protein C in the absence of TM (13,14). Based on these and other observations, it has been postulated that binding of Ca 2ϩ to the 70-loop of protein C changes the conformation of the activation peptide of the zymogen in a way that promotes cleavage by thrombin in the presence but not the absence of TM (1, 15). However, no direct evidence for the Ca 2ϩ -induced conformational change in the activation peptide of protein C has been presented so far.In addition to a Ca 2ϩ binding site in the 70-loop, the catalytic domain of activated protein C possesses a functiona...

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Modeling Zymogen Protein C

Cited by 30 publications

References 80 publications

Predicted solution structure of zymogen human coagulation FVII

Predicted solution structure of zymogen human coagulation FVII

Multifunctional Specificity of the Protein C/Activated Protein C Gla Domain

The Conformation of the Activation Peptide of Protein C Is Influenced by Ca2+ and Na+ Binding

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