Identification of the Factor VIIa Binding Site on Tissue Factor by Homologous Loop Swap and Alanine Scanning Mutagenesis

Gibbs, Craig S.; McCurdy, Sarah N.; Leung, Lawrence L.K.; Paborsky, Lisa R.

doi:10.1021/bi00251a007

Cited by 45 publications

(49 citation statements)

References 50 publications

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“…Several years ago, the solution of the crystal structure of the TF extracellular portion (sTF) by two groups (29, 30) allowed a structure-based reinterpretation (31) of previous TF-FVIIa studies using a variety of techniques including antibody mapping (32), alanine-scanning mutagenesis (8,33,34), proteolytic fragmentation (35), and chemical cross-linking (36). A large interfacial area of binding was predicted, likely to involve multiple domains in the two proteins.…”

Section: Discussionmentioning

confidence: 99%

Coagulation Factor VII Gln100 → Arg

Kemball‐Cook¹,

Johnson²,

Takahashi³

et al. 1998

Journal of Biological Chemistry

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We have used recombinant mammalian expression and purification of the factor VII (FVII) variant Gln 100 3 Arg (Q100RFVII) to study FVII deficiency in subjects with this mutation. Q100RFVII was secreted poorly in comparison with wild-type FVII (WTFVII) in a stable mammalian expression system, and purified variant protein was found to have undetectable clotting activity. Following activation by immobilized factor Xa, Q100RFVIIa had amidolytic activity similar to WTFVIIa in the absence of soluble tissue factor (sTF); however, unlike WTFVIIa no typical increase in activity was seen after addition of sTF. In a factor X activation assay using relipidated transmembrane truncated tissue factor (residues 1-243), Q100RFVIIa showed less than 5% of the ability of WTFVIIa to activate factor X.We performed direct binding analysis of WT and Q100RFVII/FVIIa to immobilized sTF using surface plasmon resonance, and severely reduced binding of both non-activated and activated Q100RFVII to sTF was seen, indicating a pronounced defect in tissue factor (TF) interaction with this variant.In the sTF-FVIIa crystal structure the candidate residue Gln 100 is not in contact with TF but is at the epidermal growth factor 2-protease domain interface. We suggest that the mutation results in a global fold change severely reducing tissue factor interaction; mutation of FVII residues not directly involved in the interaction with TF may still result in variant FVII unable to take part in the initiation of coagulation.

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Section: Discussionmentioning

confidence: 99%

Coagulation Factor VII Gln100 → Arg

Kemball‐Cook¹,

Johnson²,

Takahashi³

et al. 1998

Journal of Biological Chemistry

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“…Antibodies against Mip/LIN-9, mAb and rabbit polyclonal (Gibbs et al, 1994), were previously described (Sandoval et al, 2006). The following antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA): anti-B-Myb (N-19, sc-724); -cyclin B (sc-245), -cyclin A (sc-751), -E2F4 (sc-866) and -E2F3 (sc-879).…”

Section: Immunoprecipitations and Immunoblottingmentioning

confidence: 99%

Mammalian Mip/LIN-9 interacts with either the p107, p130/E2F4 repressor complex or B-Myb in a cell cycle-phase-dependent context distinct from the Drosophila dREAM complex

2007

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Mammalian Mip/LIN-9 is a cell cycle regulatory protein that is negatively regulated by CDK4/cyclin D. It has been demonstrated that Mip/LIN-9 collaborates with B-Myb during S and G 2 /M in the induction of cyclins A and B, and CDK1. The ortholog of Mip/LIN-9 in Drosophila, Mip130, is part of a large multisubunit protein complex that includes RBF, repressor E2Fs and Myb, in what was termed the dREAM complex. A similar complex, although lacking B-Myb, was also described in Caenorhabditis elegans. Here, we demonstrate that unlike Drosophila, Mip/LIN-9 has mutually exclusive and cell cycle-phasespecific interactions with the mammalian orthologs of the dREAM complex. In G 0 /early G 1 , Mip/LIN-9 forms a complex with E2F4 and p107 or p130, while in late G 1 /S phase, it associates with B-Myb. The separation of Mip/LIN-9 from p107,p130/E2F4 is likely driven by phosphorylation of the pocket proteins by CDK4 since Mip/LIN-9 fails to interact with phosphorylated forms of p107,p130. Importantly, the repressor complex that Mip/LIN-9 forms with p107 takes functional precedence over the transcriptional activation linked to the Mip/LIN-9 and B-Myb interaction since expression of p107 blocks the activation of the cyclin B promoter triggered by B-Myb and Mip/LIN-9.

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“…The methodology includes functional titration (11)(12)(13) or direct biophysical evaluation using surface plasmon resonance (14). Soluble TF has a 20-fold reduced affinity for FVIIa as compared to full-length IF when analyzed with similar methodology under identical conditions (15).…”

Section: The Macromolecular Ligands For Tfmentioning

confidence: 99%

Tissue Factor: molecular recognition and cofactor function

Martin¹,

Boys

Ruf

1995

FASEB j.

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One aspect of the inflammatory response is the activation of the coagulation protease cascade resulting from the expression of tissue factor (TF) on vascular cells. TF is the cell-surface receptor for the coagulation serine protease factor VIIa, providing cofactor function by "switching on" the catalytic site of the bound enzyme and by contributing to the assembly with macromolecular substrate. The recently determined crystal structure of the TF extracellular domain shows two beta-strand modules of C2 immunoglobulin-like topology that align at a 125 degrees angle with an extensive intermodule interface. Mutagenesis studies have identified residues in both modules that are important for the binding of ligand. The deduced ligand interface extends from the convex side of the molecule into the concave side of the elbow angle. Specific binding residues control the catalytic activity of the bound protease. At the lower end of the carboxyl-terminal module, basic residues form part of a region that is important for both recognition and activation of macromolecular substrate and, potentially, for modulation of proteolytic function. After combining the biochemical data with the crystal structure, a model of TF function can be proposed in which the catalytic activity of the active site of the protease and the extended recognition of macromolecular substrates are separately controlled by distinct structural sites of the cofactor.

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Identification of the Factor VIIa Binding Site on Tissue Factor by Homologous Loop Swap and Alanine Scanning Mutagenesis

Cited by 45 publications

References 50 publications

Coagulation Factor VII Gln100 → Arg

Coagulation Factor VII Gln100 → Arg

Mammalian Mip/LIN-9 interacts with either the p107, p130/E2F4 repressor complex or B-Myb in a cell cycle-phase-dependent context distinct from the Drosophila dREAM complex

Tissue Factor: molecular recognition and cofactor function

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