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http://purl.uniprot.org/citations/24960590http://www.w3.org/1999/02/22-rdf-syntax-ns#typehttp://purl.uniprot.org/core/Journal_Citation
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Background

Protein Z (PZ) has been reported to promote the inactivation of factor Xa (FXa) by PZ-dependent protease inhibitor (ZPI) by about three orders of magnitude. Previously, we prepared a chimeric PZ in which its C-terminal pseudo-catalytic domain was grafted on FX light-chain (Gla and EGF-like domains) (PZ/FX-LC). Characterization of PZ/FX-LC revealed that the ZPI interactive-site is primarily located within PZ pseudo-catalytic domain. Nevertheless, the cofactor function and apparent Kd of PZ/FX-LC for interaction with ZPI remained impaired ~6-7-fold, suggesting that PZ contains a ZPI interactive-site outside pseudo-catalytic domain. X-ray structural data indicates that Tyr-240 of ZPI interacts with EGF2-domain of PZ. Structural data further suggests that 3 other ZPI surface loops make salt-bridge interactions with PZ pseudo-catalytic domain. To identify ZPI interactive-sites on PZ, we grafted the N-terminal EGF2 subdomain of PZ onto PZ/FX-LC chimera (PZ-EGF2/FX-LC) and also generated two compensatory charge reversal mutants of PZ pseudo-catalytic domain (Glu-244 and Arg-212) and ZPI surface loops (Lys-239 and Asp-293).

Methods

PZ chimeras were expressed in mammalian cells and ZPI derivatives were expressed in Escherichia coli.

Results

The PZ EGF2 subdomain fusion restored the defective cofactor function of PZ/FX-LC. The activities of PZ and ZPI mutants were all impaired if assayed individually, but partially restored if the compensatory charge reversal mutants were used in the assay.

Conclusions

PZ EGF2 subdomain constitutes an interactive-site for ZPI. Data with compensatory charge reversal mutants validates structural data that the identified residues are part of interactive-sites.

General significance

Insight is provided into mechanisms through which specificity of ZPI-PZ-FXa complex formation is determined."xsd:string
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http://purl.uniprot.org/citations/24960590http://purl.uniprot.org/core/author"Lu Q."xsd:string
http://purl.uniprot.org/citations/24960590http://purl.uniprot.org/core/author"Yang L."xsd:string
http://purl.uniprot.org/citations/24960590http://purl.uniprot.org/core/author"Rezaie A.R."xsd:string
http://purl.uniprot.org/citations/24960590http://purl.uniprot.org/core/author"Manithody C."xsd:string
http://purl.uniprot.org/citations/24960590http://purl.uniprot.org/core/author"Qureshi S.H."xsd:string
http://purl.uniprot.org/citations/24960590http://purl.uniprot.org/core/date"2014"xsd:gYear
http://purl.uniprot.org/citations/24960590http://purl.uniprot.org/core/name"Biochim Biophys Acta"xsd:string
http://purl.uniprot.org/citations/24960590http://purl.uniprot.org/core/pages"1631-1637"xsd:string
http://purl.uniprot.org/citations/24960590http://purl.uniprot.org/core/title"Characterization of the protein Z-dependent protease inhibitor interactive-sites of protein Z."xsd:string
http://purl.uniprot.org/citations/24960590http://purl.uniprot.org/core/volume"1844"xsd:string
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