Conformational stability of factor VIIa: biophysical studies of thermal and guanidine hydrochloride-induced denaturation.


Abstract

The binding of the multidomain protein factor VIIa (fVIIa) to tissue factor provides the interprotein communication necessary to make fVIIa an efficient catalyst of the initial event in the extrinsic pathway of blood coagulation. We have investigated the stability of individual domains in fVIIa and the influence of Ca2+ and an irreversible active-site inhibitor (FFR-chloromethyl ketone). Equilibrium guanidine hydrochloride (GuHCl)-induced unfolding monitored by tryptophan fluorescence and far-UV circular dichroism (CD) demonstrated that the gamma-carboxyglutamic acid (Gla) domain unfolds at 0.3 M GuHCl and the serine protease (SP) domain at 3 M GuHCl and that Ca2+ is a prerequisite for the formation of an ordered, compact structure in the Gla domain. The loss of amidolytic activity coincides with the first transition, which is stabilized by the active-site inhibitor, and a change in the environment of the active site is demonstrated using a fluorescent inhibitor (DEGR-chloromethyl ketone). Thermal unfolding monitored by differential scanning calorimetry (DSC) reveals that Ca2+ stabilizes the SP domain slightly, increasing the unfolding temperature by 2.7 degrees C. In addition, Ca2+ is required for a large enthalpy change concomitant with unfolding of the Gla domain, and this unfolding enthalpy is only detectable in the presence of the SP domain, indicating some kind of interaction between these domains. Thermal unfolding measured by CD indicates secondary structural changes at the same temperature as the heat absorption in the DSC but only when both the Gla domain and the SP domain are present together with Ca2+ ions. Taken together, these results indicate a Ca2+-dependent interaction between the Gla domain and the SP domain, implying a high degree of flexibility of the domains in free fVIIa. It is also shown that the epidermal growth factor-like domains are stable at elevated temperatures and high GuHCl concentrations. Moreover, already at physiological temperature, subtle structural changes take place which influence the overall shape of fVIIa and are detrimental to its enzymatic activity. Study holds ProTherm entries: 9485, 9486, 9487, 9488, 9489, 9490 Extra Details: multidomain protein factor; amidolytic activity;,active-site inhibitor; unfolding enthalpy; enzymatic activity

Submission Details

ID: 5KKcCBB8

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:38 p.m.

Version: 1

Publication Details
Freskgârd PO;Petersen LC;Gabriel DA;Li X;Persson E,Biochemistry (1998) Conformational stability of factor VIIa: biophysical studies of thermal and guanidine hydrochloride-induced denaturation. PMID:9585532
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1BF9 1998-05-28T00:00:00+0000 0 N-TERMINAL EGF-LIKE DOMAIN FROM HUMAN FACTOR VII, NMR, 23 STRUCTURES
1CVW 1999-08-24T00:00:00+0000 2.28 Crystal structure of active site-inhibited human coagulation factor VIIA (DES-GLA)
1CVW 1999-08-24T00:00:00+0000 2.28 Crystal structure of active site-inhibited human coagulation factor VIIA (DES-GLA)
1DAN 1997-03-05T00:00:00+0000 2.0 Complex of active site inhibited human blood coagulation factor VIIA with human recombinant soluble tissue factor
1DAN 1997-03-05T00:00:00+0000 2.0 Complex of active site inhibited human blood coagulation factor VIIA with human recombinant soluble tissue factor
1DVA 2000-01-20T00:00:00+0000 3.0 Crystal Structure of the Complex Between the Peptide Exosite Inhibitor E-76 and Coagulation Factor VIIA
1DVA 2000-01-20T00:00:00+0000 3.0 Crystal Structure of the Complex Between the Peptide Exosite Inhibitor E-76 and Coagulation Factor VIIA
1F7E 1999-02-19T00:00:00+0000 0 THE FIRST EGF-LIKE DOMAIN FROM HUMAN BLOOD COAGULATION FVII, NMR, 20 STRUCTURES
1F7M 1999-02-19T00:00:00+0000 0 THE FIRST EGF-LIKE DOMAIN FROM HUMAN BLOOD COAGULATION FVII, NMR, MINIMIZED AVERAGE STRUCTURE
1FAK 1998-12-28T00:00:00+0000 2.1 HUMAN TISSUE FACTOR COMPLEXED WITH COAGULATION FACTOR VIIA INHIBITED WITH A BPTI-MUTANT

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
195.8 H,L COAGULATION FACTOR VIIA Q2F9P4 FA7_PANPA
198.8 H,L COAGULATION FACTOR VIIA Q2F9P2 FA7_PANTR
200.0 H,L COAGULATION FACTOR VIIA P08709 FA7_HUMAN