Activity, folding, misfolding, and aggregation in vitro of the naturally occurring human tissue factor mutant R200W.


Abstract

Tissue factor (TF), a small transmembrane receptor, binds factor VIIa (FVIIa), and the formed complex initiates blood coagulation by proteolytic activation of substrate factors IX and X. A naturally occurring mutation in the human TF gene was recently reported, where a single-base substitution results in an R200W mutation in the TF extracellular domain [Zawadzki, C., Preudhomme, C., Gaveriaux, V., Amouyel, P., and Jude, B. (2002) Thromb. Haemost. 87, 540-541]. This mutation appears to be associated with low monocyte TF expression and may protect against thrombosis but has not been associated with any pathological condition, and individuals who present the heterozygous trait appear healthy. Here, we report the activity, folding, and aggregation behavior of the R200W mutant of the 219-residue soluble extracellular domain of TF (sTF(R200W)) compared to that of the wild-type protein (sTF(wt)). No differences in stability or FVIIa cofactor activity but an impaired ability to promote FX activation at physiological conditions between the sTF(R200W) mutant and sTF(wt) were evident. Increased binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to sTF(R200W) indicated a population of partially folded intermediates during denaturation. sTF(R200W) showed a dramatically increased propensity for aggregate formation compared to sTF(wt) at mildly acidic pHs, with an increased rate of aggregation during conditions, promoting the intermediate state. The lowered pH resistance could explain the loss of sTF(R200W) in vivo because of aggregation of the mutant. The intrinsic structure of the sTF aggregates appears reminiscent of amyloid fibrils, as revealed by thioflavin T fluorescence, atomic force microscopy, and transmission electron microscopy. We conclude that the lowered activity for FX activation and the propensity of the mutant protein to misfold and aggregate will both contribute to decreased coagulation activity in TF(R200W) carriers, which could protect from thrombotic disease. Study holds ProTherm entries: 19169, 19170 Extra Details: transmembrane receptor; proteolytic activation; thrombosis; partially folded intermediates

Submission Details

ID: qtzCiUfR

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
Wiréhn J;Carlsson K;Herland A;Persson E;Carlsson U;Svensson M;Hammarström P,Biochemistry (2005) Activity, folding, misfolding, and aggregation in vitro of the naturally occurring human tissue factor mutant R200W. PMID:15865421
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
1AHW 1997-04-10T00:00:00+0000 3.0 A COMPLEX OF EXTRACELLULAR DOMAIN OF TISSUE FACTOR WITH AN INHIBITORY FAB (5G9)
1BOY 1996-01-11T00:00:00+0000 2.2 EXTRACELLULAR REGION OF HUMAN 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
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
1FAK 1998-12-28T00:00:00+0000 2.1 HUMAN TISSUE FACTOR COMPLEXED WITH COAGULATION FACTOR VIIA INHIBITED WITH A BPTI-MUTANT
1J9C 2001-05-24T00:00:00+0000 2.9 Crystal Structure of tissue factor-factor VIIa complex
1JPS 2001-08-03T00:00:00+0000 1.85 Crystal structure of tissue factor in complex with humanized Fab D3h44
1O5D 2003-09-09T00:00:00+0000 2.05 Dissecting and Designing Inhibitor Selectivity Determinants at the S1 site Using an Artificial Ala190 Protease (Ala190 uPA)
1TFH 1997-04-10T00:00:00+0000 2.4 EXTRACELLULAR DOMAIN OF HUMAN TISSUE FACTOR
1UJ3 2003-07-25T00:00:00+0000 2.1 Crystal structure of a humanized Fab fragment of anti-tissue-factor antibody in complex with tissue factor

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Tissue factor P13726 TF_HUMAN