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
2CEZ 2007-02-20 Phosphorylation of the Cytoplasmic Tail of Tissue Factor and its Role in Modulating Structure and Binding Affinity
2CFJ 2007-03-20 Phosphorylation of the Cytoplasmic Tail of Tissue Factor and its Role in Modulating Structure and Binding Affinity
2CEF 2007-02-13 Phosphorylation of the Cytoplasmic Tail of Tissue Factor and its Role in Modulating Structure and Binding Affinity.
2CEH 2007-02-13 Phosphorylation of the Cytoplasmic Tail of Tissue Factor and its Role in Modulating Structure and Binding Affinity
4YLQ 2015-12-30 1.4 Crystal Structure of a FVIIa-Trypsin Chimera (FT) in Complex with Soluble Tissue Factor
2HFT 1996-01-29 1.69 THE CRYSTAL STRUCTURE OF THE EXTRACELLULAR DOMAIN OF HUMAN TISSUE FACTOR AT 1.7 ANGSTROMS RESOLUTION
2C4F 2006-10-18 1.72 crystal structure of factor VII.stf complexed with pd0297121
3TH2 2012-08-22 1.72 Mg2+ Is Required for Optimal Folding of the Gamma-Carboxyglutamic Acid (Gla) Domains of Vitamin K-Dependent Clotting Factors At Physiological Ca2+
2A2Q 2006-07-04 1.8 Complex of Active-site Inhibited Human Coagulation Factor VIIa with Human Soluble Tissue Factor in the Presence of Ca2+, Mg2+, Na+, and Zn2+
3TH4 2012-08-22 1.8 Mg2+ Is Required for Optimal Folding of the Gamma-Carboxyglutamic Acid (Gla) Domains of Vitamin K-Dependent Clotting Factors At Physiological Ca2+
4IBL 2014-04-16 1.8 Rubidium Sites in Blood Coagulation Factor VIIa
1JPS 2002-02-03 1.85 Crystal structure of tissue factor in complex with humanized Fab D3h44
2AER 2006-07-04 1.87 Crystal Structure of Benzamidine-Factor VIIa/Soluble Tissue Factor complex.
2FLB 2006-04-04 1.95 Discovery of a Novel Hydroxy Pyrazole Based Factor IXa Inhibitor
1Z6J 2005-05-03 2.0 Crystal Structure of a ternary complex of Factor VIIa/Tissue Factor/Pyrazinone Inhibitor
2EC9 2008-02-19 2.0 Crystal structure analysis of human Factor VIIa , Souluble tissue factor complexed with BCX-3607
2FIR 2006-07-11 2.0 Crystal structure of DFPR-VIIa/sTF
1DAN 1997-09-04 2.0 Complex of active site inhibited human blood coagulation factor VIIA with human recombinant soluble tissue factor
1O5D 2004-09-21 2.05 Dissecting and Designing Inhibitor Selectivity Determinants at the S1 site Using an Artificial Ala190 Protease (Ala190 uPA)
2PUQ 2007-05-22 2.05 Crystal structure of active site inhibited coagulation factor VIIA in complex with soluble tissue factor
1UJ3 2004-07-25 2.1 Crystal structure of a humanized Fab fragment of anti-tissue-factor antibody in complex with tissue factor
1FAK 1999-12-03 2.1 HUMAN TISSUE FACTOR COMPLEXED WITH COAGULATION FACTOR VIIA INHIBITED WITH A BPTI-MUTANT
1WTG 2005-11-23 2.2 Human Factor Viia-Tissue Factor Complexed with ethylsulfonamide-D-biphenylalanine-Gln-p-aminobenzamidine
3ELA 2008-11-04 2.2 Crystal structure of active site inhibited coagulation factor VIIA mutant in complex with soluble tissue factor
2ZWL 2009-01-20 2.2 Human factor viia-tissue factor complexed with highly selective peptide inhibitor
1BOY 1996-06-10 2.2 EXTRACELLULAR REGION OF HUMAN TISSUE FACTOR
2B7D 2006-02-14 2.24 Factor VIIa Inhibitors: Chemical Optimization, Preclinical Pharmacokinetics, Pharmacodynamics, and Efficacy in a Baboon Thrombosis Model
4Z6A 2015-12-30 2.25 Crystal Structure of a FVIIa-Trypsin Chimera (YT) in Complex with Soluble Tissue Factor
4ZMA 2015-12-30 2.3 Crystal Structure of a FVIIa-Trypsin Chimera (ST) in Complex with Soluble Tissue Factor
2FLR 2007-01-23 2.35 Novel 5-Azaindole Factor VIIa Inhibitors
1WUN 2005-12-08 2.4 Human Factor Viia-Tissue Factor Complexed with ethylsulfonamide-D-Trp-Gln-p-aminobenzamidine
1TFH 1998-02-25 2.4 EXTRACELLULAR DOMAIN OF HUMAN TISSUE FACTOR
2ZZU 2009-03-24 2.5 Human Factor VIIA-Tissue Factor Complexed with ethylsulfonamide-D-5-(3-carboxybenzyloxy)-Trp-Gln-p-aminobenzamidine
1W0Y 2005-01-21 2.5 tf7a_3771 complex
1WQV 2005-10-02 2.5 Human Factor Viia-Tissue Factor Complexed with propylsulfonamide-D-Thr-Met-p-aminobenzamidine
2AEI 2006-08-01 2.52 Crystal structure of a ternary complex of factor VIIa/tissue factor and 2-[[6-[3-(aminoiminomethyl)phenoxy]-3,5-difluro-4-[(1-methyl-3-phenylpropyl)amino]-2-pyridinyl]oxy]-benzoic acid
2F9B 2006-02-28 2.54 Discovery of Novel Heterocyclic Factor VIIa Inhibitors
5W06 2017-06-14 2.6 HUMAN TISSUE FACTOR IN COMPLEX WITH ANTIBODY M1587
1WSS 2005-11-10 2.6 Human Factor Viia-Tissue Factor in Complex with peptide-mimetic inhibitor that has two charged groups in P2 and P4
2ZP0 2008-07-15 2.7 Human factor viia-tissue factor complexed with benzylsulfonamide-D-ile-gln-P-aminobenzamidine
1WV7 2005-12-11 2.7 Human Factor Viia-Tissue Factor Complexed with ethylsulfonamide-D-5-propoxy-Trp-Gln-p-aminobenzamidine
3TH3 2012-08-22 2.7 Mg2+ Is Required for Optimal Folding of the Gamma-Carboxyglutamic Acid (Gla) Domains of Vitamin K-Dependent Clotting Factors At Physiological Ca2+
2B8O 2007-02-13 2.8 Crystal Structure of Glu-Gly-Arg-Chloromethyl Ketone-Factor VIIa/Soluble Tissue Factor Complex
1J9C 2004-07-27 2.9 Crystal Structure of tissue factor-factor VIIa complex
1W2K 2005-06-20 3.0 tf7a_4380 complex
1AHW 1998-02-25 3.0 A COMPLEX OF EXTRACELLULAR DOMAIN OF TISSUE FACTOR WITH AN INHIBITORY FAB (5G9)
4M7L 2014-08-13 3.4 Crystal structure of the complex between human tissue factor extracellular domain and antibody 10H10 FAB fragment

Relevant UniProtKB Entries

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