The formation of a native-like structure containing eight conserved hydrophobic residues is rate limiting in two-state protein folding of ACBP.

The acyl-coenzyme A-binding proteins (ACBPs) contain 26 highly conserved sequence positions. The majority of these have been mutated in the bovine protein, and their influence on the rate of two-state folding and unfolding has been measured. The results identify eight sequence positions, out of 24 probed, that are critical for fast productive folding. The residues are all hydrophobic and located in the interface between the N- and C-terminal helices. The results suggest that one specific site dominated by conserved hydrophobic residues forms the structure of the productive rate-determining folding step and that a sequential framework model can describe the protein folding reaction.

Involvement of the hydrophobic stack residues 39-44 of factor VIIa in tissue factor interactions.

Des(1-38) factor VIIa and des(1-44) factor VIIa were obtained by limited proteolysis. The binding of tissue factor to these factor VIIa-derivatives was assessed from its stimulation of the proteolytic activity on chromogenic oligopeptide substrates. Compared to native factor VIIa (KTF = 0.6 +/- 0.1 nM), Tissue factor binds to des(1-38) factor VIIa with a lower, but still significant affinity (KTF = 4.8 +/- 0.3 nM). The activity of des(1-44) factor VIIa was only slightly stimulated by TF (KTF approximately 200 nM). Binding of TF depends critically on the presence of Ca2+ ions. Ca2+ ions stimulated the activity of factor VIIa/TF with an apparent KCa = 0.16 +/- 0.02 mM. Factor VIIa in the absence of tissue factor was stimulated by Ca2+ with an apparent KCa = 0.05 +/- 0.01 mM, and similar KCa values were obtained for the truncated derivatives of factor VIIa. Measurements of Ca(2+)-induced changes in intrinsic protein fluorescence suggest a conformational change. The Ca2+ ion concentration at which this change occurred was higher for des(1-44) factor VIIa (apparent KCa = 0.14 mM) than for des(1-38)- and native factor VIIa (apparent KCa = 0.04 mM). The Tb3+ ion luminescence technique was used to further investigate the Ca2+ binding sites. Tb3+ ions bound with a lower affinity to des(1-44) factor VIIa than to des(1-38)-and native factor VIIa. The observed drastic decrease in affinity for tissue factor as a result of truncation of the 'hydrophobic stack' residues 39-44, suggest that this region of factor VIIa provides a structural determinant that together with other regions participates in tissue factor binding.

Identification of a calcium binding site in the protease domain of human blood coagulation factor VII: evidence for its role in factor VII-tissue factor interaction.

Previous studies have identified a putative calcium binding site involving two glutamic acid residues located in the protease domain of coagulation factor IX. Amino acid sequence homology considerations suggest that factor VII (FVII) possesses a similar site involving glutamic acid residues 210 and 220. In the present study, we have constructed site-specific mutants of human factor VII in which Glu-220 has been replaced with either a lysine (E220K FVII) or an alanine (E220A FVII). These mutants were indistinguishable from wild-type factor VII by SDS-PAGE but only possessed 0.1% the coagulant activity of factor VII. Incubation of E220K/E220A FVII with factor Xa resulted in a slower than normal activation rate which eventually yielded a two-chain factor VIIa molecule possessing a coagulant activity of approximately 10% that of wild-type rFVIIa. Amidolytic activity measurements indicated that E220K/E220A FVIIa, unlike wild-type factor VIIa, possessed no measurable amidolytic activity toward the chromogenic substrate S-2288, even at high CaCl2 concentrations. Addition of tissue factor apoprotein, however, induced the amidolytic activity of the mutant molecule to a level 30% of that observed for wild-type factor VIIa. This tissue factor dependent enhancement of E220K/E220A FVIIa amidolytic activity was calcium dependent and required a CaCl2 concentration in excess of 5 mM for maximal rate enhancement. This was in sharp contrast to wild-type factor VIIa which required CaCl2 levels of 0.5 mM for maximal enhancement of tissue factor dependent amidolytic activity. Competition binding experiments suggest that the decrease in amidolytic and coagulant activity observed in the factor VII mutants is a direct result of impaired tissue factor binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Two different Ca2+ ion binding sites in factor VIIa and in des(1-38) factor VIIa.

The Ca2+ ion binding of factor VIIa and the derivative lacking the gamma-carboxyglutamic acid domain, des(1-38) factor VIIa, was investigated using intrinsic protein fluorescence and Tb3+ ion phosphorescence methods. Binding of Ca2+ ions giving rise to a decrease in the intrinsic protein fluorescence (approximately 50% at saturating conditions) is seen with both proteins. Each of the saturation curves is in accordance with the formation of a 1:1 complex of factor VIIa-Ca2+ (KD approximately 30 microM) and des(1-38) factor VIIa-Ca2+ (KD approximately 40 microM)). Yet another Ca2+ ion binding site reveals itself in each protein in Tb3+ ion phosphorescence experiments. Ca2+ ion competition studies have showed 1:1 complexes (KD's approximately 2 mM). The results are interpreted in terms of two different Ca2+ ion binding sites, one in the EGF-1 domain and one in the Gly-209-Gln-221 loop of the serine proteinase part.