High-resolution NMR studies of fibrinogen-like peptides in solution: structural basis for the bleeding disorder caused by a single mutation of Gly(12) to Val(12) in the A.alpha. chain of human fibrinogen Rouen

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Abstract
In human fibrinogen Rouen, which is the origin of a bleeding disorder, a single amino acid is mutated from Gly(12) to Val(12) in the A.alpha. chain. In the previous paper of this series, this mutation was predicted to disrupt the structure of fibrinogen-like peptides bound to bovine thrombin. The structural basis of this bleeding disorder has been further assessed by studying the interaction of the following Val(12)- substituted human fibrinogen-like peptides with bovine thrombin in aqueous solution by use of two-dimensional NMR spectroscopy (including TRNOE): Ala-Asp-Ser-Gly-Asp(7)-Phe-Leu-Ala-Glu-Val(12)- Gly-Gly-Val-Arg(16)-Gly(17)-Pro-Arg-Val-NH2 (F16), Ala-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala- Glu-Val(12)-Gly-Gly-Val-Arg(16) (tF16), Ala-Asp-Ser-Gly-Glu-Cys(Acm)-Asp(7)-Phe-Leu-Ala-GluVal(12)-Gly-Gly-Val-Arg(16)-Gly(17)-Pro-Arg-Val-Cys(Acm)-NH2 (F17), and Ala-Asp-Ser-Gly-Glu- Cys(Acm)-Asp(7)-Phe-Leu-Ala-Glu-Val(12)-Gly-Gly-Val-Arg(16) (tF17). Binding of thrombin to peptides F16 and F17, and hence to tF16 and tF17 as a result of the cleavage of the Arg(16)-Gly(17) peptide bond, broadens the proton resonances of residues Asp(7) to Arg(16), suggesting that thrombin interacts specifically with this sequence of residues. Medium- and long-range TRNOE''s were observed between the NH proton of Asp(7) and the C.beta.H protons of Ala(10) and between the ring protons of Phe(8) and the C.gamma.H protons of Val(12) and Val(15) in complexes of thrombin with both tF16 and tF17. A strong TRNOE, in peptides tF16 and tF17, between the C.beta.H protons of Glu(11) and the backbone NH proton of Val(12) was also observed. However, TRNOE''s between the ring protons of Phe(8) and the C.alpha.H protons of Gly (14) and between the C.alpha.H proton of Glu(11) and the NH proton of Gly(13), previously observed in the complex of thrombin with FpA, were absent in both peptides tF16 and tF17. From incorporation of TRNOE information into distance geometry calculations. Val(12)was found to disrupt the type II .beta.-turn involving Glu(11) and Gly(12) that is present in complexes of thrombin with normal fibrinogen-like peptides. The positions of Gly(13) and Gly(14) in the complex are also displaced, relative to the aromatic ring of Phe(8), by the Val(12) substitution. This altered geometry presumably affects the positioning of the Arg(16)-Gly(17) bond in the active site of thrombin. This suggestion was supported by the observation that the cleavage of the Arg(16)-Gly(17) peptide bond in both F16 and F17 by thrombin is much slower than the cleavage of the same bond in normal fibrinogen-like peptides. Thus, the bleeding disorder caused by the single mutation of Gly(12) to Val(12) may be due to the structural alteration of human fibrinogen Rouen identified here.