Transient hydrogen bonds identified on the surface of the NMR solution structure of Hirudin.

Recombinant desulfatohirudin retains largely the thrombin-inhibitory activity of natural hirudin from Hirudo medicinalis and causes at most minimal immune response in humans. With regard to potential pharmaceutical applications it is of interest to further investigate the structural basis of hirudin functions. In this paper transient hydrogen bonds between backbone amide protons and side-chain carboxylates on the protein surface of desulfatohirudin (variant 1) have been identified using two-dimensional 1H NMR experiments and site-directed mutagenesis. The analysis of pH titration curves measured with NMR enabled the determination of the pK values of all 13 carboxylates, and downfield shifts larger than 0.2 ppm arising from weak bonding interactions with carboxylates were observed for the amide protons of Gly 25, Ser 32, Glu 35, and Cys 39. For these backbone amide protons virtually identical titration parameters were observed in intact desulfatohirudin and the mutant, truncated hirudin(1-51), demonstrating that the hydrogen bond acceptors are located in the N-terminal polypeptide segment 1-51. The hydrogen bonds Gly 25 NH-Glu 43 delta COO-, Ser 32 NH-Glu 35 delta COO-, Glu 35 NH-Asp 33 gamma COO-, Glu 35 NH-Glu 35 delta COO-, and Cys 39 NH-Glu 17 delta COO- were identified by considering spatial proximity in the NMR solution structure of hirudin(1-51), and comparing the pK values for the amide protons and the carboxylates in desulfatohirudin and the mutants hirudin(E43Q), hirudin(E35Q), hirudin(D33N) and hirudin(E17A).(ABSTRACT TRUNCATED AT 250 WORDS)