TRANSIENT HYDROGEN-BONDS IDENTIFIED ON THE SURFACE OF THE NMR SOLUTION STRUCTURE OF HIRUDIN

Recombinant desulfatohirudin retains largely the thrombin-inhibitory a ctivity of natural hirudin from Hirudo medicinalis and causes at most minimal immune response in humans. With regard to potential pharmaceut ical applications it is of interest to further investigate the structu ral basis of hirudin functions. In this paper transient hydrogen bonds between backbone amide protons and side-chain carboxylates on the pro tein surface of desulfatohirudin (variant 1) have been identified usin g two-dimensional H-1 NMR experiments and site-directed mutagenesis. T he analysis of pH titration curves measured with NMR enabled the deter mination of the pK values of all 13 carboxylates, and downfield shifts larger than 0.2 ppm arising from weak bonding interactions with carbo xylates were observed for the amide protons of Gly 25, Ser 32, Glu 35, and Cys 39. For these backbone amide protons virtually identical titr ation parameters were observed in intact desulfatohirudin and the muta nt, truncated hirudin(1-51), demonstrating that the hydrogen bond acce pters are located in the N-terminal polypeptide segment 1-51. The hydr ogen bonds Gly 25 NH-Glu 43 delta COO-, Ser 32 NH-Glu 35 delta COO-, G lu 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 val ues for the amide protons and the carboxylates in desulfatohirudin and the mutants hirudin(E43Q), hirudin(E35Q), hirudin(D33N) and hirudin(E 17A). Comparative structure calculations with and without distance con straints for these hydrogen bonds showed that although they are all co mpatible with the NMR solution structure, these hydrogen bonds are tra nsient dynamic features of the protein surface which, with the sole ex ception of Cys 39 NH-Glu 17 delta COO-, would not have been detected i n a conventional NMR structure determination. Of special interest is t he clear-cut information obtained on the fact that the lifetimes of th e dynamic ''bifurcated'' hydrogen-bonding interactions of the amide pr oton of Glu 35 are in the millisecond time range or shorter.