3-DIMENSIONAL SOLUTION STRUCTURE OF HUMAN ANGIOGENIN DETERMINED BY H-1,N-15-NMR SPECTROSCOPY - CHARACTERIZATION OF HISTIDINE PROTONATION STATES AND PK(A) VALUES

Human angiogenin is a member of the pancreatic ribonuclease superfamil y that induces blood vessel formation. Its three-dimensional solution structure has been determined to high resolution by heteronuclear NMR spectroscopy, 30 structures were calculated, based on a total of 1441 assigned NOE correlations, 64 coupling constants and 50 hydrogen bonds , The backbone atomic rms difference from the mean coordinates is 0.06 7 +/- 0.012 nm and 0.13 nm from the previously determined crystal stru cture, The side-chain of Gln117 was found to obstruct the active site as observed in the crystal state. There was no evidence of an alternat ive open form of angiogenin, although two sets of chemical shifts were observed for some residues, mainly around the active site and in the C-terminal segment. The topology of the ribonucleolytic active site is described with a particular emphasis on the conformation and protonat ion of active-site His residues. The side-chain of His114 adopts two m ain conformations in solution. In contrast to pancreatic ribonuclease A, His13 was shown to be more basic than His114, with pK(a) values of 6.65 and 6.05 respectively. The His47 residue is located in an environ ment very resistant to protonation with a pk(a) lower than 4.