3-DIMENSIONAL STRUCTURE OF THE COMPLEXES OF RIBONUCLEASE-A WITH 2',5'-CPA AND 3',5'-D(CPA) IN AQUEOUS-SOLUTION, AS OBTAINED BY NMR AND RESTRAINED MOLECULAR-DYNAMICS

The three-dimensional structure of the complexes of ribonuclease A wit h cytidyl-2',5'-adenosine (2',5'-CpA) and deoxycytidyl-3',5'-deoxyaden osine [3',5'-d(CpA)] in aqueous solution has been determined by H-1 NM R methods in combination with restrained molecular dynamics calculatio ns. Twenty-three intermolecular NOE crosscorrelations for the 3',5'-d( CpA) complex and 19 for the 2',5'-CpA, together with about 1,000 intra molecular NOEs assigned for each complex, were translated into distanc e constraints and used in the calculation. No significant changes in t he global structure of the enzyme occur upon complex formation. The si de chains of His 12, Thr 45, His 119, and the amide backbone group of Phe 120 are involved directly in the binding of the ligands at the act ive site. The conformation of the two bases is anti in the two complex es, but differs from the crystal structure in the conformation of the two sugar rings in 3',5'-d(CpA), shown to be in the S-type region, as deduced from an analysis of couplings between the ribose protons. His 119 is found in the two complexes in only one conformation, correspond ing to position A in the free protein. Side chains of Asn 67, Gln 69, Asn 71, and Glu 111 form transient hydrogen bonds with the adenine bas e, showing the existence of a pronounced flexibility of these enzyme s ide chains at the binding site of the downstream adenine. All other ge neral features on the structures coincide clearly with those observed in the crystal state.