The structures of rnase a complexed with 3′‐CMP and d(CpA): Active site conformation and conserved water molecules

Abstract

The interactions of RNase A with cytidine 3'-monophosphate (3"CMP) and deoxycytidyl-3',5'-deoxyadenosine (d(CpA)) were analyzed by X-ray crystallography. The 3'-CMP complex and the native structure were determined from trigonal crystals, and the d(CpA) complex from monoclinic crystals. The differences between the overall structures are concentrated in loop regions and are relatively small. The protein-inhibitor contacts are interpreted in terms of the catalytic mechanism. The general base His 12 interacts with the 2' oxygen, as does the electrostatic catalyst Lys 41. The general acid His 119 has 2 conformations (A and B) in the native structure and is found in, respectively, the A and the B conformation in the d(CpA) and the 3'-CMP complex. From the present structures and from a comparison with RNase T1, we propose that His 119 is active in the A conformation. The structure of the d(CpA) complex permits a detailed analysis of the downstream binding site, which includes His 119 and Asn 71. The comparison of the present RNase A structures with an inhibitor complex of RNase T1 shows that there are important similarities in the active sites of these 2 enzymes, despite the absence of any sequence homology. The water molecules were analyzed in order to identify conserved water sites. Seventeen water sites were found to be conserved in RNase A structures from 5 different space groups. It is proposed that 7 of those water molecules play a role in the binding of the N-terminal helix to the rest of the protein and in the stabilization of the active site.

The structure of the endoribonuclease XendoU: From small nucleolar RNA processing to severe acute respiratory syndrome coronavirus replication

“…Interestingly, we notice that this site is reminiscent of the active site of both (i) RNaseT1 (15), where the phosphate of 3Ј GMP is held in place by H40, H92, Y38, and R77; and (ii) bovine RNaseA (16), where the phosphate of 3Ј CMP is surrounded by H119, H12, Q11, and K41. It seems that all these RNA-processing enzymes share some common residues in the active site (namely two His and one basic, either Arg or Lys).…”

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

“…In the wild-type trigonal structure, N ε2 of Gln11 forms a 2.6 Å hydrogen bond with the conserved active-site water and a 3.0 Å hydrogen bond with a sulfate oxygen bound in the active site (49). The conserved active-site water is displaced by C 1 of an acetate ion in the D121A variant.…”

“…It has also been argued that His119 is in position A during catalysis of transphosphorylation and in position B during catalysis of hydrolysis (69). In position A, N ε2 of His119 forms a hydrogen bond with O δ1 of Asp121, which directs N δ1 of His119 toward the active site where it can form a hydrogen bond with a ligand (49). In the absence of a ligand, N δ1 of His119 forms no apparent hydrogen bonds to water molecules (48).…”

Molecular mechanics calculations on rous sarcoma virus protease with peptide substrates

“…The minimized structure for the RSV protease complex with unsubstituted substrate was compared to the starting crystal structure with modeled flaps for the atoms of the protease dimer. The protease-substrate model and starting structure had an RMS deviation of 0.44 A on C a atoms, 0.67 A for backbone atoms, 0.78 8, for side-chain atoms, and These values were well within the range of 0.16-0.79 8, for RMS differences observed between main-chain atoms in different crystal forms of the same protein (Wlodawer et al, 1987;Zegers et al, 1994) and close to the mean of 0.40 8, observed for C a atoms (mores et al, 1993). The differences were spread throughout the structure, except for RMS differences of more than 1 A for residues 64-66,70,71,64', and 65' in the flaps.…”