Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis.
The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.
Department of Physics, UNESP/IBILCE, S. J. do Rio Preto-SP, Brazil.
R K
RK
Arni
L
L
Watanabe
R J
RJ
Ward
R J
RJ
Kreitman
K
K
Kumar
F G
FG
Walz
D001236:Aspergillus oryzae / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000737:chemistry; D015226:Dinucleoside Phosphates / Q000737:chemistry*; D006157:Guanosine Monophosphate / Q000737:chemistry; D008024:Ligands; D046911:Macromolecular Substances; D008958:Models, Molecular; D063065:Organophosphonates / Q000737:chemistry*; D006163:Ribonuclease T1 / Q000737:chemistry*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013816:Thermodynamics
D015226:Dinucleoside Phosphates; D008024:Ligands; D046911:Macromolecular Substances; D063065:Organophosphonates; C117628:guanylyl(3'-6')-6'-deoxyhomouridine; C018310:guanylyl-(3'-5')-uridine; C068152:3'-guanylic acid; D006157:Guanosine Monophosphate; D006163:Ribonuclease T1; D003857:Deoxyuridine