1B2M

THREE-DIMENSIONAL STRUCTURE OF RIBONULCEASE T1 COMPLEXED WITH AN ISOSTERIC PHOSPHONATE ANALOGUE OF GPU: ALTERNATE SUBSTRATE BINDING MODES AND CATALYSIS.

Summary for 1B2M

• Entry DOI: 10.2210/pdb1b2m/pdb
• Descriptor: 5'-R(*GP*(U34))-3', RIBONUCLEASE T1 (3 entities in total)
• Functional Keywords: hydrolase, endoribonuclease, hydrolase/rna, hydrolase-rna complex
• Biological source: Aspergillus oryzae
• Total number of polymer chains: 5
• Total formula weight: 24048.61

Authors

Arni, R.K.,Watanabe, L.,Ward, R.J.,Kreitman, R.J.,Kumar, K.,Walz Jr., F.G. (deposition date: 1998-11-27, release date: 1999-03-25, Last modification date: 2024-10-30)

Primary citation

Arni, R.K.,Watanabe, L.,Ward, R.J.,Kreitman, R.J.,Kumar, K.,Walz Jr., F.G.
Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis.
Biochemistry, 38:2452-2461, 1999

PubMed Abstract: 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.

PubMed: 10029539
DOI: 10.1021/bi982612q

Experimental method

X-RAY DIFFRACTION (2 Å)