2o1p

From Proteopedia

Revision as of 13:03, 23 January 2008

Structure of yeast Poly(A) Polymerase in a somewhat closed state

Overview

Polyadenylate polymerase (PAP) catalyzes the synthesis of poly(A) tails on, the 3'-end of pre-mRNA. PAP is composed of three domains: an N-terminal, nucleotide-binding domain (homologous to the palm domain of DNA and RNA, polymerases), a middle domain (containing other conserved, catalytically, important residues), and a unique C-terminal domain (involved in, protein-protein interactions required for 3'-end formation). Previous, X-ray crystallographic studies have shown that the domains are arranged in, a V-shape such that they form a central cleft with the active site located, at the base of the cleft at the interface between the N-terminal and, middle domains. In the previous studies, the nucleotides were bound, directly to the N-terminal domain and exhibited a conspicuous lack of, adenine-specific interactions that would constitute nucleotide, recognition. Furthermore, it was postulated that base-specific contacts, with residues in the middle domain could occur either as a result of a, change in the conformation of the nucleotide or domain movement. To, address these issues and to better characterize the structural basis of, substrate recognition and catalysis, we report two new crystal structures, of yeast PAP. A comparison of these structures reveals that the N-terminal, and C-terminal domains of PAP move independently as rigid bodies along two, well defined axes of rotation. Modeling of the nucleotide into the most, closed state allows us to deduce specific nucleotide interactions, involving residues in the middle domain (K215, Y224 and N226) that are, proposed to be involved in substrate binding and specificity. To further, investigate the nature of PAP domain flexibility, 2-aminopurine labeled, molecular probes were employed in steady state fluorescence and acrylamide, quenching experiments. The results suggest that the closed domain, conformation is stabilized upon recognition of the correct subtrate, MgATP, in an enzyme-substrate ternary complex. The implications of these, results on the enzyme mechanism of PAP and the possible role for domain, motion in an induced fit mechanism are discussed.

About this Structure

2O1P is a Single protein structure of sequence from Saccharomyces cerevisiae. Active as Polynucleotide adenylyltransferase, with EC number 2.7.7.19 Full crystallographic information is available from OCA.

Reference

X-ray crystallographic and steady state fluorescence characterization of the protein dynamics of yeast polyadenylate polymerase., Balbo PB, Toth J, Bohm A, J Mol Biol. 2007 Mar 9;366(5):1401-15. Epub 2006 Dec 19. PMID:17223131

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