Unusual conformational pathways of mismatched dNTP incorporation by DNA Polβ

Abstract

Poster Sessions 4 4 metabolism. AT reverbly catalyzes the transamination reaction in which the alpha-amino acid of amino acid 1 is transferred to the 2oxo acid of amino acid 2 to produce the 2-oxo-acid of amino acid 1 and amino acid 2. Thus, AT recognizes two different kind of amino acid 1 and 2 (Dual substrate recognition). psi-BLAST search showed that TTHA0173 is a dual functional AT which works at serine and alanine metabolic pathway. TTHA0173 is serine:pyruvate AT and at the same time, alanine:glyoxylate AT. Three-dimensional structure of unliganded TTH0173 was determined by MAD method using Se-Met tSPAT at 1.45 Å resolution. TTH0173 is a homodimer and the polypeptide chain is folded into the small and large domains. The active-site pocket is formed at the subunit interface and the domain interface with the coenzyme PLP forming a Schiff base with the catalytic lysine residue. Program DALI was used to search PDB for proteins possessing structures similar to that of TTHA0173, therefore the highest Z-score was calculated to be 47.4 with sequence identity of 33% for human serine pyruvate-alanine:glyoxylate AT. The kcat and Kd values of tSPAT for various amino acid and their 2-oxo acid measured. Unexpectedly, glutamate and its 2-oxo acid which have bulky side chain, were good substrate. In order to elucidate the substrate recognition mechanism, we have determined the crystal structure of complex with 2-methylalanine and 2-methyl glutamine at 1.55 and 1.50 Å resolution, respectively.Understanding how DNA polymerases control fidelity requires elucidation of the mechanisms of matched and mismatched dNTP incorporations. Little is known about the latter because mismatched complexes do not crystallize readily. In this report, we employed small-angle X-ray scattering (SAXS), X-ray crystallography and structural modeling to probe the conformations of different intermediate states of mammalian DNA polymerase ß (Pol ß) in its wild-type and an error-prone variant, I260Q. Our structural results indicate that the mismatched ternary complex lies in-between the open and the closed forms, but more closely resembles the open form for WT and the closed form for I260Q. On the basis of molecular modeling, this over-stabilization of mismatched ternary complex of I260Q is likely caused by formation of a hydrogen bonding network between the side chains of Gln260, Tyr296, Glu295 and Arg258, freeing up Asp192 to coordinate MgdNTP. These results argue against recent reports suggesting that mismatched dNTP incorporations follow a conformational path distinctly different from that of matched dNTP incorporation, or that its conformational closing is a major contributor to fidelity.