Vaccinia topoisomerase mutants illuminate conformational changes during closure of the protein clamp and assembly of a functional active site Journal Article
| Authors: | Krogh, B. O.; Shuman, S. |
| Article Title: | Vaccinia topoisomerase mutants illuminate conformational changes during closure of the protein clamp and assembly of a functional active site |
| Abstract: | We present a mutational analysis of vaccinia topoisomerase that highlights the contributions of five residues in the catalytic domain (Phe-88 and Phe-101 in helix α1, Ser-204 in α5, and Lys-220 and Asn-228 in α6) to the DNA binding and transesterification steps. When augmented by structural information from exemplary type IB topoisomerases and tyrosine recombinases in different functional states, the results suggest how closure of the protein clamp around duplex DNA and assembly of a functional active site might be orchestrated by internal conformational changes in the catalytic domain. Lys-220 is a constituent of the active site, and a positive charge at this position is required for optimal DNA cleavage. Ser-204 and Asn-228 appear not to be directly involved in reaction chemistry at the scissile phosphodiester. We propose that (i) Asn-228 recruits the Tyr-274 nucleophile to the active site by forming a hydrogen bond to the main chain of the tyrosine-containing α8 helix and that (ii) contacts between Ser-204 and the DNA backbone upstream of the cleavage site trigger a separate conformational change required for active site assembly. Mutations of Phe-88 and Phe-101 affect DNA binding, most likely at the clamp closure step, which we posit to entail a distortion of helix α1. |
| Keywords: | genetics; mutation; dose response; nonhuman; molecular genetics; protein conformation; protein domain; proteins; metabolism; serine; protein assembly; protein binding; enzymology; dose-response relationship, drug; tyrosine; time; time factors; chemistry; dna; double stranded dna; amino acid sequence; molecular sequence data; sequence homology, amino acid; dna viruses; nucleotide sequence; vaccinia virus; base sequence; binding site; hydrogen bond; mutagenesis, site-directed; binding sites; conformational transition; catalysis; sequence homology; dna binding; site directed mutagenesis; biochemistry; catalytic domain; sodium chloride; asparagine; transesterification; phenylalanine; dna cleavage; dna topoisomerase; dna topoisomerases, type i; enzyme active site; catalytic domains; vaccinia; hydrogen bonds; conformations; alpha helix; priority journal; article; magnesium chloride |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 276 |
| Issue: | 39 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2001-09-28 |
| Start Page: | 36091 |
| End Page: | 36099 |
| Language: | English |
| DOI: | 10.1074/jbc.M102609200 |
| PUBMED: | 11441004 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 21 May 2015 -- Source: Scopus |
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