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 alpha1, Ser-204 in alpha5, and Lys-220 and Asn-228 in alpha6) 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 alpha8 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 alpha1.
authors: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors: B O
authors: BO
authors: Krogh
authors: S
authors: S
authors: Shuman
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D014616:Vaccinia virus / Q000201:enzymology*
chemical_list: D015636:Magnesium Chloride; D014443:Tyrosine; D012965:Sodium Chloride; D012694:Serine; D010649:Phenylalanine; D001216:Asparagine; D004247:DNA; D004264:DNA Topoisomerases, Type I