title: Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition.
abstract: Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp(203)/Trp(205)/Trp(206)) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan 'anchor' was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.
authors.affiliation: Laboratory of Biochemistry, Lake Erie College of Osteopathic Medicine, Bradenton, FL 34211, USA. glaco@lecom.edu
authors.forename: Gary S
authors.initials: GS
authors.lastname: Laco
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D018411:Spodoptera; D055672:Static Electricity; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
chemical_list: D014364:Tryptophan; D004247:DNA; D004264:DNA Topoisomerases, Type I