title: The role of tryptophan 314 in the conformational changes of beta1,4-galactosyltransferase-I.
abstract: beta1,4-Galactosyltransferase-I (beta4Gal-T1) undergoes critical conformational changes upon substrate binding from an open conformation (conf-I) to the closed conformation (conf-II). This change involves two flexible loops: the small (residues 313-316) and the long loop (residues 345-365). Upon substrate binding, Trp314 in the small flexible loop moves towards the catalytic pocket and interacts with the donor and the acceptor substrates. For a better understanding of the role played by Trp314 in the conformational changes of beta4Gal-T1, we mutated it to Ala and carried out substrate-binding, proteolytic and crystallographic studies. The W314A mutation reduces the enzymatic activity, binding to substrates and to the modifier protein, alpha-lactalbumin (LA), by over 99%. The limited proteolysis with Glu-C or Lys-C proteases shows differences in the rate of cleavage of the long loop of the wild-type and mutant W314A, indicating conformational differences in the region between the two proteins. Without substrate, the mutant crystallizes in a conformation (conf-I') (1.9A resolution crystal structure), that is not identical with, but close to an open conformation (conf-I), whereas its complex with the substrates and alpha-lactalbumin, crystallizes in a conformation (2.3A resolution crystal structure) that is identical with the closed conformation (conf-II). This study shows the crucial role Trp314 plays in the conformational state of the long loop, in the binding of substrates and in the catalytic mechanism of the enzyme.
authors.affiliation: Structural Glycobiology Section, LECB, CCR, NCI-Frederick, Building 469, Room 221, 21702, Frederick, MD, USA.
authors.forename: Velavan
authors.initials: V
authors.lastname: Ramasamy
authors.forename: Boopathy
authors.initials: B
authors.lastname: Ramakrishnan
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Boeggeman
authors.forename: Pradman K
authors.initials: PK
authors.lastname: Qasba
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002846:Chromatography, Affinity; D018360:Crystallography, X-Ray; D005700:Galactosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D007768:Lactalbumin / Q000378:metabolism; D046911:Macromolecular Substances; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry*; D014530:Uridine Diphosphate / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D011994:Recombinant Proteins; D008345:Manganese; D014530:Uridine Diphosphate; D014364:Tryptophan; D007768:Lactalbumin; D005700:Galactosyltransferases; C119701:beta-1,4-galactosyltransferase I; D000117:Acetylglucosamine