1PZT

CRYSTAL STRUCTURE OF W314A-BETA-1,4-GALACTOSYLTRANSFERASE (B4GAL-T1) CATALYTIC DOMAIN WITHOUT SUBSTRATE

Summary for 1PZT

• Entry DOI: 10.2210/pdb1pzt/pdb
• Related: 1FGX 1O0R
• Descriptor: Beta-1,4-galactosyltransferase 1, SULFATE ION (3 entities in total)
• Functional Keywords: beta1, 4-galactosyltransferase-i tryptophan mutant, flexible loop conformation, protease digestion, substrate binding, catalytic mechanism, transferase
• Biological source: Bos taurus (cattle)
• Cellular location: Isoform Long: Golgi apparatus, Golgi stack membrane; Single-pass type II membrane protein. Isoform Short: Golgi apparatus, Golgi stack membrane; Single-pass type II membrane protein. Processed beta-1,4-galactosyltransferase 1: Secreted: P08037
• Total number of polymer chains: 1
• Total formula weight: 32830.51

Authors

Ramasamy, V.,Ramakrishnan, B.,Boeggeman, E.,Qasba, P.K. (deposition date: 2003-07-14, release date: 2003-09-16, Last modification date: 2024-10-30)

Primary citation

Ramasamy, V.,Ramakrishnan, B.,Boeggeman, E.,Qasba, P.K.
The role of tryptophan 314 in the conformational changes of beta1,4-galactosyltransferase-I
J.Mol.Biol., 331:1065-1076, 2003

PubMed 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.

PubMed: 12927542
DOI: 10.1016/S0022-2836(03)00790-3

Experimental method

X-RAY DIFFRACTION (1.92 Å)