1PZY

W314A-BETA1,4-GALACTOSYLTRANSFERASE-I COMPLEXED WITH ALPHA-LACTALBUMIN IN THE PRESENCE OF N-ACETYLGLUCOSAMINE, UDP AND MANGANESE

Summary for 1PZY

• Entry DOI: 10.2210/pdb1pzy/pdb
• Related: 1NKH 1NMM
• Descriptor: ALPHA-LACTALBUMIN, BETA-1,4-GALACTOSYLTRANSFERASE, CALCIUM ION, ... (7 entities in total)
• Functional Keywords: beta1, 4-galactosyltransferase-i tryptophan mutant, flexible loop conformation, protease digetion, substrate binding, catalytic mechanism, transferase activator-transferase complex, transferase activator/transferase
• Biological source: Mus musculus (house mouse); More
• Total number of polymer chains: 4
• Total formula weight: 94941.34

Authors

Ramasamy, V.,Ramakrishnan, B.,Boeggeman, E.,Qasba, P.K. (deposition date: 2003-07-14, release date: 2003-09-02, Last modification date: 2024-11-06)

Primary citation

Ramasamy, V.,Ramakrishnan, B.,Boeggeman, E.,Qasba, P.K.
The Role of Tryptophan 314 in the Conformational Changes of beta 1,4-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 (2.3 Å)