Investigating the Structural Dynamics of 伪-1,4-Galactosyltransferase C from Neisseria meningitidis by Nuclear Magnetic Resonance Spectroscopy
详细信息 查看全文
- 作者：Patrick H. W. Chan ; Adrienne H. Cheung ; Mark Okon ; Hong-Ming Chen ; Stephen G. Withers ; Lawrence P. McIntosh
- 刊名：Biochemistry
- 出版年：2013
- 出版时间：January 15, 2013
- 年：2013
- 卷：52
- 期：2
- 页码：320-332
- 全文大小：797K
- 年卷期：v.52,no.2(January 15, 2013)
- ISSN：1520-4995
文摘
Neisseria meningitidis 伪-1,4-galactosyltransferase C (LgtC) is responsible for the transfer of 伪-galactose from donor UDP-galactose to the lipooligosaccharide terminal acceptor lactose. Crystal structures of its substrate analogue complexes have provided key insights into the galactosyl transfer mechanism, including a hypothesized need for active site mobility. Accordingly, we have used nuclear magnetic resonance spectroscopy to probe the structural dynamics of LgtC in its apo form and with bound substrate analogues. More than the expected number of signals were observed in the methyl-TROSY spectra of apo LgtC, indicating that the protein adopts multiple conformational states. Magnetization transfer experiments showed that the predominant states, termed 鈥渁鈥?and 鈥渂鈥? are in equilibrium on a time scale of seconds. Their relative populations change with temperature and mutations, and only the 鈥渂鈥?state is competent for substrate binding. For both states, relaxation dispersion studies also revealed substantial millisecond time scale motions of isoleucine side chains within and distal to the active site. Although altered, these motions were still detected in LgtC with a noncovalently bound donor analogue. A mutant, LgtC-Q189E, which forms an unexpected glycosyl鈥揺nzyme intermediate via a residue (Asp190) distal from its active site, was also investigated. Apo LgtC-Q189E did not show any enhanced motions that might account for the dramatic structural change required for the galactosylation of Asp190, yet formation of a trapped glycosyl鈥揺nzyme intermediate substantially reduced its millisecond time scale conformational mobility. Although further studies are required to link the detected motions of LgtC with its enzymatic mechanism, this work clearly demonstrates the complex structural dynamics of a model glycosyltransferase.