Synchrotron radiation circular dichroism spectroscopy-defined structure of the C-terminal domain of NaChBac and its role in channel assembly.
Extramembranous domains play important roles in the structure and function of membrane proteins, contributing to protein stability, forming association domains, and binding ancillary subunits and ligands. However, these domains are generally flexible, making them difficult or unsuitable targets for obtaining high-resolution X-ray and NMR structural information. In this study we show that the highly sensitive method of synchrotron radiation circular dichroism (SRCD) spectroscopy can be used as a powerful tool to investigate the structure of the extramembranous C-terminal domain (CTD) of the prokaryotic voltage-gated sodium channel (Na(V)) from Bacillus halodurans, NaChBac. Sequence analyses predict its CTD will consist of an unordered region followed by an alpha-helix, which has a propensity to form a multimeric coiled-coil motif, and which could form an association domain in the homotetrameric NaChBac channel. By creating a number of shortened constructs we have shown experimentally that the CTD does indeed contain a stretch of approximately 20 alpha-helical residues preceded by a nonhelical region adjacent to the final transmembrane segment and that the efficiency of assembly of channels in the membrane progressively decreases as the CTD residues are removed. Analyses of the CTDs of 32 putative prokaryotic Na(V) sequences suggest that a CTD helical bundle is a structural feature conserved throughout the bacterial sodium channel family.
Powl
Andrew M
AM
Department of Crystallography, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom.
O'Reilly
Andrias O
AO
Miles
Andrew J
AJ
Wallace
B A
BA
D001407:Bacillus / Q000737:chemistry*; D002942:Circular Dichroism / Q000379:methods*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism; D017356:Synchrotrons
D062554:NAV1.5 Voltage-Gated Sodium Channel; D015222:Sodium Channels