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Gas Phase Characterization of the Noncovalent Quaternary Structure of Cholera Toxin and the Cholera Toxin B Subunit Pentamer

Jonathan P. Williams ^*, Daniel C. Smith ^*, Brian N. Green , Brian D. Marsden , Keith R. Jennings ^*, Lynne M. Roberts ^* and James H. Scrivens ^*

^* Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom; Waters MS Technologies Centre, Micromass UK Ltd., Manchester M22 5PP, United Kingdom; and Structural Genomics Consortium, University of Oxford, Botnar Research Centre, Oxford OX3 7LD, United Kingdom

Correspondence: Address reprint requests to Jonathan P. Williams, Dept. of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK. Tel.: 44-2476-522157; Fax: 44-2476-523701; E-mail: j.p.williams{at}warwick.ac.uk.

Cholera toxin (CTx) is an AB₅ cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the noncovalent homopentameric complex of CTx B chain (CTx B₅) using electrospray ionization triple quadrupole mass spectrometry and tandem mass spectrometry and the analysis of the noncovalent hexameric holotoxin usingelectrospray ionization time-of-flight mass spectrometry over a range of pH values that correlate with those encountered by this toxin after cellular uptake. We show that noncovalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related Escherichia coli Shiga-like toxin B₅ pentamer (SLTx B), the CTx B₅ pentamer was stable at low pH, indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional -helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilization of CTx B relative to SLTx B.