Structure of the Toxic Domain of the Escherichia coli Heat-Stable Enterotoxin ST I

Jean Gariépy, Andrew Lane, Felix Frayman, David Wilbur, Wolfgang Robien, Gary K. Schoolnik, Oleg Jardetzky

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38 Scopus citations

Abstract

Active fragments of the heat-stable enterotoxin ST I of Escherichia coli were chemically synthesized with the sequence Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-(Tyr) and studied by proton (1H NMR) and carbon-13 (13C NMR) nuclear magnetic resonance spectroscopy as a function of pH and temperature. All of the nonexchangeable protons in the 1H NMR spectrum were assigned. Although all amide protons were present at temperatures below 25 °C and pH values below 6, some of the resonances are broad and could not be assigned. The temperature dependence of these broad resonances indicates a change in conformation that is localized in the N-terminus. Other amide protons disappear at higher temperatures owing to chemical exchange with the solvent. Sufficient resonance assignments can be made at high and low temperatures to permit structural conclusions to be made. The chemical shifts of the a-carbon protons indicate the presence of substantial structure, which was further defined with the observed pattern of nuclear Overhauser enhancements (NOEs), coupling constants, and exchange rates. The NMR data identify a turn from Ala-14 to Cys-18. A second likely turn is centered around the proline residue. An interresidue NOE between the a-carbon protons of Asn-12 and Gly-17 indicates that the molecule folds back on itself. The NMR information is sufficient to define the structure of the C-terminal region of ST I. Manual model building then indicated that one arrangement of the three disulfides is particularly compatible with the NMR data and van der Waals constraints. A model incorporating the disulfide arrangement proposed by Houghten and his co-workers [Houghten, R. A., Ostresh, J. M., & Klipstein, F. A. (1984) Eur. J. Biochem. 145, 157–162] and the NMR constraints was derived with the programs PROTO [Frayman, F. (1985) Ph.D. Thesis, Northwestern University] and Noemot [Lane, A. N., Lefevre, J.-F., & Jardetsky, O. (1986) Biochim. Biophys. Acta 867, 45–56].

Original languageFrench
Pages (from-to)7854-7866
Number of pages13
JournalBiochemistry
Volume25
Issue number24
DOIs
StatePublished - Dec 1986

ASJC Scopus subject areas

  • Biochemistry

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