Protein Science, Vol 6, Issue 7 1516-1528, Copyright © 1997 by Cold Spring Harbor Laboratory Press

ARTICLE

Structural studies of receptor binding by cholera toxin mutants

E. A. MERRITT, S. SARFATY, M. G. JOBLING, T. CHANG, R. K. HOLMES, T. R. HIRST and WGJ. HOL
Department of Biological Structure, University of Washington, Seattle, Washington 98195-7742

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33 -> Arg complexed with the G(M1) oligosaccharide, as well as the 2.2-A refinement of a Gly 33 -> Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33 -> Arg CTB mutant are occupied by bound G(M1) oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:G(M1) oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33 -> Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33 -> Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33 -> Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.
CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				W. Y. Chung, R. Carter, T. Hardy, M. Sack, T. R. Hirst, and R. F. L. James Inhibition of Escherichia coli Heat-labile Enterotoxin B Subunit Pentamer (EtxB5) Assembly in Vitro Using Monoclonal Antibodies J. Biol. Chem., December 22, 2006; 281(51): 39465 - 39470. [Abstract] [Full Text] [PDF]

				M. Nagae, N. Nishi, T. Murata, T. Usui, T. Nakamura, S. Wakatsuki, and R. Kato Crystal Structure of the Galectin-9 N-terminal Carbohydrate Recognition Domain from Mus musculus Reveals the Basic Mechanism of Carbohydrate Recognition J. Biol. Chem., November 24, 2006; 281(47): 35884 - 35893. [Abstract] [Full Text] [PDF]

				H. Attrill, A. Imamura, R. S. Sharma, M. Kiso, P. R. Crocker, and D. M. F. van Aalten Siglec-7 Undergoes a Major Conformational Change When Complexed with the {alpha}(2,8)-Disialylganglioside GT1b J. Biol. Chem., October 27, 2006; 281(43): 32774 - 32783. [Abstract] [Full Text] [PDF]

				I. Moustafa, H. Connaris, M. Taylor, V. Zaitsev, J. C. Wilson, M. J. Kiefel, M. von Itzstein, and G. Taylor Sialic Acid Recognition by Vibrio cholerae Neuraminidase J. Biol. Chem., September 24, 2004; 279(39): 40819 - 40826. [Abstract] [Full Text] [PDF]

				A. L. Horstman, S. J. Bauman, and M. J. Kuehn Lipopolysaccharide 3-Deoxy-D-manno-octulosonic Acid (Kdo) Core Determines Bacterial Association of Secreted Toxins J. Biol. Chem., February 27, 2004; 279(9): 8070 - 8075. [Abstract] [Full Text] [PDF]

				L. de Haan, A. R. Hearn, A. J. Rivett, and T. R. Hirst Enhanced Delivery of Exogenous Peptides into the Class I Antigen Processing and Presentation Pathway Infect. Immun., June 1, 2002; 70(6): 3249 - 3258. [Abstract] [Full Text] [PDF]

				M. G. Jobling and R. K. Holmes Mutational Analysis of Ganglioside GM1-Binding Ability, Pentamer Formation, and Epitopes of Cholera Toxin B (CTB) Subunits and CTB/Heat-Labile Enterotoxin B Subunit Chimeras Infect. Immun., March 1, 2002; 70(3): 1260 - 1271. [Abstract] [Full Text] [PDF]

				M. Soriani, N. A. Williams, and T. R. Hirst Escherichia coli Enterotoxin B Subunit Triggers Apoptosis of CD8+ T Cells by Activating Transcription Factor c-Myc Infect. Immun., August 1, 2001; 69(8): 4923 - 4930. [Abstract] [Full Text] [PDF]

				T. O. Nashar, Z. E. Betteridge, and R. N. Mitchell Evidence for a role of ganglioside GM1 in antigen presentation: binding enhances presentation of Escherichia coli enterotoxin B subunit (EtxB) to CD4+ T cells Int. Immunol., April 1, 2001; 13(4): 541 - 551. [Abstract] [Full Text] [PDF]

				A. Imberty, C. Gautier, J. Lescar, S. Perez, L. Wyns, and R. Loris An Unusual Carbohydrate Binding Site Revealed by the Structures of Two Maackia amurensis Lectins Complexed with Sialic Acid-containing Oligosaccharides J. Biol. Chem., June 2, 2000; 275(23): 17541 - 17548. [Abstract] [Full Text] [PDF]