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Diagnostic cross-linking of paired cysteine pairs demonstrates homologous structures for two chemoreceptor domains with low sequence identity

¹ Department of Biochemistry, University of Missouri–Columbia, Columbia, Missouri 65211, USA
² Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA
³ Departments of Chemistry and Pharmacology and the Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37235-1822, USA

(RECEIVED August 25, 2005; FINAL REVISION September 29, 2005; ACCEPTED September 29, 2005)

Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand-recognition domains of approximately the same size, but little or no sequence identity. The only structure determined is for the periplasmic domain of chemoreceptor Tar from Salmonella and Escherichia coli. Do sequence-divergent but similarly sized chemoreceptor periplasmic domains have related structures? We addressed this issue for the periplasmic domain of chemoreceptor Trg_E from E. coli, which has a low level of sequence similarity to Tar, by combining homology modeling and diagnostic cross-linking between pairs of introduced cysteines. A homology model of the Trg_E domain was created using the homodimeric, four-helix bundle structure of the Tar_S domain from Salmonella. In this model, we chose four pairs of positions at which introduced cysteines would be sufficiently close to form disulfides across each of four different helical interfaces. For each pair we chose a second pair, in which one cysteine of the original pair was shifted by one position around the helix and thus would be less favorably placed for disulfide formation. We created genes coding for proteins containing four such pairs of cysteine pairs and investigated disulfide formation in vivo as well as functional consequences of the substitutions and disulfides between neighboring helices. Results of the experimental tests provided strong support for the accuracy of the model, indicating that the Trg_E periplasmic domain is very similar to the Tar_S domain. Diagnostic cross-linking of paired pairs of introduced cysteines could be applied generally as a stringent test of homology models.

Keywords: bacterial chemotaxis; transmembrane receptors; homology modeling; ligand-binding domains; cysteine cross-linking

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051802806.

Reprint requests to: Gerald L. Hazelbauer, Department of Biochemistry, 117 Schweitzer Hall, University of Missouri–Columbia, Columbia, MO 65211, USA; e-mail: hazelbauerg{at}missouri.edu; fax: (573) 882-5635.

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