The Receptor Binding Site for the Methyltransferase of Bacterial Chemotaxis Is Distinct from the Sites of Methylation

Abstract

The principal locus for binding interactions between the aspartate and serine receptors of escherichia coli and the methyltransferase was found to be in the last five amino acids of the receptor. The thermodynamic parameters of transferase-receptor interactions were determined by isothermal titration calorimetry. the serine receptor and three C-terminal fragments (C-fragments) of the aspartate receptor consisting of ether the last 297, 88, or 38 amino acids gave comparable values for binding (n=1, deltaH approximately 13 kcal/mol, and Ka approximately 4 x 10(5)M-1). Truncating either 16 or 36 amino acids form the C-terminus eliminated observable interactions. Finally the pentapeptide Asn-Trp-Glu-Thr-Phe, which corresponds to the last five amino acids of the receptor and is strictly conserved among E. coli serine amd aspartate receptors and the Salmonella typhimurium aspartate receptor, was found to have all the binding activity of the full-length receptor and the C-fragments. An in vitro methylation assay was used to obtain evidence for the physiological significance of this interaction in which excess peptide was able to completely block receptor methylation. The location of the binding site far from the methylation sites in the primary structure of the receptor suggests that the principle role of this interaction may be to hold the transferase in close proximity to all the methylation sites. Intersubunit methylation implication is proposed as plausible consequence of this "controlled proximity" mechanism since the ribose-galactose and dipeptide receptors lack the transferase binding sequence, and appear unable to bind transferase. Intersubunit methylation implies that transferase bound to eother the serine or aspartate receptor subunit may catalyze methylation of receptor subunits in a neighboring dimer, including those that have ligand specificity.

Bacterial chemoreceptors: high-performance signaling in networked arrays

“…Conformational signals in the kinase control module are transmitted by rearrangements of helix-helix contacts at the subunit interface (see text for details). The carboxyl terminus of some receptors carries a conserved pentapeptide sequence (NWETF or NWESF in the single-letter amino acid code) that interacts with the two enzymes of adaptational modification (CheR and CheB) and enhances the rates of the reactions they catalyze [69,70].…”

“…Conformational signals in the kinase control module are transmitted by rearrangements of helix-helix contacts at the subunit interface (see text for details). The carboxyl terminus of some receptors carries a conserved pentapeptide sequence (NWETF or NWESF in the single-letter amino acid code) that interacts with the two enzymes of adaptational modification (CheR and CheB) and enhances the rates of the reactions they catalyze [69,70].The sequence and 3D structure of kinase control modules are conserved across the diversity of bacteria and archaea [7]. Sequence conservation is more subtle among HAMP modules [42,45] but 3D structure is probably shared for the approximately two-thirds of all chemoreceptors that contain the module [43].…”

Structure of a conserved receptor domain that regulates kinase activity: the cytoplasmic domain of bacterial taxis receptors

“…Finally, recent biochemical studies have revealed evidence of limited cooperative interactions between chemotaxis receptors during kinase regulation, and chemoreceptors are known to cluster at the poles of E. coli in living cells [53,54,55••,65••,66••]. Such clustering is critical to the adaptation of certain receptors that lack the C-terminal docking site for adaptation enzymes that is present on other receptors, including the aspartate and serine receptors [67][68][69]. It is not yet clear, however, whether clustering plays an important role in transmembrane kinase regulation.…”

Interaction of CheY with the C-Terminal Peptide of CheZ

“…The use of Phe as an anchor is not unique to CheY-CheZ C recognition in bacterial chemotaxis. The pentapeptide motif (NWETF) present at the C termini of high-copy-number E. coli methylaccepting proteins (Tar and Tsr) also uses the C-terminal Phe to tether methyltransferase CheR for methylation (50).…”