Identification of functionally important helical faces in transmembrane segments by scanning mutagenesis.

Abstract

We applied mutational analysis to a protein domain that functions in neither catalysis nor binding but, rather, in transmembrane signaling. The domain is part of chemoreceptor Trg from Escherichia coli. It contains four transmembrane segments, two from each subunit of the homodimer. We used cysteine scanning to investigate the functional importance of each of54 residues in the two transmembrane segments. Cysteines at some positions resulted in subtle but significant reductions in tactic response. Those positions defined a specific helical face on each segment, implying that the segments function as helices. The functionally important faces corresponded to structural, helical packing faces identified independently by biochemical studies. All functionally impaired receptors exhibited altered signaling properties, either reduced signaling upon stimulation or induced signaling in the absence of stimulation. The distribution of substitutions creating these two phenotypes implied that conformational signaling involves movement between the two transmembrane helices within a subunit and that signaling is optimal when stable interactions are maintained across the interface between subunits.Four transmembrane receptors mediate chemotaxis by Escherichia coli (1-3). Recognition of positive stimuli generates counterclockwise (CCW) rotation of bacterial flagella, which in turn results in coordinated swimming. Recognition of negative stimuli generates clockwise (CW) rotation and uncoordinated tumbles. Chemoreceptor Trg recognizes sugaroccupied galactose-and ribose-binding proteins and thereby mediates chemotactic response to those sugars. Chemoreceptors are homodimers (4-6). Receptor monomers, "60 kDa, have a transmembrane domain consisting of two hydrophobic segments, a periplasmic, ligand-binding domain and a highly conserved cytoplasmic domain that mediates intracellular signaling by controlling a noncovalently associated protein kinase and that mediates adaptation through dynamic methylation and demethylation of several glutamyl residues. An understanding of transmembrane signaling in chemoreceptors of E. coli should be relevant not only to methyl-accepting taxis proteins in other bacteria (7) but also to the many related environmental sensor proteins in prokaryotes (1, 3) and eukaryotes (8).The monomer-dimer equilibrium for chemoreceptors strongly favors dimers and does not appear to shift in the course of the sensory cycle (4), implying that transmembrane signaling involves conformational changes within the dimer. It is not known what features of the domain are important for or involved in transmembrane signaling. We approached these issues with mutational analysis by "cysteine scanning." The approach has many of the features and advantages of "alaninescanning mutagenesis" (9). Cysteine is an attractive choice for scanning mutagenesis of transmembrane segments because itsThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisemen...

Transmembrane signaling in bacterial chemoreceptors

“…Because mutational substitutions usually perturb interactions, the clustering of mutations that mimic the effects of attractant binding along the TM1-TM2 interface implies that this interface is perturbed by conformational signaling. Similarly, clustering along the TM1-TM1′ interface of substitutions that reduce attractant-induced methylation implies that signaling is optimal when native subunit interactions are maintained 25 .…”

“…Other studies examined the functional effects of point mutations at specific helix interfaces 25 . Cys substitutions at any one of the positions in TM1 and TM2 of chemoreceptor …”

“…Other studies examined the functional effects of point mutations at specific helix interfaces 25 . Cys substitutions at any one of the positions in TM1 and TM2 of chemoreceptor Trg did not eliminate receptor function, but ~40% had effects on receptor signaling in vivo, as assessed by altered methylation at the receptor adaptation sites.…”

“…These studies revealed that the aspartate-induced signal is generated at a specific half-site and is transmitted through only one subunit to the cytoplasmic domain. The signaling half-site includes residues at the periplasmic end of the α4/TM2 helix that interact with the amino group of aspartate 2,6 , thereby providing a simple mechanism by which attractant binding could displace the α4/TM2 helix and perturb the α1/TM1-α4/TM2 interface in the subunit that carries the transmembrane signal 2 .Other studies examined the functional effects of point mutations at specific helix interfaces 25 . Cys substitutions at any one of the positions in TM1 and TM2 of chemoreceptor …”

Energy sensors for aerotaxis in Escherichia coli : Something old, something new

“…But the chemoreceptors have periplasmic sensor domains and cytoplasmic signaling domains, whereas both domains of Aer are predicted to be located in the cytoplasm. In the chemoreceptors, the importance of the transmembrane regions in transmitting signals between the periplasmic ligand-binding domains and the cytoplasmic signaling domains has been clearly established (18)(19)(20). The topological arrangement of Aer raises some interesting questions.…”