Mutational analysis of the connector segment in the HAMP domain of Tsr, the Escherichia coli serine chemoreceptor

Abstract

HAMP domains are approximately 50-residue motifs, found in many bacterial signaling proteins, that consist of two amphiphilic helices joined by a nonhelical connector segment. The HAMP domain of Tsr, the serine chemoreceptor of Escherichia coli, receives transmembrane input signals from the periplasmic serine binding domain and in turn modulates output signals from the Tsr kinase control domain to elicit chemotactic responses. We created random amino acid replacements at each of the 14 connector residues of Tsr-HAMP to identify those that are critical for Tsr function. In all, we surveyed 179 connector missense mutants and identified three critical residues (G235, L237, and I241) at which most replacements destroyed Tsr function and another important residue (G245) at which most replacements impaired Tsr function. The region surrounding G245 tolerated 1-residue deletions and insertions of up to 10 glycines, suggesting a role as a relatively nonspecific, flexible linker. The critical connector residues are consistent with a structural model of the Tsr-HAMP domain based on the solution structure of an isolated thermophile HAMP domain (M. Hulko, F. Berndt, M. Gruber, J. U. Linder, V. Truffault, A. Schultz, J. Martin, J. E. Schultz, A. N. Lupas, and M. Coles, Cell 126:929-940, 2006) in which G235 defines a critical turn at the C terminus of the first helix and L237 and I241 pack against the helices, perhaps to stabilize alternative HAMP signaling conformations. Most I241 lesions locked Tsr signal output in the kinase-on mode, implying that this residue is responsible mainly for stabilizing the kinase-off signaling state. In contrast, lesions at L237 resulted in a variety of aberrant output patterns, suggesting a role in toggling output between signaling states.

FIG. 1.

(A) Functional architecture of Tsr and other MCP molecules. Native Tsr is a homodimer; each subunit is 551 residues in length. Most of the secondary structure elements are alpha-helices (shown as thickened cylinders). Structural elements of the TM sensing and kinase control domain are shown in their native arrangement; those of the HAMP domain are not. The kinase control domain is organized as a four-helix bundle of coiled coils. Several of the adaptation (methylation) sites are initially synthesized as glutamate residues (white circles); others are synthesized as glutamine residues (black circles) and subsequently converted to glutamates by an irreversible CheB-mediated deamidation. The C terminus of each subunit ends in a pentapeptide (NWETF) to which the adaptation enzymes CheR and CheB can bind. This tethering site is joined to the kinase control domain through a ∼30-residue segment that is thought to serve as a flexible linker. (B) Structure of the Tsr-HAMP domain modeled on the Af1503-HAMP coordinates (24). HAMP connects to the TM helices at the top of the structure; its connection to the kinase control helices is at the bottom of the structure. Note that each connector segment interacts with the helices from the same subunit (most easily seen in the lower view). This shading convention (light gray, AS1; dark gray, AS2; black and white, CTR) is maintained in subsequent figures (Fig. 3, 6, and 7).

FIG. 2.

Representative chemotaxis phenotypes of Tsr-HAMP connector mutants. Tryptone soft agar plates were toothpick inoculated from fresh transformant colonies of UU1250 cells carrying pRR53 (wild-type Tsr), its mutant derivatives, or pRR48 (vector control). Plates contained 50 μg/ml ampicillin and were incubated at 32.5°C for 8 h. The outer (serine) ring of the G235W and I241P colonies has a wild-type morphology, whereas that of the G234T mutant has a thinner, sharper edge. The G235S and G235Y colonies also have sharp edges, which are indicative of some chemotactic ability. The I241T and I241N colonies form diffuse discs with a ring near their perimeter, most likely reflecting some residual chemotactic ability. In contrast, the G234P and G235P mutants exhibit a fully null phenotype. In UU1250, these mutant transducers produce both CCW and CW flagellar rotation (Fig. 4), which enables the cells to spread through the agar more readily than the vector-containing cells, which are CCW only.

FIG. 3.

Mutational survey of Tsr-HAMP connector residues. The mutations isolated at each position and their phenotypes are indicated by circles: white, Tsr⁺; gray, Tsr^+/−; black, Tsr⁻. See the text for an explanation of these phenotypes. Plus symbols indicate the wild-type residues. Where no symbol is given, that particular replacement was not isolated. The D236H mutant protein (open diamond) was poorly expressed or unstable (∼14% of the wild-type steady-state level). All other mutant proteins were present at 50% or more of the wild-type level (data not shown). Amino acid replacements are grouped by side chain properties to facilitate comparisons among the sites. At the top, critical connector residues are circled: G235, L237, and I241. At these positions, a majority of replacements impaired function, and a majority of those were null defects. G245 (dashed circle) is a less critical site: a majority of replacements at this residue impaired function, but none of them caused null defects.

FIG. 4.

Examples of signal output patterns of loss-of-function (null) Tsr-HAMP connector mutants. Mutant pRR53 derivatives were transferred to UU1535 (adaptation-defective) and UU1250 (adaptation-proficient), and their resultant flagellar rotation patterns were assessed by cell tethering. Each panel shows the rotation profile of 100 cells of each strain, distributed across five rotation classes, from exclusively CCW on the left through frequently reversing in the center to exclusively CW on the right. The wild-type pattern is shown at the top. The criteria used to define the various mutant output categories are described in Materials and Methods. Where two examples are given for an output class, they represent the most different patterns of that class.

FIG. 5.

(A) Scheme for testing dominance and signal asymmetry of Tsr-HAMP connector lesions. Plasmid-borne connector mutations were tested for Tsr function in host strains with a binding site defect (R69E or T156K) in their chromosomal tsr gene. The coexpression of both Tsr subunits leads to the formation of heterodimers that are competent for serine binding in one orientation. Recessive HAMP defects should allow those heterodimers to function; dominant HAMP defects should not. If the HAMP lesion affects signaling asymmetrically, one type of heterodimer could function, whereas the other does not. × symbols represent mutational lesions, S-shaped symbols represent serine molecules, and white triangles indicate the subunit that transmits the ligand-induced piston signal to the HAMP domain. (B) Examples of recessive and dominant Tsr-HAMP connector lesions. Tryptone soft agar plates containing 50 μg/ml ampicillin and various concentrations of IPTG were inoculated with freshly transformed colonies and incubated at 32.5°C for 8 h. The host strain was UU2377 (Tsr-R69E); plasmids were pRR48 (vector control) and pRR53 derivatives expressing Tsr-I241R (recessive) or Tsr-G235C (dominant). Note that the Tsr-R69E receptor, although defective in serine sensing, is able to mediate a slow migratory response, most likely via aerotaxis or pH taxis (22, 35). In combination with a recessive HAMP lesion (I241R), this behavior is supplanted by a more robust chemotactic response to serine (note differences in colony morphology at 50 or 100 μM IPTG). In combination with a dominant HAMP lesion (G235C), this behavior is suppressed, and the strain exhibits a null phenotype (e.g., 100 μM IPTG).

FIG. 6.

Structure-function features in the Tsr-HAMP connector. Critical and important residues are circled. Amino acid replacements that cause jamming behavior are shown in boldface type. The connector exhibits three subsegments with different inferred roles. G234 to D236 are small residues that may comprise a turn. Nearly every type of amino acid replacement at G235 destroys function. Most replacements at L237 and I241 also destroy Tsr function but with a variety of signal output patterns. These residues may contribute side chain packing interactions that stabilize alternative HAMP signaling conformations. The E242-to-N247 segment contains mostly small residues and may serve as a flexible linker. Single-residue deletions at position 244, 245, or 246 had no deleterious effect on Tsr function. Similarly, the indicated 5- and 10-residue insertions between G245 and S246 did not interfere with Tsr function. Other insertions and larger deletions in this segment impaired or destroyed Tsr function, respectively.

FIG. 7.

Critical connector residues in the modeled Tsr-HAMP structure. For clarity, only one HAMP subunit is shown. The AS1 and AS2 helices are shaded as described in the legend of Fig. 1. All structures are oriented as shown above (A), with AS1 labeled at its N terminus and AS2 labeled at its C terminus. (A) All residues of the AS1 and AS2 helices and the functionally important residues of the connector are shown in a space-filled representation. In the modeled structure, G235 lies at the center of a tight turn (see C), and the side chains of L237 and I241 interact primarily with residues in the AS2 helix (see B). I241 is moderately exposed; L237 is more buried. G245 and neighboring residues (not shown as space filled) comprise a noncritical linker segment that packs loosely against AS2 residues oriented toward the interior of the four-helix bundle. (B) Side chain contacts between AS2 residues and functionally important connector residues. L263 lies against L237. The aliphatic portion of the R257 side chain abuts I241, but this interaction is probably not critical to Tsr function; the actual orientation of the I241 side chain may be different (see text). (C) G235 lies at a sharp turn at the end of AS1. Both of its neighboring residues (G234 and D236) have small side chains, but, unlike G235, they tolerate a variety of amino acid replacements with no loss of Tsr function.