Structure of the conserved HAMP domain in an intact, membrane-bound chemoreceptor: a disulfide mapping study

Abstract

The HAMP domain is a conserved motif widely distributed in prokaryotic and lower eukaryotic organisms, where it is often found in transmembrane receptors that regulate two-component signaling pathways. The motif links receptor input and output modules and is essential to receptor structure and signal transduction. Recently, a structure was determined for a HAMP domain isolated from an unusual archeal membrane protein of unknown function [Hulko, M., et al. (2006) Cell 126, 929-940]. This study uses cysteine and disulfide chemistry to test this archeal HAMP model in the full-length, membrane-bound aspartate receptor of bacterial chemotaxis. The chemical reactivities of engineered Cys residues scanned throughout the aspartate receptor HAMP region are highly correlated with the degrees of solvent exposure of corresponding positions in the archeal HAMP structure. Both domains are homodimeric, and the individual subunits of both domains share the same helix-connector-helix organization with the same helical packing faces. Moreover, disulfide mapping reveals that the four helices of the aspartate receptor HAMP domain are arranged in the same parallel, four-helix bundle architecture observed in the archeal HAMP structure. One detectable difference is the packing of the extended connector between helices, which is not conserved. Finally, activity studies of the aspartate receptor indicate that contacts between HAMP helices 1 and 2' at the subunit interface play a critical role in modulating receptor on-off switching. Disulfide bonds linking this interface trap the receptor in its kinase-activating on-state, or its kinase inactivating off-state, depending on their location. Overall, the evidence suggests that the archeal HAMP structure accurately depicts the architecture of the conserved HAMP motif in transmembrane chemoreceptors. Both the on- and off-states of the aspartate receptor HAMP domain closely resemble the archeal HAMP structure, and only a small structural rearrangement occurs upon on-off switching. A model incorporating HAMP into the full receptor structure is proposed.

Figure 1

Previous studies of the conserved HAMP motif. (A) Recent NMR structure of a HAMP domain isolated from an archeal transmembrane protein of unknown function (6), illustrating in cartoon form the parallel, four-helix bundle architecture generated by the association of two identical subunits (yellow and gray) in a symmetric homodimer. (B) Sequence alignment of representative HAMP domains (5, 6), indicating their putative helix–loop–helix regions. Bold residues are most widely conserved in these sequences; underlined residues are conserved except in the archeal and Aer sequences. The blue horizontal line highlights a change of helix register in the aspartate receptor (4); the empty triangle indicates the location of the membrane–water interface (4); and blue band and filled triangle denote the location of an arginine side chain, conserved in some but not all receptors, which is the major site of proteolysis in the S. typhimurium aspartate receptor (25). The latter position may represent the border between HAMP amphiphilic helix 2 (AH2) and cytoplasmic domain helix 1 (CD1) (see the text). (C) Comparison of cysteine chemical reactivities, previously measured for the aspartate receptor HAMP [thick line, ●, (−) Asp; ○, (+) Asp] (4), to the solvent accessibilities calculated for the corresponding positions in the archeal HAMP NMR structure (thin line, □) (6). The correlation is strong in the helical regions, but not in the connector region. (D) Comparison of symmetric disulfide dimer formation, previously measured for the aspartate receptor HAMP (thick line, ●) (4), to spatial proximities calculated for the corresponding cysteine pairs in the archeal HAMP NMR structure (thin line, □) (6). The correlation is strong only in the region of the second helix. Spatial proximities were calculated using the indicated equation where x is the distance in Å between β-carbons.

Figure 2

Cys pairs selected for analysis of the HAMP AH1–AH2′ helical interface. (A) Summary of pairs of positions selected in the archeal HAMP structure (6), and the corresponding pairs in the S. typhimurium aspartate receptor. Also shown are the four β-carbon–β-carbon distances measured for each pair in the homodimeric, archeal HAMP structure. Each proximal pair (P) possesses an asymmetric, intersubunit distance of < 7 Å, while the other three distances simultaneously exceed 10 Å. In each distal pair (D), all four distances exceed 10 Å. Parentheses indicate low-yield di-Cys mutants not suitable for further analysis (see the text). (B and C) Locations of the proximal (B) and distal (C) Cys pairs employed for disulfide mapping. Ovals highlight the asymmetric, intersubunit separation for each pair.

Figure 3

Comparison of disulfide-linked dimer formation for proximal and distal Cys pairs, and assignment of products. (A) Oxidation of four representative di-Cys mutant aspartate receptors, and the corresponding single-Cys mutants, in isolated E. coli membranes. Reactions were initiated by addition of the redox catalyst Cu(II)(1,10-phenanthroline)₃ (1 mM final) to receptor (2.5 µM dimer) in 160 mM KCl and 50 mM Tris (pH 7.2 with HCl), 5 mM MgCl₂, and 1.5 mM EDTA, followed by incubation for 1 min at 30 °C, quenching, and analysis by Coomassie SDS–PAGE. The I224C/T253C and A231C/S260C Cys pairs are predicted to be proximal, while the A221C/S260C and I224C/S260C pairs are predicted to be distal (Figure 2A). (B) Extent of disulfide-linked dimer formation for all 11 di-Cys mutants and the corresponding single-Cys mutants. Oxidation reactions were carried out and analyzed as described for panel A. The I224C/E249C, I224C/T253C, A231C/T253C, A231C/H256C, and A231C/S260C pairs are predicted to be proximal, while the A221C/S260C, I224C/S260C, T225C/G252C, T225C/H256C, A231C/N245C, and S232C/E255C pairs are predicted to be distal. (C) Kinetic product analysis for a representative di-Cys mutant, A231C/T253C, and the corresponding single-Cys mutants. Oxidation at 30 °C as in panel A, except that oxidation strength and time were varied to examine early (+), middle (++), and late (++++) products as follows: (+) 0.5 mM redox catalyst, 5 mM EDTA, 1 min; (++) 1 mM redox catalyst, 0.8 mM EDTA, 5 min; (++++) 1 mM redox catalyst, no EDTA, 10 min. Reactions were quenched with 5-fluoresceinmaleimide (5FM, 500 µM) to visualize the unreacted, free Cys residues remaining after oxidation. The expanded view of the dimer region compares fluorescent (top) and all (bottom) dimeric products. The most fluorescent band is an early oxidation product containing one asymmetric disulfide and two free cysteines modified with 5FM during quenching. (D) Oxidized product analysis for a representative tri-Cys mutant, N36C/A231C/T253C, and the corresponding di-Cys mutant. Oxidation as in panel A, except that oxidation strength and time were varied to examine middle (+++) and late (+++++) products as follows: (+++) 1 mM redox catalyst, no EDTA, 5 min at 30 °C; (+++++) 2 mM redox catalyst, no EDTA, 20 min at 37 °C. Reactions were quenched with 5FM as in panel C. Images show the expanded dimer region comparing fluorescent (top) and all (bottom) dimeric products. Following oxidation, the uppermost fluorescent dimer contained one asymmetric disulfide and either two free cysteines for the di-Cys mutant, or four free cysteines for the tri-Cys mutant. The latter product was labeled by as many as four 5FM molecules during quenching and thus exhibits the highest ratio of fluorescence/Coomassie stain. (E) Same as panel D but showing the entire oligomer region for the Coomassie gel. No oligomers larger than dimers are detected. In panels C and E, single-Cys mutants were overloaded relative to di-Cys mutants to ensure visibility of their relatively rare dimeric products.

Figure 4

Effect of Cys substitutions and disulfide bonds on receptor activity in vivo and in vitro. (A) Comparison of reduced WT and mutant receptor activities in the standard in vivo swim plate assay of bacterial chemotaxis in soft agar (11, 14). (B) Comparison of wildtype and mutant receptor activites following receptor reduction (left) or oxidation (right) in the standard in vitro assay measuring receptor-regulated CheA kinase activity in the reconstituted signaling complex (12). Shown are kinase activities in the absence and presence of attractant aspartate. (C) Comparison of wild-type and mutant receptor activities following reduction (left) or oxidation (right) in the standard in vitro assay measuring the extent of receptor methylation by CheR (12), in the absence and presence of aspartate.

Figure 5

Model for HAMP structure in the full-length aspartate receptor. (A) The model is built using disulfide mapping constraints (4) to define the geometry of the AH2–AH2′ helix pair, then building in the AH1–AH2′ interaction based on additional disulfide mapping constraints (Figure 3), and finally using the known C2 symmetry of the homodimer to complete the parallel, four-helix bundle (see the text). (B and C) Schematic model incorporating HAMP into the full-length receptor, illustrating a new helix nomenclature. For clarity, the model is shown in space-filling, ribbon, and cylinder formats which together portray the overall shape, subunit supercoiling, and helical structure of the homodimer. Highlighted is (i) the junction between the end of helix TM2 and the beginning of HAMP helix HD1, where there is a helical phase change (see the text), and (ii) the major proteolysis site, Arg259. The protease accessibility of the latter site suggests that position 259 lies at or near C-terminus of the distinct HAMP structural domain. The model of the full-length homodimer illustrates the three distinct modules (transmembrane signaling, signal conversion, and kinase control) of the receptor, as well as the three functional regions (adaptation, flexible, and protein interaction) of the latter module (3, 27).