Self-association of the histidine kinase CheA as studied by pulsed dipolar ESR spectroscopy

Abstract

Biologically important protein complexes often involve molecular interactions that are low affinity or transient. We apply pulsed dipolar electron spin resonance spectroscopy and site-directed spin labeling in what to our knowledge is a new approach to study aggregation and to identify regions on protein surfaces that participate in weak, but specific molecular interactions. As a test case, we have probed the self-association of the chemotaxis kinase CheA, which forms signaling clusters with chemoreceptors and the coupling protein CheW at the poles of bacterial cells. By measuring the intermolecular dipolar interactions sensed by spin-labels distributed over the protein surface, we show that the soluble CheA kinase aggregates to a small extent through interactions mediated by its regulatory (P5) domain. Direct dipolar distance measurements confirm that a hydrophobic surface at the periphery of P5 subdomain 2 associates CheA dimers in solution. This result is further supported by differential disulfide cross-linking from engineered cysteine reporter sites. We suggest that the periphery of P5 is an interaction site on CheA for other similar hydrophobic surfaces and plays an important role in structuring the signaling particle.

Figure 1

Interfaces that mediate CheA to CheA and CheA to CheW contacts in crystals. In crystal structures, a symmetric contact between the P5 domains mediates dimer associations. ESR structures of the complex between CheA (P3, dark-blue ribbons; P4, gray ribbons; P5, light-blue ribbons) and CheW (green) are shown associated through the P5-P5 interface found in crystal contacts. (Insets) Interfaces formed between CheW and P5 subdomain 1 (purple and magenta ribbons) and between the two P5 domains (orange and red ribbons) have similar hydrophobic character.

Figure 2

Minor CheA aggregation evident from size-exclusion chromatography. Overlay of gel filtration profiles of wild-type CheAΔ289 (solid line) and CheAΔ289 D579C (dotted line) show the dominant elution peak corresponds to the molecular mass of dimer, whereas a small shoulder at 163 ml indicates higher-order associations. The SDS PAGE analysis of selected fractions close to the peak shoulder from CheAΔ289 variant D579C (below) produces higher molecular-mass bands (∼84 kDa), which correspond to the two cross-linked monomer subunits of CheAΔ289D579C from two different dimers.

Figure 3

Spin-label positions on the CheAΔ289. Position of spin-label sites on the crystal structure of CheAΔ289 that were tested in cross-linking and magnetic dilution experiments. Sites on the P4 and P5 domain used for cross-linking experiments (spheres) are shown on different subunits. From a set of 14 sites, only E646C and D579C on P5 domain (dark-pink spheres) were found to form disulphide bonds readily. Magnetic dilution experiments were performed with four spin-labeled sites (E301C, Q545C, D508C, and D579C) on CheAΔ289. Sites D508C and D579C (spheres marked with solid black circles) are located at the periphery of the complex and show drastic reduction in baseline slope when unlabeled CheAΔ289 protein is added. In contrast, only minor changes were observed for E301C and Q545C (spheres marked with dashed black circle), as they are located near the core of the protein.

Figure 4

Magnetic dilution experiments on spin-labeled CheA. Magnetic dilution experiments were carried out with four (E301C, D508C, Q545C, and D579C) spin-labeled sites on CheAΔ289. Whereas the first two sites belong to domain P3 and P4, respectively, the latter two are located on different ends of P5 domain. In all samples, the concentration of spin-labeled protein was kept constant at 25 μM. The solid line in each plot shows the DEER signal obtained from spin-labeled CheAΔ289 without dilution. Wild-type protein was added in three (dashed line) and five times (dotted line) concentration excess of spin-labeled CheAΔ289. Stoichiometric amounts of wild-type CheW were added in all samples.

Figure 5

Distance distributions for spin-labels at the 579 position. Short reconstructed distances of spin-label separation in both CheAΔ289-D579C (500 μM, 10% Gly; solid line) and P4P5-D579C (500 μM, 10% Gly; dashed line) are consistent with intermolecular interactions between P5 subdomain 2. Corresponding time domain spectra shown in the panel below. For comparison, the highly distributed signal of P4P5-D579CP4P5 is shown at lower protein and higher glycerol concentration (100 μM, 40% Gly; dash-dotted line). The distribution of for CheAΔ289-D646C is dominated by the broad, intradimer distance at ∼60 Å, but also shows some features in the short distance range (25 μM; CheW, 50 μM, 30% Gly; dotted line). CheW was added to block self-associations and cross-linking through subdomain 1 (see Fig. 6). For ease of comparison, the heights of the distance distributions are scaled to 1.

Figure 6

CheA cross-linking. Fourteen cysteine-substituted variants of CheAΔ289 were tested for their ability to form disulphide bonds or cross-link in presence of an oxidizing reagent. (a) None of the seven cysteine substitutions on P4 domain successfully cross-linked. (b) Out of seven positions on P5 domain, D579C and E646C (lanes 2 and 5, respectively) cross-linked. Disulfide formation by E646C was blocked by CheW (lane 6) whereas that by D579C was unaffected (lane 3).