Hydrogen exchange differences between chemoreceptor signaling complexes localize to functionally important subdomains.

Membrane proteins perform key life processes, including transmitting information into cells to allow responses to the environment. Bacterial chemotaxis receptors make up an ideal system for investigations of the mechanistic details of transmembrane signaling. Bacteria sense chemicals in the environment and relay a signal through the chemoreceptors that ultimately controls the swimming response of the cell. The membrane-spanning receptors form ternary complexes (Figure ​(Figure1A)1A) with two cytoplasmic proteins, a scaffolding protein CheW and a histidine kinase CheA; these complexes are found in large hexagonal arrays primarily at the poles of the cell. On the basis of a wide variety of studies, including Cys cross-linking, mutagenesis, electron paramagnetic resonance (EPR), and solid-state nuclear magnetic resonance, ligand binding to the receptor is thought to cause an ∼2 A piston motion of an α helix in the periplasmic and transmembrane domains.1 It is less clear how the signal is then propagated through the cytoplasmic domain to inhibit the kinase bound ∼200 A away at the membrane-distal tip of the receptor, and how methylation of Glu residues in this domain restores kinase activation as part of receptor adaptation to ongoing stimuli. Figure 1 HDX-MS approach for testing proposed changes in dynamics of chemoreceptors within functional complexes during signaling. (A) Model of a single-transmembrane chemoreceptor dimer (gray surface) superimposed on one cytoplasmic fragment dimer within a model ... A number of studies have suggested that changes in dynamics in the cytoplasmic domain may play a role in signaling, but it is difficult to measure dynamics within membrane proteins and large functional multiprotein complexes. Previous studies from our laboratory have shown that the cytoplasmic fragment is highly dynamic in solution,2 and small changes (mutation of a single residue or protonation of a few residues) can lead to significant stabilization of a large fraction of the protein.3 Proposals that receptor signaling involves changes in dynamics3,4 have been corroborated by mutagenesis and cross-linking results. For example, electrostatic interactions are thought to affect the conformational dynamics of the methylation subdomain, with charge-neutralizing mutations at the acidic methylation sites and other sites favoring a less dynamic, kinase-activating state.5 On the basis of cysteine cross-linking and alanine knob mutational studies, Falke and co-workers have recently proposed that signaling involves antisymmetric changes in dynamics in different subdomains of the cytoplasmic domain: the kinase-inhibited state is proposed to be destabilized in the methylation subdomain and stabilized in the signaling subdomain that interacts with CheA and CheW, relative to the kinase-activating state.6 Also on the basis of mutagenesis studies, Parkinson and co-workers have proposed a compatible model involving changes in dynamics in which the kinase-inhibited state again has a destabilized methylation subdomain and also has a stabilized HAMP subdomain.7 What has been lacking until now is a means for direct measurement of dynamics in functional receptor complexes to test these proposals (listed in Figure ​Figure1A,1A, left) and delineate the role of changing dynamics in the signaling mechanism. There has been tremendous recent progress in mass spectrometry of membrane proteins,8,9 including hydrogen exchange mass spectrometry (HDX-MS) studies of dynamics of membrane proteins in detergent micelles10−15 and nanodiscs.16 We have developed an alternate approach, summarized in Figure ​Figure1B, that1B, that can be applied for HDX-MS studies of a soluble domain of a membrane protein in membrane-bound functional complexes with its partner proteins. We have shown that vesicle template assembly can be combined with HDX-MS to measure dynamics in membrane-bound functional complexes.17 The vesicle template assembly method18 employs vesicles containing a nickel-chelating lipid to bind a His-tagged chemoreceptor cytoplasmic fragment (CF) and assemble functional complexes with CheA and CheW in different signaling states.19 The potential generality of this assembly method has been demonstrated by its use to assemble functional complexes of other systems such as eukaryotic receptor tyrosine kinases.20−23 With HDX-MS, we have demonstrated that the rapid global hydrogen exchange rates (exchange throughout the entire protein) observed for the Asp receptor CF in solution are reduced by assembly of functional CF complexes, but there are no large changes in CF global exchange rates between samples representative of different signaling states.17 Here we report measurements of local exchange in functional CF complexes that mimic two signaling states. Functional complexes are exchanged in D2O for various times before quenching at low pH, pepsin digestion, and analysis by LC–ESI-MS. This localizes the exchange information to the individual peptide products of the pepsin digest. Our data show that stabilization of the CF upon formation of functional complexes extends throughout most of the protein, but that changes in dynamics between complexes that mimic the signaling states localize to key regions of the receptor critical to excitation (control of the CheA kinase) and adaptation (mediated by methylation of specific Glu residues). In both subdomains, there is some reduction in the exchange rate in the kinase-activating state, which suggests that the mechanism of kinase activation involves stabilization of two key features of the chemoreceptor, its methylation subdomain involved in adaptation and its interactions between the signaling subdomain and CheA and CheW involved in kinase activation in the signaling array. These changes are measured within arrays that are nativelike, with ∼100 nm dimensions24 and 12 nm hexagonal spacing,25 comparable to array properties observed in cells. These results illustrate the promise of combining HDX-MS with vesicle template assembly to reveal differences in structure and dynamics between physiologically relevant states of membrane-bound multiprotein complexes and gain insights into the role of dynamics in mechanisms of complex systems.