Hydrogen Exchange Reveals Differences between Bacterial Chemoreceptor Signaling States

Bacterial chemotaxis receptors form membrane-bound nanoarrays that sense and adapt to ligands from the environment. These arrays consist of receptors interacting at their cytoplasmic tips with a “baseplate” formed by the CheA kinase and CheW coupling protein, ≈300 A away from the periplasmic ligand binding sites. Receptor methylation at 4 glutamate residues in the cytoplasmic domain mediates adaptation to ongoing stimuli. To determine what ligand- and methylation-induced changes in the receptor cytoplasmic domain control the kinase activity, we have developed (1) methods to reconstitute native-like arrays of receptor cytoplasmic fragments, CheA, and CheW, and (2) a hydrogen exchange mass spectrometry (HDX-MS) method applicable to membrane-bound, multi-protein complexes. HDX-MS comparison of complexes with high and low kinase activity shows that differences localize to two functionally important subdomains of the receptor. Changes in the methylation subdomain that mediates adaptation are complex. The uniform exchange behavior of these peptides in the kinase-off complexes splits roughly in half in the kinase-on complexes, one fraction with slower exchange and the other with extremely rapid exchange (complete in 3 min). For the signaling subdomain that binds CheA and CheW, peptides exhibit protection from exchange at long times (16 hours) that is greater in the kinase-on state. HDX-MS of complexes prepared using different means of shifting the signaling state will reveal which changes correlate with kinase activity and will quantify stabilization of receptor subdomains and binding interfaces that contribute to receptor control of kinase activity. Thus HDX-MS provides an important tool in a hybrid approach for understanding structure and mechanism in membrane-bound, multi-protein complexes.This research supported by GM085288, and a University of Massachusetts fellowship to Seena Koshy as part of the Chemistry-Biology Interface Training Program (NRSA T32 GM08515).