Bacterial chemotaxis proteins are both potential targets for novel antibiotics and a key model system for understanding transmembrane signaling mechanisms. Molecules in the environment are detected by a remarkable sensor array of membrane-bound chemoreceptors with two cytoplasmic partner proteins. Signaling through this complex begins as a ligand-induced 2 Å displacement of a receptor alpha helix that extends from the periplasm through the membrane. How is the signal propagated an additional ~200 Å from the membrane to the cytoplasmic tip of the receptor, and how does it control the activity of the kinase bound to the tip of the receptor? To address this question, we assemble homogeneous, native-like functional complexes of an E. coli Asp receptor cytoplasmic fragment (CF) with the kinase CheA and coupling protein CheW. A synergistic combination of solid-state NMR and hydrogen exchange mass spectrometry (HDX-MS) has identified highly dynamic regions of CF and CheA in these complexes. Furthermore, the CF is partially disordered within functional complexes and has a small, well-ordered protein interaction region. Reduced dynamics and slower exchange in CF in the kinase-activating state suggest signaling inputs modulate the disorder of the cytoplasmic domain to control the kinase activity. HDX-MS of CheA alone and in these functional complexes indicate that stabilization of the kinase core domains is key to kinase activation. NMR experiments are in progress to study protein-protein and domain-domain interactions in these complexes. This study reveals the mechanistic role of protein disorder and stabilization in signaling and control of catalysis.

Protein stabilization plays a key role in signaling by bacterial chemoreceptor complexes

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