Structural Basis of Bacterial Transmembrane Signaling

Abstract

Bacteria have evolved membrane-bound receptors and signaling proteins to detect and respond to external signals, regulating essential processes such as nutrient acquisition, stress adaptation, and cell-to-cell communication. However, the molecular mechanisms of cross-membrane signaling remain incompletely understood. My research focuses on two distinct bacterial signaling systems: the methyl-accepting chemotaxis protein McpZ in Sinorhizobium meliloti and the signaling histidine kinase GacS in Pseudomonas aeruginosa. What made this work particularly challenging is that the ligands that induce signal transduction across the inner cell membrane in either protein remain unidentified. We solved the crystal structure of the McpZ periplasmic domain, revealing a novel tri-modular helical fold and an unconventional dimerization interface. Our molecular dynamics simulation data suggest that transmembrane signaling in McpZ involves both piston-type and scissoring movements. These findings contribute to a broader understanding of bacterial chemotaxis signaling. GacS is a central component of the GacS/GacA phosphorelay system, positioned at the heart of perhaps the most complex known multikinase network. GacS upregulates virulence factors associated with biofilm formation and chronic infections. Sensor histidine kinase-like RetS directly inhibits GacS to promote the expression of virulence factors linked to acute infection and suppress those required for chronic disease. Important gaps remain in our understanding of how the activities of RetS and GacS are regulated through inter-extracellular signals. Both proteins feature essential periplasmic sensory domains, but the specific ligands remain unknown. Mediated through their respective DHp domains, RetS and GacS form a domain-swapped oligomer. According to this partial structural model, GacS undergoes a significant conformational transition upon RetS binding. In this study, because the natural ligand is unknown, we designed and constructed the CitAGacS chimeric protein. Having an active chimeric CitAGacS allowed us to investigate GacS cross-membrane signaling. We discovered that GacS ligand binding dissociates RetS:GacS complex inside the cytoplasm. Strikingly, we were able to demonstrate that the presence of RetS primes GacS ligand binding in the periplasm, using a novel inside-out signaling mechanism.