Biochemical and structural characterization of Myxococcus xanthus Frz chemosensory receptor complex

Abstract

In bacterial chemotaxis, methyl-accepting chemotaxis proteins (MCPs) detect environmental cues and initiate a phosphorelay signal. In Myxococcus xanthus, the Frz pathway regulates motility in response to these cues. MCPs form organized hexagonal arrays on the bacterial membrane, composed of receptors, adaptor proteins, and histidine kinase. These arrays amplify, integrate, and transmit signals cooperatively, but the precise mechanism of signal propagation remains unclear. Unlike canonical membrane-bound MCP arrays, Frz pathway has a cytoplasmic MCP FrzCD, which uses the nucleoid as a scaffold for oligomerization. In M. xanthus, FrzA and FrzB are adaptor proteins linking FrzCD to the histidine kinase FrzE. We identified di-HAMP domains between the DNA-binding and signalling domains of FrzCD. In vitro, the signalling domain promotes the stable oligomerization of FrzCD in the presence of DNA. The di-HAMP domains influence DNA binding and regulate FrzCD oligomerization. Together with in vivo studies, we found that the di-HAMP domains control nucleoid array formation and regulates downstream signalling, influencing bacterial motility and development. To understand the significance of di-HAMP domains observed in FrzCD and identify a possible signalling input module in FrzCD, we analyzed MCP sequences from the Pfam database. We observed that cytoplasmic receptors predominantly lacked HAMP domains and identified the prevalence of certain LBDs for cytoplasmic MCPs that can sense small diffusible molecules or intracellular metabolites. Next, we reconstituted the receptor-adaptor complex in vitro. Through cryoEM studies of the FrzCD-FrzA complex, combined with AlphaFold-generated models, we have identified the interacting interface. Mutational analysis confirmed that hydrophobic and salt-bridge interactions mediate receptor-adaptor complexes. FrzA and FrzB interact at the same interface on FrzCD, but FrzCD-FrzA forms a more stable complex than FrzCD-FrzB. This may explain a mechanism for regulating the number and size of clusters on the nucleoid. Additionally, the dimeric receptor in the complex has a curvature mediated by a glycine hinge. We believe that the curvature induces an asymmetric interface on the dimeric FrzCD signalling tip, potentially resulting in separate preferred binding sites for adaptor proteins, histidine kinase or oligomerization. Interestingly, we observed a rotational shift of the adaptor protein at the signalling tip in the absence of HAMP2 domain, which suggests a mechanism of signal transduction in chemosensory complexes. Our work supports that this cytoplasmic chemosensory system can serve as a model for studying the mechanism of structural changes in signal transduction within bacterial chemosensory complexes.