RomX, a novel prokaryotic regulator, links the response receiver domain of RomR with GTP-bound MglA for establishingMyxococcus xanthuspolarity

Abstract

AbstractCell polarity specification and reversals are distinctive features of motility of the soil bacteriumMyxococcus xanthus. The bacterial small Ras-like GTPase, MglA, serves as a key player orchestrating these polarity oscillations. RomR, a response regulator, along with its partner RomX, has been identified as a guanine nucleotide exchange factor (GEF) for MglA, crucial for its polar recruitment. In this study, we determine the crystal structure of RomX, a protein of a hitherto unknown fold. RomX consists of a three-helix bundle, identified to be the same fold as the stalk domain of atlastin, a member of the dynamin family of GTPases. From our structure-based sequence analysis for proteins of similar fold, we observe the co-occurrence of the RomX fold with response receiver domains in several bacterial response regulators. We demonstrate that the binding between MglA and RomX is exclusively in the presence of GTP. Based on mutational analysis and affinity measurements, we conclude that the helix-1 of RomX mediates the interaction with MglA-GTP, while helix-3 of RomX interacts with the RomR N-terminal receiver (REC) domain. Absence of additional stimulation of RomX GEF activity in the presence of RomR-REC supports the mutually exclusive interface on RomX for RomR and MglA interaction. Collectively, our findings validate the positioning of RomX between MglA and RomR-REC, providing insights into the concerted action of the bipolarly localized RomR/RomX complex in driving MglA localization within polarized cells.