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An array of cytosol guarding factors impede bacterial invasion and preserve cellular sterility. Amongst them, proteasomal degradation of ubiquitinated pathogens has emerged as a critical mechanism ensuring cytosolic sanctity. But smaller size of the proteasomal barrel and its inability to extract membrane-bound proteins, questions this paradigm. Our study unveiled a unique mechanical force-based strategy, employed by VCP/p97, an AAA-ATPase, which by extracting ubiquitinated bacterial surface proteins, eliminates pathogens. Using molecular dynamic simulation along with in-vitro and ex-vivo experiments, we demonstrated that p97's segregase activity is central to its bactericidal effect. Assisted by cofactors NPLOC4 and UFD1, p97 causes extensive lysis of phylogenetically diverse microbes and triggers leaching of bacterial cytosolic contents. In-vivo, p97 abrogated bacterial proliferation in host tissues and protected animals from lethal infections. Overall, we unravelled a distinct innate immune function of p97, which is critical for host protection against bacterial infections.

Bacteria surviving in high xenobiotic polluted environment have evolved specialized sensing mechanisms that identify their cognate ligand with exquisite selectivity and subsequently exploit it as an energy source. Here, by employing MopR protein, a phenol sensor as a model system and via an integrated all-atom computer simulation, statistical model and biochemical assay approaches we determine the molecular basis of this complex selection process of ligand entry. Multiple simulations followed by experiment demonstrate that a set of three strategically placed selectivity filters placed along the entry pathway, serve as checkpoints, where diverse aromatic ligands are first screened at the surface, gauged according to respective chemical features or sizes and finally allowed to enter the sensing site in an orientation-dependent fashion. Sequence and structural analysis show that the ligand entry mechanism is conserved across this sensor class and to achieve selectivity, fine tuning of individual amino acids along the selectivity filter path, in each sensor protein, determines ligand selection. Together, the investigation impresses that key to ligand selectivity not only rest on interaction of the sensor molecule within the resting pocket, rather subtle interaction with the path of ligand entry is a decisive factor for selective biological sensing. This study opens doors to rational expansion of biosensor repertoire for biotechnologically important aromatic pollutant class of compounds.

NtrC family of proteins sense external stimuli and accordingly stimulate stress and virulence pathways via activation of associated σ54-dependent RNA polymerases. Here, we establish that MopR, an NtrC protein, harbors a dynamic bi-directional electrostatic network that connects the phenol pocket to two distal regions, namely the “G-hinge” and the “allo-steric-linker”. While G-hinge influences the entry of phenol, the allosteric-linker passes the signal to the downstream ATPase domain. Phenol binding induces a rewiring of the electrostatic connections by eliciting dynamic allostery, and it was demonstrated that perturbation of the core relay residues results in a complete loss of ATPase stimulation. A mutation of the G-hinge,∼20Å from the phenol pocket, demonstrated altered flexibility by shifting the pattern of conformational states accessed, leading to a protein with 7-fold enhanced phenol binding ability and enhanced transcriptional activation. A global analysis illustrates that dynamic allostery-driven conserved community networks are universal and evolutionarily conserved across species.