Biology subjects

Sahil, M.

Publications and source records attributed to Sahil, M..

4 recordsLinked to original sources

bioRxiv2023

A host AAA-ATPase exhibits bacteriolytic activity for clearance of microbial infection

An array of host cytosol guarding factors impede bacterial proliferation and preserve cellular sterility. Amongst them, proteasomal degradation of ubiquitinated pathogens has emerged as a critical mechanism for ensuring cytosolic sanctity. We wondered how proteasomes, with their small size and inability to extract membrane-bound proteins, can eradicate pathogens. Here, we unveil a unique strategy, wherein VCP/p97, a host AAA-ATPase, eliminates pathogens by exerting mechanical force that physically unfolds and pulls out ubiquitinated proteins from bacterial membrane. Combining a single-molecule approach along with molecular dynamic simulation and in-vitro reconstitution, we demonstrate that protein extraction by p97 causes extensive membrane lysis and release of cytosolic contents from phylogenetically diverse microbes. Additionally, in an in-vivo mouse sepsis model, this segregase-dependent bactericidal effect of p97 abrogated microbial proliferation in host tissues. Overall, we discovered a distinct innate antimicrobial function of p97, that protects the host against lethal bacterial infections. One Sentence SummaryA host AAA-ATPase exhibits bacteriolytic activity.

microbiology↗

bioRxiv2023

Identifying Selectivity Filters in Protein Biosensor for Ligand Screening

Specialized sensing mechanisms in bacteria enable the identification of cognate ligands with remarkable selectivity in highly xenobiotic-polluted environments, where these ligands are utilized as energy sources. Here, via an integrated all-atom computer simulation, biochemical assay and isothermal titration calorimetry approaches we determine the molecular basis of MopR, a phenol biosensors complex selection process of ligand entry. Our results reveal a set of strategically placed selectivity filters along the ligand entry pathway of MopR. These filters act as checkpoints, screening diverse aromatic ligands at the protein surface based on their chemical features and sizes. Ligands meeting specific criteria are allowed to enter the sensing site in an orientation-dependent manner. Sequence and structural analyses demonstrate the conservation of this ligand entry mechanism across the sensor class, with individual amino acids along the selectivity filter path playing a critical role in ligand selection. Together, this investigation highlights the importance of interactions with the ligand entry pathway, in addition to interactions within the binding pocket, for achieving ligand selectivity in biological sensing. The findings enhance our understanding of ligand selectivity in bacterial phenol biosensors and provide insights for the rational expansion of the biosensor repertoire, particularly for the biotechnologically relevant class of aromatic pollutants.

biochemistry↗

bioRxiv2023

Discovery of a Novel 3site State as the Multi-Substrate Bound State of P450cam

Archetypal metalloenzyme Cytochrome P450cam (CYP101A1) catalyzes regioselective hydroxylation of its native substrate camphor in heme active site. However, the proposal of potential existence of additional substrate binding modes distal from the active site in P450cam and their concomitant roles in regulating recognition at active site have remained a matter of recurring discourse. Herein we report the discovery of a novel 3site state in P450cam, where three substrate molecules were observed to simultaneously bind to P450cam at three distinct sites including the heme active site. These three binding modes, hereby referred as catalytic, waiting and allosteric binding modes in 3site state, are allosterically inter-linked and function in mutually synergistic fashion. The 3site state possesses regio-selective conformations of substrate essential for catalysis and establishes substrate-ingress and product exit process to and from the active site via two distinct channels. The ensemble of three-state binding modes are found to be self-consistent with NMR pseudo-contact shift data obtained from TROSY-HSQC measurements and DEER based predictions. Binding of redox partner Putidaredoxin with 3site model retains closed conformation of 3site state, siding with NMR based hypothesis that the catalysis would take place in closed insulation of P450cam even in presence of its redox partner. Statement of SignificanceUbiquitous superfamily of mono-oxygenases cytochrome P450s are involved in broad range of metabolic process in all domains of life and are also important drug targets. Apart from the well known and established binding mode in heme active site, the substrate bindings at additional distal sites have been postulated in multitude of P450s. Using the archetypal bacterial cytochrome P450 i.e., P450cam, a novel 3site state of cytochrome P450 is elucidated in this work. The novel 3site state has two additional binding modes namely waiting and allosteric (also postulated previously), apart from known binding mode catalytic in the active site. The known functions of P450cam are found to be most optimally explained by this 3site state, instead of single substrate bound catalytic state. This state can be of critical importance for CYP superfamily at large and potentially be useful in understanding the non-Michaelis behaviour, observed in many P450s.

biochemistry↗

bioRxiv2022

Phenol Sensing in Nature Modulated via a Conformational Switch Governed by Dynamic Allostery

NtrC family of proteins sense external stimuli and accordingly stimulate stress and virulence pathways via activation of associated {sigma}54-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,[~]20[A] 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.

biochemistry↗