Biology subjects

Izadi-Pruneyre, N.

Publications and source records attributed to Izadi-Pruneyre, N..

4 recordsLinked to original sources

bioRxiv2022

Structure and dynamic association of an assembly platform subcomplex of the bacterial type II secretion system

Type II secretion systems (T2SS) allow diderm bacteria to secrete hydrolytic enzymes, adhesins or toxins important for growth and virulence. In T2SS, secretion of folded proteins from the periplasm to the cell surface requires assembly of periplasmic filaments called pseudopili. Like the related type IV pili, pseudopili are polymerized in the inner membrane through addition of subunits at the filament base, mediated by the essential assembly platform (AP). To understand the structure and molecular role of the AP, we focused on its components PulL and PulM from the Klebsiella oxytoca T2SS. By combining biophysical methods, NMR and X-ray crystallography we studied the structure and associations of their periplasmic domains. We describe the first structure of the heterodimer complex formed by the PulL and PulM ferredoxin-like domains and show how their structural complementarity and plasticity favor their association during the secretion process. Cysteine scanning and cross-linking of transmembrane segments provided additional constraints to build a structural model of the PulL-PulM complex and assembly in the cellular context. Together with the relative abundance of PulL, PulM and their partners our findings suggest a model of the AP as a dynamic hub that orchestrates pseudopilus polymerization.

microbiology↗

bioRxiv2021

Interaction of the TonB dependent transporter HasR with its cognate TonB-like protein HasB in a membrane environment

The envelope of Gram-negative bacteria is composed of a double membrane separated by the periplasmic space. This organization imposes geometrical and distance constraints that are key for the mechanism of action of multicomponent systems spanning the envelope. However, consideration of all three compartments by experimental approaches is still elusive. Here we used the state-of-the-art molecular dynamics simulation in an Escherichia coli envelope model to obtain a dynamic view of molecular interactions between the outer membrane heme transporter HasR and the inner membrane TonB-like protein HasB. Their interaction allows the transfer of the inner membrane proton motive force derived energy to the transporter for heme internalization. The simulations which incorporate both membranes show the key role of periplasmic domains of both proteins, and their dynamics in the complex formation and stability. They revealed a previously unidentified atomic network of interactions, as well as the sequences of the interactions and their variations with the presence of external substrates. Experimental validation (mutations, phenotypic and in vitro assays) confirms the robustness of our approach and provides verification of the simulation-predicted interactions. Based on structural and sequence conservation, the network of interaction revealed in this study is expected to occur in other nutrient import systems. The integrative approach presented here is highly useful to study the dynamic interplay between components of any other bacterial transmembrane systems.

biophysics↗

bioRxiv2021

Functional and structural characterization of Serratia marcescens ExbB: determinants of the interaction with HasB/TonB

ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbBSm has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbBs are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbBSm and HasB. We determined the cryo-EM structures of ExbBSm and of the ExbB-ExbDSm complex from S. marcescens. ExbBSm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbBSm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbBSm and ExbBEc, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the new class of inner membrane energy machinery formed by ExbB,ExbD and HasB.

microbiology↗

bioRxiv2021

Computational and biochemical analysis of type IV Pilus dynamics and stability

Type IV pili (T4P) are distinctive dynamic filaments at the surface of many bacteria that can rapidly extend, retract and withstand strong forces. T4P are important virulence factors in many human pathogens, including Enterohemorrhagic Escherichia coli (EHEC). The structure of the EHEC T4P has been determined by integrating Nuclear Magnetic Resonance (NMR) and cryo-electron microscopy data. To better understand pilus assembly, stability and function, we performed a total of 108 s all-atom molecular dynamics simulations of wild-type and mutant T4P. Extensive characterization of the conformational landscape of T4P in different conditions of temperature, pH and ionic strength was complemented by targeted mutagenesis and biochemical analyses. Our simulations and NMR experiments revealed a conserved set of residues defining a novel calcium-binding site at the interface between three pilin subunits. Calcium binding enhanced T4P stability ex vivo and in vitro, supporting the role of this binding site as a potential pocket for drug design.

biochemistry↗