Biology subjects

Stansfeld, P.

Publications and source records attributed to Stansfeld, P..

6 recordsLinked to original sources

bioRxiv2023

Structure of the Native Chemotaxis Core Signalling Unit from E-gene lysed E. coli cells

Motile bacteria detect ambient chemical gradients and control their locomotion via conserved chemotaxis signaling networks, enabling cells to locate nutrients, potential hosts and other important biological niches. The sensory apparatus of the chemotaxis pathway is an array of core-signaling units (CSU) composed of transmembrane chemoreceptors, the histidine kinase CheA and an adaptor protein CheW. Although chemotaxis pathways represent the best understood signaling systems, a detailed mechanistic understanding of signal transduction has been hindered by the lack of a complete structural picture of the CSU and extended array. In this study, we present the structure of the complete CSU from phage E-gene lysed E. coli cells, determined using cryo-electron tomography and sub-tomogram averaging to 12 [A] resolution. Using AlphaFold2, we further predict the atomic structures of the CSUs constituent proteins as well as key protein-protein interfaces, enabling the assembly an all-atom CSU model, which we conformationally refine using our cryoET map. Molecular dynamics simulations of the resulting model provide new insight into the periplasmic organization of the complex, including novel interactions between neighboring receptor ligand binding domains. Our results further elucidate previously unresolved interactions between individual CheA domains, including an anti-parallel P1 dimer and non-productive binding mode between P1 and P4, enhancing our understanding of the structural mechanisms underlying CheA signaling and regulation.

microbiology↗

bioRxiv2022

Inhibited KdpFABC resides in an E1 off-cycle state

KdpFABC is a high-affinity prokaryotic K+ uptake system that forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB). At high K+ levels, KdpFABC needs to be inhibited to prevent excessive K+ accumulation to the point of toxicity. This is achieved by a phosphorylation of the serine residue in the TGES162 motif in the A domain of the pump subunit KdpB (KdpBS162-P). Here, we explore the structural basis of inhibition by KdpBS162 phosphorylation by determining the conformational landscape of KdpFABC under inhibiting and non-inhibiting conditions. Under turnover conditions, we identified a new inhibited KdpFABC conformation that we termed E1-P tight, which is not part of the canonical Post-Albers transport cycle of P-type ATPases. It likely represents the biochemically described stalled E1-P state adopted by KdpFABC upon KdpBS162 phosphorylation. The E1-P tight state exhibits a compact fold of the three cytoplasmic domains and is likely adopted when the transition from high-energy E1-P states to E2-P states is unsuccessful. This study represents a structural characterization of a biologically relevant off-cycle state in the P-type ATPase family and supports the emerging discussion of P-type ATPase regulation by such conformations.

molecular biology↗

bioRxiv2021

Structural, functional and computational studies of membrane recognition by Plasmodium Perforin-Like Proteins 1 and 2.

Perforin-like proteins (PLPs) play key roles in the mechanisms associated with parasitic disease caused by apicomplexans such as Plasmodium (malaria) and Toxoplasma. The T. gondii PLP1 (TgPLP1) mediates tachyzoite egress from cells, while the five Plasmodium PLPs carry out various roles in the life cycle of the parasite and with respect to the molecular basis of disease. Here we focus on Plasmodium vivax PLP1 and PLP2 (PvPLP1 and PvPLP2) compared to TgPLP1; PvPLP1 is important for invasion of mammalian hosts by the parasite and establishment of a chronic infection, PvPLP2 is important during the symptomatic blood stage of the parasite life cycle. Determination of the crystal structure of the membrane-binding APC{beta} domain of PvPLP1 reveals notable differences with that of TgPLP1, which are reflected in its inability to bind lipid bilayers in the way that TgPLP1 and PvPLP2 can be shown to. Molecular dynamics simulations combined with site-directed mutagenesis and functional assays allow a dissection of the binding interactions of TgPLP1 and PvPLP2 on lipid bilayers, and reveal a similar tropism for lipids found enriched in the inner leaflet of the mammalian plasma membrane. In addition to this shared mode of membrane binding PvPLP2 displays a secondary synergistic interaction side-on from its principal bilayer interface. This study underlines the substantial differences between the biophysical properties of the APC{beta} domains of Apicomplexan PLPs, which reflect their significant sequence diversity. Such differences will be important factors in determining the cell targeting and membrane-binding activity of the different proteins, in their different developmental roles within parasite life cycles.

biophysics↗

bioRxiv2020

Modified Bacterial Lipids Which Alter Membrane Surface Charge Reduce Binding of Antimicrobial Peptides

Antimicrobial peptides (AMPs) initiate killing of bacteria by binding to and destabilizing their membranes. The multiple peptide resistance factor (MprF) provides a defence mechanism for bacteria against a broad range of AMPs. MprF reduces the negative charge of both Gram-positive and Gram--negative bacterial membranes through enzymatic conversion of the anionic lipid phosphatidyl glycerol (PG) to either zwitterionic alanyl-phosphatidyl glycerol (Ala-PG) or cationic lysylphosphatidyl glycerol (Lys-PG). The resulting change in membrane charge is suggested to reduce AMP-membrane binding and hinder downstream AMP activity. Using molecular dynamics to investigate the effects of these modified lipids on AMP-binding to model membranes, we show that AMPs have substantially reduced affinity for model membranes containing Ala-PG or Lys-PG. A total of ~7000 simulations are used to define the relationship between bilayer composition and binding for 5 different membrane active peptides. The reduction of degree of interaction of a peptide with the membrane is shown to correlate with the change in membrane surface charge density. Free energy profile (potential of mean force) calculations reveal that these lipid modifications alter the energy barrier to peptide helix penetration of the bilayer. These results will enable us to guide design of novel peptides which address the issue of resistance via MprF-mediated membrane modification.

biophysics↗

bioRxiv2019

Insights into Membrane Protein-Lipid Interactions from Free Energy Calculations

Integral membrane proteins are regulated by specific interactions with lipids from the surrounding bilayer. The structures of protein-lipid complexes can be determined through a combination of experimental and computational approaches, but the energetic basis of these interactions is difficult to resolve. Molecular dynamic simulations provide the primary computational technique to estimate the free energies of these interactions. We demonstrate that the energetics of protein-lipid interactions may be reliably and reproducibly calculated using three simulation-based approaches: potential of mean force calculations. alchemical free energy perturbation, and well-tempered metadynamics. We employ these techniques within the framework of a coarse-grained force field, and apply them to both bacterial and mammalian membrane protein-lipid systems. We demonstrate good agreement between the different techniques, providing a robust framework for their automated implementation within a pipeline for annotation of newly determined membrane protein structures.

biochemistry↗

bioRxiv2017

Substrate-triggered position-switching of TatA and TatB is an essential step in the Escherichia coli Tat protein export pathway.

The twin arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. Here we use disulfide crosslinking and molecular modelling to show there are two binding sites for TatA/B proteins on TatC. TatA and TatB are each able to occupy both sites if they are the only TatA/B protein present. However, under resting conditions the sites are differentially occupied with TatB occupying the polar cluster site while TatA binds adjacently at the TatC transmembrane helix 6 binding site. When the Tat system is activated by the overproduction of a substrate, TatA and TatB switch their binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

microbiology↗