31 results found
Matthews S, 2022, Secondary structure and X-ray crystallographic analysis of the Glideosome-Associated Connector (GAC) from Toxoplasma gondii, Crystals, Vol: 12, Pages: 1-6, ISSN: 2073-4352
A model for parasitic motility has been proposed in which parasite filamentous actin (F-actin) is attached to surface adhesins by a large component of the glideosome, known as the glideosome-associated connector protein (GAC). This large 286 kDa protein interacts at the cytoplasmic face of the plasma membrane with the phosphatidic acid-enriched inner leaflet and cytosolic tails of surface adhesins to connect them to the parasite actomyosin system. GAC is observed initially to the conoid at the apical pole and re-localised with the glideosome to the basal pole in gliding parasite. GAC presumably functions in force transmission to surface adhesins in the plasma membrane and not in force generation. Proper connection between F-actin and the adhesins is as important for motility and invasion as motor operation itself. This notion highlights the need for new structural information on GAC interactions, which has eluded the field since its discovery. We have obtained crystals that diffracted to 2.6–2.9 Å for full-length GAC from Toxoplasma gondii in native and selenomethionine-labelled forms. These crystals belong to space group P212121; cell dimensions are roughly a = 119 Å, b = 123 Å, c = 221 Å, α = 90°, β = 90° and γ = 90° with 1 molecule per asymmetric unit, suggesting a more compact conformation than previously proposed
Rattu P, Glencross F, Mader SL, et al., 2021, Atomistic level characterisation of ssDNA translocation through the E. coli proteins CsgG and CsgF for nanopore sequencing, COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL, Vol: 19, Pages: 6417-6430, ISSN: 2001-0370
Matthews-Palmer T, Gonzalez-Rodriguez N, Calcraft T, et al., 2021, Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism, Journal of Structural Biology, Vol: 213, ISSN: 1047-8477
Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A ‘gatekeeper’ complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region’s structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.
Murphy P, Xu Y, Rouse SL, et al., 2020, Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer, NATURE COMMUNICATIONS, Vol: 11, ISSN: 2041-1723
Khalid S, Rouse SL, 2020, Simulation of subcellular structures, CURRENT OPINION IN STRUCTURAL BIOLOGY, Vol: 61, Pages: 167-172, ISSN: 0959-440X
Chorev DS, Tang H, Rouse SL, et al., 2020, The use of sonicated lipid vesicles for mass spectrometry of membrane protein complexes, NATURE PROTOCOLS, Vol: 15, Pages: 1690-1706, ISSN: 1754-2189
Matthews S, McKenna S, Malito E, et al., 2020, Structure, dynamics and immunogenicity of a catalytically inactive CXC Chemokine-degrading Protease SpyCEP from Streptococcus pyogenes, Computational and Structural Biotechnology Journal, Vol: 18, Pages: 650-660, ISSN: 2001-0370
Over 18 million disease cases and half a million deaths worldwide are estimated to be caused annually by Group A Streptococcus. A vaccine to prevent GAS disease is urgently needed. SpyCEP (Streptococcus pyogenes Cell-Envelope Proteinase) is a surface-exposed serine protease that inactivates chemokines, impairing neutrophil recruitment and bacterial clearance, and has shown promising immunogenicity in preclinical models. Although SpyCEP structure has been partially characterized, a more complete and higher resolution understanding of its antigenic features would be desirable prior to large scale manufacturing. To address these gaps and facilitate development of this globally important vaccine, we performed immunogenicity studies with a safety-engineered SpyCEP mutant, and comprehensively characterized its structure by combining X-ray crystallography, NMR spectroscopy and molecular dynamics simulations. We found that the catalytically-inactive SpyCEP antigen conferred protection similar to wild-type SpyCEP in a mouse infection model. Further, a new higher-resolution crystal structure of the inactive SpyCEP mutant provided new insights into this large chemokine protease comprising nine domains derived from two non-covalently linked fragments. NMR spectroscopy and molecular simulation analyses revealed conformational flexibility that is likely important for optimal substrate recognition and overall function. These combined immunogenicity and structural data demonstrate that the full-length SpyCEP inactive mutant is a strong candidate human vaccine antigen. These findings show how a multi-disciplinary study was used to overcome obstacles in the development of a GAS vaccine, an approach applicable to other future vaccine programs. Moreover, the information provided may also facilitate the structure-based discovery of small-molecule therapeutics targeting SpyCEP protease inhibition.
Miliara X, Tatsuta T, Berry J-L, et al., 2019, Structural determinants of lipid specificity within Ups/PRELI lipid transfer proteins, Nature Communications, Vol: 10, Pages: 1-15, ISSN: 2041-1723
Conserved lipid transfer proteins of the Ups/PRELI family regulate lipid accumulation in mitochondria by shuttling phospholipids in a lipid-specific manner across the intermembrane space. Here, we combine structural analysis, unbiased genetic approaches in yeast and molecular dynamics simulations to unravel determinants of lipid specificity within the conserved Ups/PRELI family. We present structures of human PRELID1–TRIAP1 and PRELID3b–TRIAP1 complexes, which exert lipid transfer activity for phosphatidic acid and phosphatidylserine, respectively. Reverse yeast genetic screens identify critical amino acid exchanges that broaden and swap their lipid specificities. We find that amino acids involved in head group recognition and the hydrophobicity of flexible loops regulate lipid entry into the binding cavity. Molecular dynamics simulations reveal different membrane orientations of PRELID1 and PRELID3b during the stepwise release of lipids. Our experiments thus define the structural determinants of lipid specificity and the dynamics of lipid interactions by Ups/PRELI proteins.
Chorev DS, Baker LA, Wu D, et al., 2018, Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry, SCIENCE, Vol: 362, Pages: 829-+, ISSN: 0036-8075
Rouse SL, Matthews SJ, Dueholm MS, 2018, Ecology and biogenesis of functional amyloids in pseudomonas, Journal of Molecular Biology, Vol: 430, Pages: 3685-3695, ISSN: 0022-2836
Functional amyloids can be found in the extracellular matrix produced by many bacteria during biofilm growth. They mediate the initial attachment of bacteria to surfaces and provide stability and functionality to mature biofilms. Efficient amyloid biogenesis requires a highly coordinated system of amyloid subunits, molecular chaperones and transport systems. The functional amyloid of Pseudomonas (Fap) represents such a system. Here, we review the phylogenetic diversification of the Fap system, its potential ecological role and the dedicated machinery required for Fap biogenesis, with a particular focus on the amyloid exporter FapF, the structure of which has been recently resolved. We also present a sequence covariance-based in silico model of the FapC fiber-forming subunit. Finally, we highlight key questions that remain unanswered and we believe deserve further attention by the scientific community.
Darvill N, Blake T, Rouse S, et al., 2018, Structural basis of phosphatidic acid sensing by APH in apicomplexan parasites, Structure, Vol: 26, Pages: 1059-1071.e6, ISSN: 0969-2126
Plasmodium falciparum and Toxoplasma gondii are obligate intracellular parasites that belong to the phylum of Apicomplexa and cause major human diseases. Their access to an intracellular lifestyle is reliant on the coordinated release of proteins from the specialized apical organelles called micronemes and rhoptries. A specific phosphatidic acid effector, the acylated pleckstrin homology domain-containing protein (APH) plays a central role in microneme exocytosis and thus is essential for motility, cell entry, and egress. TgAPH is acylated on the surface of the micronemes and recruited to phosphatidic acid (PA)-enriched membranes. Here, we dissect the atomic details of APH PA-sensing hub and its functional interaction with phospholipid membranes. We unravel the key determinant of PA recognition for the first time and show that APH inserts into and clusters multiple phosphate head-groups at the bilayer binding surface.
Rouse S, Stylianou F, wu G, et al., 2018, The FapF amyloid secretion transporter possesses an atypical asymmetric coiled coil, Journal of Molecular Biology, Vol: 430, Pages: 3863-3871, ISSN: 0022-2836
Gram-negative bacteria possess specialized biogenesis machineries that facilitate the export of amyloid subunits, the fibers of which are key components of their biofilm matrix. The secretion of bacterial functional amyloid requires a specialized outer-membrane protein channel through which unfolded amyloid substrates are translocated. We previously reported the crystal structure of the membrane-spanning domain of the amyloid subunit transporter FapF from Pseudomonas. However, the structure of the periplasmic domain, which is essential for amyloid transport, is yet to be determined. Here, we present the crystal structure of the N-terminal periplasmic domain at 1.8-Å resolution. This domain forms a novel asymmetric trimeric coiled coil that possesses a single buried tyrosine residue as well as an extensive hydrogen-bonding network within a glutamine layer. This new structural insight allows us to understand this newly described functional amyloid secretion system in greater detail.
rouse S, hawthorne, berry, et al., 2017, A new class of hybrid secretion system is employed in Pseudomonas amyloid biogenesis, Nature Communications, Vol: 8, ISSN: 2041-1723
Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix. The secretion of bacterial functional amyloid requires a bespoke outer-membrane protein channel through which unfolded amyloid substrates are translocated. Here, we combine X-ray crystallography, native mass spectrometry, single-channel electrical recording, molecular simulations and circular dichroism measurements to provide high-resolution structural insight into the functional amyloid transporter from Pseudomonas, FapF. FapF forms a trimer of gated β-barrel channels in which opening is regulated by a helical plug connected to an extended coil-coiled platform spanning the bacterial periplasm. Although FapF represents a unique type of secretion system, it shares mechanistic features with a diverse range of peptide translocation systems. Our findings highlight alternative strategies for handling and export of amyloid protein sequences.
Hedger G, Rouse SL, Domanski J, et al., 2017, Molecular simulations of cardiolipin interactions with the adenine nucleotide translocase, Publisher: SPRINGER, Pages: S287-S287, ISSN: 0175-7571
Otzen DE, Vad BS, Dueholm MS, et al., 2017, Self-organizing amyloid in bacteria, 19th IUPAB Congress / 11th EBSA Congress, Publisher: SPRINGER, Pages: S341-S341, ISSN: 0175-7571
Otzen DE, Vad BS, Dueholm MS, et al., 2017, Self-organizing amyloid in bacteria, 19th IUPAB Congress / 11th EBSA Congress, Publisher: SPRINGER, Pages: S98-S98, ISSN: 0175-7571
Rouse SL, Hawthorne W, Berry J, et al., 2017, Structural and Mechanistic Insights into Transport of Functional Amyloid Subunits across the Pseudomonas Outer Membrane, 61st Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 188A-188A, ISSN: 0006-3495
Hawthorne W, Rouse S, Sewell L, et al., 2016, Structural insights into functional amyloid inhibition in Gram –ve bacteria, Biochemical Society Transactions, Vol: 44, Pages: 1643-1649, ISSN: 1470-8752
Amyloids are proteinaceous aggregates known for their role in debilitating degenerative diseases involving protein dysfunction. Many forms of functional amyloid are also produced in nature and often these systems require careful control of their assembly to avoid the potentially toxic effects. The best-characterised functional amyloid system is the bacterial curli system. Three natural inhibitors of bacterial curli amyloid have been identified and recently characterised structurally. Here, we compare common structural features of CsgC, CsgE and CsgH and discuss the potential implications for general inhibition of amyloid.
Matthews SJ, rouse S, hawthorne, et al., 2016, Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter, Acta Crystallographica Section F: Structural Biology Communications, Vol: F72, Pages: 892-896, ISSN: 2053-230X
Bacteria often produce extracellular amyloid fibresviaa multi-componentsecretion system. Aggregation-prone, unstructured subunits cross the periplasmand are secreted through the outer membrane, after which they self-assemble.Here, significant progress is presented towards solving the high-resolutioncrystal structure of the novel amyloid transporter FapF fromPseudomonas,which facilitates the secretion of the amyloid-forming polypeptide FapC acrossthe bacterial outer membrane. This represents the first step towards obtainingstructural insight into the products of thePseudomonas fapoperon. Initialattempts at crystallizing full-length and N-terminally truncated constructs byrefolding techniques were not successful; however, after preparing FapF106–430from the membrane fraction, reproducible crystals were obtained using thesitting-drop method of vapour diffusion. Diffraction data have been processedto 2.5 A ̊resolution. These crystals belonged to the monoclinic space groupC121,with unit-cell parametersa= 143.4,b= 124.6,c= 80.4 A ̊, = = 90, = 96.32 and three monomers in the asymmetric unit. It was found that the switch tocomplete detergent exchange into C8E4 was crucial for forming well diffractingcrystals, and it is suggested that this combined with limited proteolysis is apotentially useful protocol for membrane -barrel protein crystallography. Thethree-dimensional structure of FapF will provide invaluable information on themechanistic differences of biogenesis between the curli and Fap functionalamyloid systems.
Hedger G, Rouse SL, Domanski J, et al., 2016, Lipid-loving ANTs: molecular simulations of cardiolipin interactions and the organization of the adenine nucleotide translocase in model mitochondrial membranes, Biochemistry, Vol: 55, Pages: 6238-6249, ISSN: 1520-4995
The exchange of ADP and ATP across the inner mitochondrial membrane is a fundamental cellular process. This exchange is facilitated by the adenine nucleotide translocase, the structure and function of which are critically dependent on the signature phospholipid of mitochondria, cardiolipin (CL). Here we employ multiscale molecular dynamics simulations to investigate CL interactions within a membrane environment. Using simulations at both coarse-grained and atomistic resolutions, we identify three CL binding sites on the translocase, in agreement with those seen in crystal structures and inferred from nuclear magnetic resonance measurements. Characterization of the free energy landscape for lateral lipid interaction via potential of mean force calculations demonstrates the strength of interaction compared to those of binding sites on other mitochondrial membrane proteins, as well as their selectivity for CL over other phospholipids. Extending the analysis to other members of the family, yeast Aac2p and mouse uncoupling protein 2, suggests a degree of conservation. Simulation of large patches of a model mitochondrial membrane containing multiple copies of the translocase shows that CL interactions persist in the presence of protein–protein interactions and suggests CL may mediate interactions between translocases. This study provides a key example of how computational microscopy may be used to shed light on regulatory lipid–protein interactions.
Rouse SL, Sansom MSP, 2015, Interactions of Lipids and Detergents with a Viral Ion Channel Protein: Molecular Dynamics Simulation Studies, JOURNAL OF PHYSICAL CHEMISTRY B, Vol: 119, Pages: 764-772, ISSN: 1520-6106
Tan J, Rouse SL, Li D, et al., 2014, A conformational landscape for alginate secretion across the outer membrane of Pseudomonas aeruginosa, ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, Vol: 70, Pages: 2054-2068, ISSN: 2059-7983
Lonsdale R, Rouse SL, Sansom MSP, et al., 2014, A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes, PLOS COMPUTATIONAL BIOLOGY, Vol: 10
Roussel G, Rouse SL, Sansom MSP, et al., 2014, The role of 2-methyl-2, 4-pentanediol in sodium dodecyl sulfate micelle dissociation unveiled by dynamic light scattering and molecular dynamics simulations, COLLOIDS AND SURFACES B-BIOINTERFACES, Vol: 114, Pages: 357-362, ISSN: 0927-7765
Rouse SL, Caffrey M, Sansom MSP, 2014, A Conformational Landscape for Alginate Secretion Across the Outer Membrane of Pseudomonas Aeruginosa, 58th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 733A-733A, ISSN: 0006-3495
Rouse SL, Marcoux J, Robinson CV, et al., 2013, Dodecyl Maltoside Protects Membrane Proteins In Vacuo, BIOPHYSICAL JOURNAL, Vol: 105, Pages: 648-656, ISSN: 0006-3495
Roussel G, Rouse SL, Sansom MS, et al., 2013, Characterization and optimization of a novel protein refolding methodology, 9th European-Biophysical-Societies-Association Congress, Publisher: SPRINGER, Pages: S69-S69, ISSN: 0175-7571
Rodriguez F, Rouse SL, Tait CE, et al., 2013, Structural model for the protein-translocating element of the twin-arginine transport system, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 110, Pages: E1092-E1101, ISSN: 0027-8424
Roussel G, Rouse SL, Sansom M, et al., 2013, Characterization and optimization of a novel protein refolding methodology, 39th Edition of the Joint European Days on Equilibrium between Phases (JEEP), Publisher: E D P SCIENCES, ISSN: 2261-236X
Rouse SL, Sansom MSP, 2011, Molecular Dynamics Simulations of the BM2 Proton Channel: Interactions with Lipids and Detergents, 55th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 346-346, ISSN: 0006-3495
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