Crystal structure of mannose specific IIA subunit of phosphotransferase system from Streptococcus pneumoniae

Streptococcus pneumoniae is a frequent bacterial pathogen of the human respiratory tract causing pneumonia, meningitis and sepsis, a serious healthcare burden in all age groups. S. pneumoniae lacks complete respiratory chain and relies on carbohydrate fermentation for energy generation. One of the essential components for this includes the mannose phosphotransferase system (Man-PTS), which plays a central role in glucose transport and exhibits a broad specificity for a range of hexoses. Importantly, Man-PTS is involved in the global regulation of gene expression for virulence determinants. We herein report the three-dimensional structure of the EIIA domain of S. pneumoniae mannose phosphotransferase system (SpEIIA-Man). Our structure shows a dimeric arrangement of EIIA and reveals a detailed molecular description of the active site. Since PTS transporters are exclusively present in microbes and sugar transporters have already been suggested as valid targets for antistreptococcal antibiotics, our work sets foundation for the future development of antimicrobial strategies against Streptococcus pneumoniae.ISSN:1420-304

Production of membrane proteins for characterisation of their pheromone-sensing and antimicrobial resistance functions

AbstractDespite the importance of membrane proteins in cellular processes, studies of these hydrophobic proteins present major technical challenges, including expression and purification for structural and biophysical studies. A modified strategy of that proposed previously by Saidijam et al. (2005) and others, for the routine expression of bacterial membrane proteins involved in environmental sensing and antimicrobial resistance (AMR), is proposed which results in purification of sufficient proteins for biophysical experiments. We report expression successes amongst a collection of enterococcal vancomycin resistance membrane proteins: VanTG, VanTG-M transporter domain, VanZ and the previously characterised VanS (A-type) histidine protein kinase (HPK). Using the same strategy, we report on the successful amplification and purification of intact BlpH and ComD2 HPKs of Streptococcus pneumoniae. Near-UV circular dichroism revealed both recombinant proteins bound their pheromone ligands BlpC and CSP2. Interestingly, CSP1 also interacted with ComD. Finally, we evaluate the alternative strategy for studying sensory HPKs involving isolated soluble sensory domain fragments, exemplified by successful production of VicKESD of Enterococcus faecalis VicK. Purified VicKESD possessed secondary structure post-purification. Thermal denaturation experiments using far-UV CD, a technique which can be revealing regarding ligand binding, revealed that: (a) VicKESD denaturation occurs between 15 and 50 °C; and (b) reducing conditions did not detectably affect denaturation profiles suggesting reducing conditions per se are not directly sensed by VicKESD. Our findings provide information on a modified strategy for the successful expression, production and/or storage of bacterial membrane HPKs, AMR proteins and sensory domains for their future crystallisation, and ligand binding studies

The Gelatinase Biosynthesis‐Activating Pheromone binds and stabilizes the FsrB membrane protein in Enterococcus faecalis quorum sensing

Quorum‐sensing mechanisms regulate gene expression in response to changing cell‐population density detected through pheromones. In Enterococcus faecalis, Fsr quorum sensing produces and responds to the gelatinase biosynthesis‐activating pheromone (GBAP). Here we establish that the enterococcal FsrB membrane protein has a direct role connected with GBAP by showing that GBAP binds to purified FsrB. Far‐UV CD measurements demonstrated a predominantly α‐helical protein exhibiting a small level of conformational flexibility. Fivefold (400 μm) GBAP stabilised FsrB (80 μm) secondary structure. FsrB thermal denaturation in the presence and absence of GBAP revealed melting temperatures of 70.1 and 60.8 °C, respectively, demonstrating GBAP interactions and increased thermal stability conferred by GBAP. Addition of GBAP also resulted in tertiary structural changes, confirming GBAP binding

Crystal structure of rhodopsin in complex with a mini-G_o sheds light on the principles of G protein selectivity

Selective coupling of G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) to specific Gα-protein subtypes is critical to transform extracellular signals, carried by natural ligands and clinical drugs, into cellular responses. At the center of this transduction event lies the formation of a signaling complex between the receptor and G protein. We report the crystal structure of light-sensitive GPCR rhodopsin bound to an engineered mini-Go protein. The conformation of the receptor is identical to all previous structures of active rhodopsin, including the complex with arrestin. Thus, rhodopsin seems to adopt predominantly one thermodynamically stable active conformation, effectively acting like a “structural switch,” allowing for maximum efficiency in the visual system. Furthermore, our analysis of the well-defined GPCR–G protein interface suggests that the precise position of the carboxyl-terminal “hook-like” element of the G protein (its four last residues) relative to the TM7/helix 8 (H8) joint of the receptor is a significant determinant in selective G protein activation

The allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity

This work reports the evolutionary relationships, amplified expression, functional characterisation and purification of the putative allantoin transport protein, PucI, from Bacillus subtilis. Sequence alignments and phylogenetic analysis confirmed close evolutionary relationships between PucI and membrane proteins of the nucleobase-cation-symport-1 family of secondary active transporters. These include the sodium-coupled hydantoin transport protein, Mhp1, from Microbacterium liquefaciens and related proteins from bacteria, fungi and plants. Membrane topology predictions for PucI were consistent with twelve putative transmembrane-spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. The pucI gene was cloned into the IPTG-inducible plasmid pTTQ18 upstream from an in-frame hexahistidine tag and conditions are described for optimal amplified expression of the PucI(His6) protein in Escherichia coli to a level of about 5% in inner membranes. Initial rates of inducible PucI-mediated uptake of 14C-allantoin into energised Escherichia coli whole cells conformed to Michaelis-Menten kinetics with an apparent affinity (Kmapp) of 24 ± 3M, therefore confirming that PucI is a medium affinity transporter of allantoin. Dependence of allantoin transport on sodium was not apparent. Competitive uptake experiments showed that PucI recognises some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides. PucI(His6) was solubilised from inner membranes using n-dodecyl-β-D-maltoside and purified. The isolated protein comprised a substantial proportion of α-helix secondary structure, consistent with the predictions, and a three-dimensional model was therefore constructed on a template of the Mhp1 structure, which aided localisation of the potential ligand binding site in PucI

Microbial expression systems for membrane proteins

Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. We provide an overview of the host strains, tags and promoters that, in our experience, are most likely to yield protein suitable for structural and functional characterization. We also catalogue the detergents used for solubilization and crystallization studies of these proteins. Here, we emphasize a combination of practical methods, not necessarily high-throughput, which can be implemented in any laboratory equipped for recombinant DNA technology and microbial cell culture

Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions

This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developedatLeedsalongsideProfessor SteveBaldwintowhomthisreviewisdedicated.Italsoreviewstwo biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins – synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerization state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs

Ultrafast structural changes direct the first molecular events of vision

視覚に関わるタンパク質の超高速分子動画 --薄暗いところで光を感じる仕組み--. 京都大学プレスリリース. 2023-03-23.Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation

Structure-activity relationships of membrane proteins:the NCS1 family of transporters and sensor kinases of two component systems

Membrane proteins perform in many critical physiological processes, constituting 25 – 30% of prokaryotic and eukaryotic genomes, and represent up to 70% of current drug targets. However, the number of three dimensional structures of membrane proteins elucidated has been modest in comparison with those of soluble proteins, and this is attributable to three major bottlenecks: expression, purification and structure determination of these hydrophobic proteins. A genomic approach was conducted to overcome these bottlenecks for two families of membrane proteins: (1) the NCS1 family of transporters and; (2) the histidine kinases of two-component signal transduction systems of E. faecalis. The genes encoding these membrane proteins were cloned separately into plasmid pTTQ18-His6 and subjected to expression trials in Escherichia coli. Purification of the recombinant intact proteins from E. coli membranes was undertaken using Ni-NTA chromatography, and for proteins that were produced in sufficient quantities crystallisation trials and investigations of structural characteristics were performed. The activities of membrane-bound and/or purified proteins were also tested, and in the case of the histidine kinases, these activity assays were expanded to successfully identify modulating signals. This is the first time that direct in vitro activity assays using intact proteins have proven successful for identification of environmental ‗signals‘ in enterococci. In this study, thirteen NCS1 transporters were cloned. Twelve of the thirteen cloned proteins were expressed in E. coli membranes, seven were successfully purified, and substrates transported by five of these were confirmed. Three of these transporters, were further characterised, including; (1) a cytosine transporter – CodB, (2) an allantoin transporter – PucI, and (3) a uracil transporter – PA0443, and their substrate specificities and kinetics were also determined. All sixteen membrane histidine kinases of E. faecalis were successfully cloned (six were assigned to me in a joint project with Dr Hayley Yuille). Fifteen of the sixteen histidine kinases were successfully expressed in E. coli, and thirteen were purified. Eleven of the fifteen membrane-bound histidine kinases and twelve of the thirteen purified proteins were active in autophosphorylation activity assays. Three histidine kinases were studied further, including; (1) EF3197, which responded to increasing concentrations of a reducing agent, suggesting a possible role in redox-sensing, (2) EF1051, which was further purified by size exclusion chromatography and showed promising results in crystallisation trials for future structural determination, and (3) EF1820 (FsrC), which was activated in in vitro assays by its pheromone signal, gelatinase biosynthesis-activating pheromone (GBAP). This study showed GBAP-induced activity of FsrC was inhibited by the anti-HIV inhibitor, siamycin I, identifying unequivocally and for the first time, the target protein for siamycin I inhibition in the Fsr pathway. This study presents a successful genomic approach for the production of milligram quantities of membrane proteins leading to characterisation and crystallisation studies. The methods can now be applied to further membrane proteins

Membrane Sensor Histidine Kinases: Insights from Structural, Ligand and Inhibitor Studies of Full-Length Proteins and Signalling Domains for Antibiotic Discovery

There is an urgent need to find new antibacterial agents to combat bacterial infections, including agents that inhibit novel, hitherto unexploited targets in bacterial cells. Amongst novel targets are two-component signal transduction systems (TCSs) which are the main mechanism by which bacteria sense and respond to environmental changes. TCSs typically comprise a membrane-embedded sensory protein (the sensor histidine kinase, SHK) and a partner response regulator protein. Amongst promising targets within SHKs are those involved in environmental signal detection (useful for targeting specific SHKs) and the common themes of signal transmission across the membrane and propagation to catalytic domains (for targeting multiple SHKs). However, the nature of environmental signals for the vast majority of SHKs is still lacking, and there is a paucity of structural information based on full-length membrane-bound SHKs with and without ligand. Reasons for this lack of knowledge lie in the technical challenges associated with investigations of these relatively hydrophobic membrane proteins and the inherent flexibility of these multidomain proteins that reduces the chances of successful crystallisation for structural determination by X-ray crystallography. However, in recent years there has been an explosion of information published on (a) methodology for producing active forms of full-length detergent-, liposome- and nanodisc-solubilised membrane SHKs and their use in structural studies and identification of signalling ligands and inhibitors; and (b) mechanisms of signal sensing and transduction across the membrane obtained using sensory and transmembrane domains in isolation, which reveal some commonalities as well as unique features. Here we review the most recent advances in these areas and highlight those of potential use in future strategies for antibiotic discovery. This Review is part of a Special Issue entitled “Interactions of Bacterial Molecules with Their Ligands and Other Chemical Agents” edited by Mary K. Phillips-Jones