David Sharples

Molecular Basis of Alternating Access Membrane Transport by the Sodium-Hydantoin Transporter Mhp1

Triangulating to Mechanism Cellular uptake and release of a variety of substrates are mediated by secondary transporters, but no crystal structures are known for all three fundamental states of the transport cycle, which has limited explanations for their proposed mechanisms. Shimamura et al. (p. 470) report a 3.8-angstrom structure of the inward-facing conformation of the bacterial sodium-benzylhydantoin transport protein, Mhp1, complementing the other two available structures. Molecular modeling for the interconversions of these structures shows a simple rigid body rotation of four helices relative to the rest of the structure in which the protein switches reversibly from outward- to inward-facing. Three complementary crystal structures reveal the mechanism of a transport protein in molecular dynamics simulations. The structure of the sodium-benzylhydantoin transport protein Mhp1 from Microbacterium liquefaciens comprises a five-helix inverted repeat, which is widespread among secondary transporters. Here, we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 angstroms resolution, complementing its previously described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations, we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward-facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8, and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.

Molecular mechanism of ligand recognition by membrane transport protein, Mhp1

The hydantoin transporter Mhp1 is a sodium‐coupled secondary active transport protein of the nucleobase‐cation‐symport family and a member of the widespread 5‐helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site‐directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5‐substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5‐substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5‐(2‐naphthylmethyl)‐L‐hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.

An ace up their sleeve: a transcriptomic approach exposes the AceI efflux protein of Acinetobacter baumannii and reveals the drug efflux potential hidden in many microbial pathogens.

The era of antibiotics as a cure-all for bacterial infections appears to be coming to an end. The emergence of multidrug resistance in many hospital-associated pathogens has resulted in “superbugs” that are effectively untreatable. Multidrug efflux pumps are well known mediators of bacterial drug resistance. Genome sequencing efforts have highlighted an abundance of putative efflux pump genes in bacteria. However, it is not clear how many of these pumps play a role in antimicrobial resistance. Efflux pump genes that participate in drug resistance can be under tight regulatory control and expressed only in response to substrates. Consequently, changes in gene expression following antimicrobial shock may be used to identify efflux pumps that mediate antimicrobial resistance. Using this approach we have characterized several novel efflux pumps in bacteria. In one example we recently identified the Acinetobacter chlorhexidine efflux protein (AceI) efflux pump in Acinetobacter. AceI is a prototype for a novel family of multidrug efflux pumps conserved in many proteobacterial lineages. The discovery of this family raises the possibility that additional undiscovered intrinsic resistance proteins may be encoded in the core genomes of pathogenic bacteria.

Probing the contacts of a low-affinity substrate with a membrane-embedded transport protein using 1H-13C cross-polarisation magic-angle spinning solid-state NMR

Abstract Solid-state NMR combined with sample deuteration was used to probe the proximity of the low-affinity substrate D-glucose to its binding site within the Escherichia coli sugar transport protein GalP. Samples of E. coli inner membranes with amplified expression of GalP were incubated in D2O with D-[13C6]glucose and 13C NMR signals from the substrate were assigned in two-dimensional dipolar-assisted rotational resonance (DARR) spectra. The signals were confirmed as representing D-glucose bound to GalP as the peaks were abolished after the substrate was displaced from the specific site with the inhibitor forskolin. The 13C chemical shift values for D-[13C6]glucose in solution revealed some differences compared to those for ligand bound to GalP, the differences being most pronounced for positions C1 and C2, and especially for C1 in the α-anomer. 13C cross-polarization build-up was measured for C1 and C2 of D-[13C6]glucose and D-[2H7, 13C6]glucose in GalP membranes suspended in D2O. The build-up curves for the deuterated substrate reflect intermolecular 1H-13C interactions between the protein and the fully deuterated substrate; the signal build-up suggests that the α-anomer is situated closer to the protein binding site than is the β-anomer, consistent with its relatively high signal intensities and more pronounced chemical shift changes in the 2D-correlation spectra. These results demonstrate the utility of solid-state NMR combined with sample deuteration for mapping the binding interface of low affinity ligands with membrane proteins.

Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity.

This work reports the evolutionary relationships, amplified expression, functional characterization 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 12 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 determined 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 energized E. coli whole cells conformed to Michaelis-Menten kinetics with an apparent affinity (Kmapp) of 24 ± 3 μM, 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 recognizes some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides. PucI(His6) was solubilized from inner membranes using n-dodecyl-β-d-maltoside and purified. The isolated protein contained a substantial proportion of α-helix secondary structure, consistent with the predictions, and a 3D model was therefore constructed on a template of the Mhp1 structure, which aided localization of the potential ligand binding site in PucI.

Topological dissection of the membrane transport protein Mhp1 derived from cysteine accessibility and mass spectrometry

Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallized in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-l-hydantoin (L-BH) does not shift this conformational equilibrium, but systematic co-addition of the two results in an attenuation of labeling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion–substrate-coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilization of structure. The methodology combines covalent labeling, mass spectrometry, and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein’s conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions.

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.

Screening of candidate substrates and coupling ions of transporters by thermostability shift assays

Substrates of most transport proteins have not been identified, limiting our understanding of their role in physiology and disease. Traditional identification methods use transport assays with radioactive compounds, but they are technically challenging and many compounds are unavailable in radioactive form or are prohibitively expensive, precluding large-scale trials. Here, we present a high-throughput screening method that can identify candidate substrates from libraries of unlabeled compounds. The assay is based on the principle that transport proteins recognize substrates through specific interactions, which lead to enhanced stabilization of the transporter population in thermostability shift assays. Representatives of three different transporter (super)families were tested, which differ in structure as well as transport and ion coupling mechanisms. In each case, the substrates were identified correctly from a large set of chemically related compounds, including stereo-isoforms. In some cases, stabilization by substrate binding was enhanced further by ions, providing testable hypotheses on energy coupling mechanisms.

Pacing across the membrane: the novel PACE family of efflux pumps is widespread in Gram-negative pathogens

The proteobacterial antimicrobial compound efflux (PACE) family of transport proteins was only recently described. PACE family transport proteins can confer resistance to a range of biocides used as disinfectants and antiseptics, and are encoded by many important Gram-negative human pathogens. However, we are only just beginning to appreciate the range of functions and the mechanism(s) of transport operating in these proteins. Genes encoding PACE family proteins are typically conserved in the core genomes of bacterial species rather than on recently acquired mobile genetic elements, suggesting that they confer important core functions in addition to biocide resistance. Three-dimensional structural information is not yet available for PACE family proteins. However, PACE proteins have several very highly conserved amino acid sequence motifs that are likely to be important for substrate transport. PACE proteins also display strong amino acid sequence conservation between their N— and C-terminal halves, suggesting that they evolved by duplication of an ancestral protein comprised of two transmembrane helices. In light of their drug resistance functions in Gram-negative pathogens, PACE proteins should be the subject of detailed future investigation.

A novel and fast purification method for nucleoside transporters

Nucleoside transporters (NTs) play critical biological roles in humans, and to understand the molecular mechanism of nucleoside transport requires high-resolution structural information. However, the main bottleneck for structural analysis of NTs is the production of pure, stable, and high quality native protein for crystallization trials. Here we report a novel membrane protein expression and purification strategy, including construction of a high-yield membrane protein expression vector, and a new and fast purification protocol for NTs. The advantages of this strategy are the improved time efficiency, leading to high quality, active, stable membrane proteins, and the efficient use of reagents and consumables. Our strategy might serve as a useful point of reference for investigating NTs and other membrane proteins by clarifying the technical points of vector construction and improvements of membrane protein expression and purification.