Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via ...PepT1 and PepT2, which belong to the proton‐dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3‐Å resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge‐like movement within the C‐terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide‐binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.
Proton‐dependent oligopeptide transporters are required for the uptake of diet‐derived peptides in all kingdoms of life. The crystal structure of a bacterial transporter in the inward open conformation, together with a published structure in an occluded conformation, reveals the peptide transport mechanism.
Proton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters. Recent crystal structures suggest the MFS fold facilitates transport through ...rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood. Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport.
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•New higher-resolution structure of PepTSo•Salt bridges stabilizing outward-facing conformations are identified•The conserved prolines in helix 8 are shown to be important•The first two helices in each inverted-topology repeat form part of a gate
Fowler et al. use biophysical and modeling approaches to identify salt bridges in two peptide transporters that stabilize their outward-facing conformations. Their results also suggest that the first two helices in each inverted-topology repeat unit forms part of either of the two gates.
The UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium) to enable the measurement of MICs ...of 14 different antituberculosis (anti-TB) compounds for >30,000 clinical
isolates. Unlike the MYCOTB plate, on which the UKMYC5 plate is based, the UKMYC5 plate includes two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds. UKMYC5 plates were tested by seven laboratories on four continents by use of a panel of 19 external quality assessment (EQA) strains, including H37Rv. To assess the optimal combination of reading method and incubation time, MICs were measured from each plate by two readers, using three methods (mirrored box, microscope, and Vizion digital viewing system), after 7, 10, 14, and 21 days of incubation. In addition, all EQA strains were subjected to whole-genome sequencing and phenotypically characterized by the 7H10/7H11 agar proportion method (APM) and by use of MGIT960 mycobacterial growth indicator tubes. We concluded that the UKMYC5 plate is optimally read using the Vizion system after 14 days of incubation, achieving an interreader agreement of 97.9% and intra- and interlaboratory reproducibility rates of 95.6% and 93.1%, respectively. The mirrored box had a similar reproducibility. Strains classified as resistant by APM, MGIT960, or the presence of mutations known to confer resistance consistently showed elevated MICs compared to those for strains classified as susceptible. Finally, the UKMYC5 plate records intermediate MICs for one strain for which the APM measured MICs close to the applied critical concentration, providing early evidence that the UKMYC5 plate can quantitatively measure the magnitude of resistance to anti-TB compounds that is due to specific genetic variation.
Abstract
Summary
Viral sequence data from clinical samples frequently contain contaminating human reads, which must be removed prior to sharing for legal and ethical reasons. To enable host read ...removal for SARS-CoV-2 sequencing data on low-specification laptops, we developed ReadItAndKeep, a fast lightweight tool for Illumina and nanopore data that only keeps reads matching the SARS-CoV-2 genome. Peak RAM usage is typically below 10 MB, and runtime less than 1 min. We show that by excluding the polyA tail from the viral reference, ReadItAndKeep prevents bleed-through of human reads, whereas mapping to the human genome lets some reads escape. We believe our test approach (including all possible reads from the human genome, human samples from each of the 26 populations in the 1000 genomes data and a diverse set of SARS-CoV-2 genomes) will also be useful for others.
Availability and implementation
ReadItAndKeep is implemented in C++, released under the MIT license, and available from https://github.com/GenomePathogenAnalysisService/read-it-and-keep.
Supplementary information
Supplementary data are available at Bioinformatics online.
Drug resistant Mycobacterium tuberculosis, which mostly results from single nucleotide polymorphisms in antibiotic target genes, poses a major threat to tuberculosis treatment outcomes. Relative ...binding free energy (RBFE) calculations can rapidly predict the effects of mutations, but this approach has not been tested on large, complex proteins. We use RBFE calculations to predict the effects of M. tuberculosis RNA polymerase and DNA gyrase mutations on rifampicin and moxifloxacin susceptibility respectively. These mutations encompass a range of amino acid substitutions with known effects and include large steric perturbations and charged moieties. We find that moderate numbers (n = 3–15) of short RBFE calculations can predict resistance in cases where the mutation results in a large change in the binding free energy. We show that the method lacks discrimination in cases with either a small change in energy or that involve charged amino acids, and we investigate how these calculation errors may be decreased.
Relative binding free energy (RBFE) calculations pose a potential avenue to predict antibiotic resistance associated with single nucleotide polymorphisms. We investigated whether RBFE calculations could be applied to two large, complex and clinically important protein targets in Mycobacterium tuberculosis. We show that RBFE calculations can predict resistance in cases where the mutation results in a large change in the binding free energy and investigate the sources of error for more complex mutations.
Cell membranes are crowded and complex environments. To investigate the effect of protein-lipid interactions on dynamic organization in mammalian cell membranes, we have performed coarse-grained ...molecular dynamics simulations containing >100 copies of an inwardly rectifying potassium (Kir) channel which forms specific interactions with the regulatory lipid phosphatidylinositol 4,5-bisphosphate (PIP
). The tendency of protein molecules to cluster has the effect of organizing the membrane into dynamic compartments. At the same time, the diversity of lipids present has a marked effect on the clustering behavior of ion channels. Sub-diffusion of proteins and lipids is observed. Protein crowding alters the sub-diffusive behavior of proteins and lipids such as PIP
which interact tightly with Kir channels. Protein crowding also affects bilayer properties, such as membrane undulations and bending rigidity, in a PIP
-dependent manner. This interplay between the diffusion and the dynamic organization of Kir channels may have important implications for channel function.
Tuberculosis (TB) is the leading cause of death from a single infectious agent and in 2019 an estimated 10 million people worldwide contracted the disease. Although treatments for TB exist, continual ...emergence of drug-resistant variants necessitates urgent development of novel antituberculars. An important new target is the lipid transporter MmpL3, which is required for construction of the unique cell envelope that shields Mycobacterium tuberculosis (Mtb) from the immune system. However, a structural understanding of the mutations in Mtb MmpL3 that confer resistance to the many preclinical leads is lacking, hampering efforts to circumvent resistance mechanisms. Here, we present the cryoelectron microscopy structure of Mtb MmpL3 and use it to comprehensively analyze the mutational landscape of drug resistance. Our data provide a rational explanation for resistance variants local to the central drug binding site, and also highlight a potential alternative route to resistance operating within the periplasmic domain.
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•Cryo-EM structure of M. tuberculosis MmpL3 determined at 3.0 Å resolution•An LMNG molecule within the periplasmic cavity suggests the TMM export pathway•Comprehensive structural mapping of resistance-conferring MmpL3 variants•Genome-mined MmpL3 mutations indicate minimal pre-existing resistance
Adams et al. report a structure of the M. tuberculosis glycolipid transporter MmpL3, a protein essential to envelope biogenesis and an emergent therapeutic target. The structure reveals the probable lipid export pathway, and mapping of resistance-conferring mutations suggests the existence of multiple routes to preclinical inhibitor evasion.
The ease with which a cell membrane can bend and deform is important for a wide range of biological functions. Peripheral proteins that induce curvature in membranes (e.g. BAR domains) have been ...studied for a number of years. Little is known, however, about the effect of integral membrane proteins on the stiffness of a membrane (characterised by the bending rigidity, K
). We demonstrate by computer simulation that adding integral membrane proteins at physiological densities alters the stiffness of the membrane. First we establish that the coarse-grained MARTINI forcefield is able to accurately reproduce the bending rigidity of a small patch of 1500 phosphatidyl choline lipids by comparing the calculated value to both experiment and an atomistic simulation of the same system. This enables us to simulate the dynamics of large (ca. 50 000 lipids) patches of membrane using the MARTINI coarse-grained description. We find that altering the lipid composition changes the bending rigidity. Adding integral membrane proteins to lipid bilayers also changes the bending rigidity, whilst adding a simple peripheral membrane protein has no effect. Our results suggest that integral membrane proteins can have different effects, and in the case of the bacterial outer membrane protein, BtuB, the greater the density of protein, the larger the reduction in stiffness.
The emergence of antimicrobial resistance threatens modern medicine and necessitates more personalized treatment of bacterial infections. Sequencing the whole genome of the pathogen(s) in a clinical ...sample offers one way to improve clinical microbiology diagnostic services, and has already been adopted for tuberculosis in some countries. A key weakness of a genetics clinical microbiology is it cannot return a result for rare or novel genetic variants and therefore predictive methods are required. Non-synonymous mutations in the
gene can be successfully classified as either conferring resistance (or not) by calculating their effect on the binding free energy of the antibiotic, trimethoprim. The underlying approach, alchemical free energy methods, requires large numbers of molecular dynamics simulations to be run. We show that a large number (
= 15) of binding free energies calculated from a series of very short (50 ps) molecular dynamics simulations are able to satisfactorily classify all seven mutations in our clinically derived testset. A result for a single mutation could therefore be returned in less than an hour, thereby demonstrating that this or similar methods are now sufficiently fast and reproducible for clinical use.
The influenza virus is surrounded by an envelope composed of a lipid bilayer and integral membrane proteins. Understanding the structural dynamics of the membrane envelope provides biophysical ...insights into aspects of viral function, such as the wide-ranging survival times of the virion in different environments. We have combined experimental data from X-ray crystallography, nuclear magnetic resonance spectroscopy, cryo-electron microscopy, and lipidomics to build a model of the intact influenza A virion. This is the basis of microsecond-scale coarse-grained molecular dynamics simulations of the virion, providing simulations at different temperatures and with varying lipid compositions. The presence of the Forssman glycolipid alters a number of biophysical properties of the virion, resulting in reduced mobility of bilayer lipid and protein species. Reduced mobility in the virion membrane may confer physical robustness to changes in environmental conditions. Our simulations indicate that viral spike proteins do not aggregate and thus are competent for multivalent immunoglobulin G interactions.
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•Microsecond timescale molecular dynamics simulations of enveloped influenza virion•Forssman glycolipid alters the biophysical properties of the influenza A virion•Spacing of the spike glycoproteins is compatible with bivalent antibody association•Mobility of viral proteins influences distribution of lipids in the viral envelope
Reddy et al. performed the first microsecond timescale coarse-grained molecular dynamics simulations of enveloped virions in explicit solvent. The simulated properties of the influenza A virion were consistent with experimental measurements, and revealed that the Forssman glycolipid affects several biophysical characteristics of the virion.
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