Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that ...utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.
Synopsis
Bacteria employ dedicated ABC transporters to secrete antibacterial peptides. X‐ray structure analysis shows that the antibacterial lasso peptide transporter McjD, in contrast to other ABC transporters, lacks a stable open cavity conformation, thus preventing the influx of the exported toxic peptide.
The structure of the ABC transporter McjD was determined in two distinct conformations, apo and nucleotide‐bound.
Both conformations show an occluded cavity at both sides of the membrane.
Pulsed electron‐electron double resonance (PELDOR) measurements in the presence of the antibacterial peptide MccJ25 did not identify a stable open‐conformation.
Cross‐linking studies in proteoliposomes show that the cavity opens transiently to release the bound substrate.
Transient opening of the antibacterial peptide transporter McjD prevents the influx of its toxic substrate MccJ25 and distinguishes it from other multi‐drug transporters.
Potassium‐coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions ...via phospho‐regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo‐EM structures of human KCC3b and KCC1, revealing structural determinants for phospho‐regulation in both N‐ and C‐termini. We show that phospho‐mimetic KCC3b is arrested in an inward‐facing state in which intracellular ion access is blocked by extensive contacts with the N‐terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho‐regulatory site in the KCC1 N‐terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP‐binding pocket in the large C‐terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.
SYNOPSIS
Chloride extrusion by transmembrane transporters is critical for regulation of cell volume and intracellular ion concentration. Here, cryo‐EM combined with functional assays identifies structural determinants of human potassium‐coupled chloride transporter (KCCs) activation.
Human KCC1 and KCC3b transporter structures solved in two different phospho‐regulatory states show substantial conformational rearrangements.
A phospho‐mimetic KCC3b mutant is auto‐inhibited by a cytoplasmic N‐terminal segment, and arrested in an inward‐facing state.
Protein dynamics assays reveal mobility differences between phospho‐inhibited and activated states.
KCC1 C‐terminal domain contains a nucleotide binding motif engaging ATP and ADP.
Cryo‐EM structures uncover phosphorylation‐dependent protein dynamics and ATP/ADP binding of human KCC membrane transporters.
Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still ...requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.
ABC transporters utilize ATP for export processes to provide cellular resistance against toxins, antibiotics, and harmful metabolites in eukaryotes and prokaryotes. Based on static structure ...snapshots, it is believed that they use an alternating access mechanism, which couples conformational changes to ATP binding (outward‐open conformation) and hydrolysis (inward‐open) for unidirectional transport driven by ATP. Here, we analyzed the conformational states and dynamics of the antibacterial peptide exporter McjD from Escherichia coli using single‐molecule Förster resonance energy transfer (smFRET). For the first time, we established smFRET for an ABC exporter in a native‐like lipid environment and directly monitor conformational dynamics in both the transmembrane‐ (TMD) and nucleotide‐binding domains (NBD). With this, we unravel the ligand dependences that drive conformational changes in both domains. Furthermore, we observe intrinsic conformational dynamics in the absence of ATP and ligand in the NBDs. ATP binding and hydrolysis on the other hand can be observed via NBD conformational dynamics. We believe that the progress made here in combination with future studies will facilitate full understanding of ABC transport cycles.
Synopsis
ABC transporters facilitate export of toxic compounds from eukaryotic and bacterial cells in an ATP‐dependent manner. While structural studies have increased our understanding of ABC transporter organization, the application of single‐molecule FRET provides the first detailed view of the dynamics associated with substrate export.
Single‐molecule FRET of ABC transporter McjD reveals conformational dynamics associated with ATP and substrate binding.
The ligand‐binding site in the transmembrane domain (TMD) is occluded in the apo form but opens up in the presence of ATP and the antibacterial peptide MccJ25.
Intrinsic conformational dynamics in the absence of ATP and ligand are observed in the Nucleotide Binding Domain (NBD) but not in the TMDs.
ATP binding is coupled to conformational dynamics in the NBDs while both ATP and peptide MccJ25 are necessary to drive conformational changes in the TMDs.
While static structural studies have increased our understanding of ABC transporter organization, the application of single‐molecule FRET provides a detailed view of the conformational dynamics associated with substrate export.
Every membrane protein is involved in close interactions with the lipid environment of cellular membranes. The annular lipids, that are in direct contact with the polypeptide, can in principle be ...seen as an integral part of its structure, akin to the first hydration shell of soluble proteins. It is therefore desirable to investigate the structure of membrane proteins and especially their conformational flexibility under conditions that are as close as possible to their native state. This can be achieved by reconstituting the protein into proteoliposomes, nanodiscs, or bicelles. In recent years, PELDOR/DEER spectroscopy has proved to be a very useful method to study the structure and function of membrane proteins in such artificial membrane environments. The technique complements both X-ray crystallography and cryo-EM and can be used in combination with virtually any artificial membrane environment and under certain circumstances even in native membranes. Of the above-mentioned membrane mimics, bicelles are currently the least often used for PELDOR studies, although they offer some advantages, especially their ease of use. Here, we provide a step-by-step protocol for studying a bicelle reconstituted membrane protein with PELDOR/DEER spectroscopy.
ABC transporters utilize ATP for export processes to provide cellular resistance against toxins, antibiotics, and harmful metabolites in eukaryotes and prokaryotes. Based on static structure ...snapshots, it is believed that they use an alternating access mechanism, which couples conformational changes to ATP binding (outward-open conformation) and hydrolysis (inward-open) for unidirectional transport driven by ATP. Here, we analyzed the conformational states and dynamics of the antibacterial peptide exporter McjD from Escherichia coli using single-molecule Forster resonance energy transfer (smFRET). For the first time, we established smFRET for an ABC exporter in a native-like lipid environment and directly monitor conformational dynamics in both the transmembrane- (TMD) and nucleotide-binding domains (NBD). With this, we unravel the ligand dependences that drive conformational changes in both domains. Furthermore, we observe intrinsic conformational dynamics in the absence of ATP and ligand in the NBDs. ATP binding and hydrolysis on the other hand can be observed via NBD conformational dynamics. We believe that the progress made here in combination with future studies will facilitate full understanding of ABC transport cycles.
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