Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters

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 phosphoregulation in both N- and C-termini. We show that phosphomimetic 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, KCC1D19, 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 Cterminal 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

Structural basis of antibacterial peptide export by ABC transporters

Under conditions of nutrient starvation bacteria produce and release antibacterial peptides like the lasso peptide microcin J25 (MccJ25). Uptake of MccJ25 by other bacteria leads to RNA polymerase inhibition and subsequent cell death. MccJ25 is also toxic to the producing organism that utilises ATP-binding cassette (ABC) transporters to provide self-immunity. The ABC transporter McjD is responsible for the efflux of MccJ25. The general architecture of an ABC transporter comprises two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Previously, the structure of McjD from Escherichia coli was determined in complex with a non-hydrolysable ATP analogue (AMP-PNP), providing some insights into the transport cycle. However, the mechanistic basis for MccJ25 secretion remained elusive. In this work, the structure of McjD has been determined in a post-ATP hydrolysis intermediate state (ADP-VO4). Using predictive cysteine cross-linking, cavity accessibility studies, transport assays and PELDOR measurements in lipid membranes, a novel mechanism for MccJ25 secretion is proposed requiring the transient opening of the McjD TMDs for substrate release. Unlike multidrug ABC exporters which display large conformational changes in the TMDs, the McjD TMDs exist in a predominantly occluded state which prevents MccJ25 reuptake upon efflux. These structural insights are complimented by the first single molecule FRET (smFRET) characterisation of an ABC exporter in a native-like environment. The smFRET findings report conformational changes in the NBDs and TMDs of McjD, demonstrating that opening of the TMDs is tightly coupled to the binding of both ATP and MccJ25. The NBDs display intrinsic conformational dynamics on the millisecond timescale whereas the TMDs do not show any dynamic behaviour. Finally, attempts are made to purify and functionally characterize two staphylococcal peptide ABC exporters Pmt and AbcA. These transporters secrete cytolytic α- helical peptides, phenol soluble modulins, that can evade the immune response.Open Acces

Conformational dynamics of the ABC transporter McjD seen by single-molecule FRET

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

Structural basis for antibacterial peptide self-immunity by the bacterial ABC transporter McjD

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

Phospho‐regulation, nucleotide binding and ion access control in potassium‐chloride cotransporters

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

Experimental phasing opportunities for macromolecular crystallography at very long wavelengths

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