high-throughput-expression-purification-human-solute-carriers-for

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

University of Oxford

Type Three Secretion Systems (T3SSs) are essential virulence determinants of many Gram-negative bacteria. The T3SS is an injection device that can transfer bacterial virulence proteins directly into host cells. The apparatus is made up of a basal body that spans both bacterial membranes and an extracellular needle that possesses a channel that is thought to act as a conduit for protein secretion. Contact with a host-cell membrane triggers the insertion of a pore into the target membrane, and effectors are translocated through this pore into the host cell. To assemble a functional T3SS, specific substrates must be targeted to the apparatus in the correct order. Recently, there have been many developments in our structural and functional understanding of the proteins involved in the regulation of secretion. Here we review the current understanding of protein components of the system thought to be involved in switching between different stages of secretion.

Timing is everything: the regulation of type III secretion.

Pore-forming proteins insert from solution into membranes to create lesions, undergoing a structural rearrangement often accompanied by oligomerization. Lysenin, a pore-forming toxin from the earthworm Eisenia fetida, specifically interacts with sphingomyelin (SM) and may confer innate immunity against parasites by attacking their membranes to form pores. SM has important roles in cell membranes and lysenin is a popular SM-labeling reagent. The structure of lysenin suggests common ancestry with other pore-forming proteins from a diverse set of eukaryotes and prokaryotes. The complex with SM shows the mode of its recognition by a protein in which both the phosphocholine headgroup and one acyl tail are specifically bound. Lipid interaction studies and assays using viable target cells confirm the functional reliance of lysenin on this form of SM recognition.

Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition.

Bacterial type III protein secretion systems inject effector proteins into eukaryotic host cells in order to promote survival and colonization of Gram-negative pathogens and symbionts. Secretion across the bacterial cell envelope and injection into host cells is facilitated by a so-called injectisome. Its small hydrophobic export apparatus components SpaP and SpaR were shown to nucleate assembly of the needle complex and to form the central "cup" substructure of a Salmonella Typhimurium secretion system. However, the in vivo placement of these components in the needle complex and their function during the secretion process remained poorly defined. Here we present evidence that a SpaP pentamer forms a 15 Å wide pore and provide a detailed map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. We further refine the current view of export apparatus assembly, consolidate transmembrane topology models for SpaP and SpaR, and present intimate interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ, indicating how export apparatus and needle filament are connected to create a continuous conduit for substrate translocation.

Structural and Functional Characterization of the Bacterial Type III Secretion Export Apparatus.

Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. Here, we present the cryoelectron microscopy structure of the Salmonella Typhimurium FliP-FliQ-FliR complex at 4.2 Å. None of the subunits adopt canonical integral membrane protein topologies, and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems, combined with cross-linking, localize the export gate as a core component of the periplasmic portion of the machinery. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.

Structure of the core of the type III secretion system export apparatus.

In the version of this article initially published, the PDB code associated with the study was given as 6F2E but should have been 6F2D in Table 1 and the data availability statement. The error has been corrected in the HTML and PDF versions of the article.

Author Correction: Structure of the core of the type III secretion system export apparatus.

Export of proteins through type III secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported through a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assembles into an extramembrane helical assembly that likely seeds correct assembly of the rod. Here we present the structure of an equivalent complex from the  virulence system at 3.5 Å by cryo-electron microscopy. This higher-resolution structure yields a more precise description of the structure and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also suggests how the SctS/FliQ subunits sequentially assemble in the complex. Although predicted on the basis of sequence conservation, the work presented here formally demonstrates that all classes of type III secretion systems, flagellar or virulence, share the same architecture at the level of the core structures. This absolute conservation of the unusual extramembrane structure of the core export gate complex now allows work to move to focusing on both mechanistic studies of type III but also on fundamental studies of how such a complex is assembled.

The Structure of an Injectisome Export Gate Demonstrates Conservation of Architecture in the Core Export Gate between Flagellar and Virulence Type III Secretion Systems.

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.

Patrizia Abrusci

Centre for Medicines Discovery,
Nuffield Department of Medicine,
Centre for Medicines Discovery, Nuffield Department of Medicine

University of Oxford

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Patrizia Abrusci

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Centre for Medicines Discovery,Nuffield Department of Medicine,Centre for Medicines Discovery, Nuffield Department of Medicine

University of Oxford

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Centre for Medicines Discovery,
Nuffield Department of Medicine,
Centre for Medicines Discovery, Nuffield Department of Medicine