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.
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.
Type III secretion systems (T3SSs) are bacterial membrane-embedded nanomachines designed to export specifically targeted proteins from the bacterial cytoplasm. Secretion through T3SS is governed by a ...subset of inner membrane proteins termed the 'export apparatus'. We show that a key member of the Shigella flexneri export apparatus, MxiA, assembles into a ring essential for secretion in vivo. The ring-forming interfaces are well-conserved in both nonflagellar and flagellar homologs, implying that the ring is an evolutionarily conserved feature in these systems. Electron cryo-tomography revealed a T3SS-associated cytoplasmic torus of size and shape corresponding to those of the MxiA ring aligned to the secretion channel located between the secretion pore and the ATPase complex. This defines the molecular architecture of the dominant component of the export apparatus and allows us to propose a model for the molecular mechanisms controlling secretion.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
•Flagellar and non-flagellar T3SS are built assembling homologous protein machineries.•Unified nomenclature for non-flagellar T3SS.•New model of the T3SS needle is consistent with the flagellar ...filament, both in terms of helical parameters and orientation.•Structural and functional implication of the new architecture of the T3SS export apparatus and ATPase complex.
To fulfill complex biological tasks, such as locomotion and protein translocation, bacteria assemble macromolecular nanomachines. One such nanodevice, the type III secretion system (T3SS), has evolved to provide a means of transporting proteins from the bacterial cytoplasm across the periplasmic and extracellular spaces. T3SS can be broadly classified into two highly homologous families: the flagellar T3SS which drive cell motility, and the non-flagellar T3SS (NF-T3SS) that inject effector proteins into eukaryotic host cells, a trait frequently associated with virulence. Although the structures and symmetries of ancillary components of the T3SS have diversified to match requirements of different species adapted to different niches, recent genetic, molecular and structural studies demonstrate that these systems are built by arranging homologous modular protein assemblies.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
► We report the structure of lysenin, a sphingomyelin (SM)-binding pore-forming protein ► Lysenin binds SM's headgroup and tail; the tail occupies the edge of a β sheet ► Lipid binding and live cell studies confirm activity dependence on SM recognition ► Lysenin is related to pore-forming proteins from disparate kingdoms
Lysenin is related to pore-forming proteins found in bacteria and fungi. De Colibus et al. describe structures of lysenin in different Apo conformations and bound to the lipid sphingomyelin. They map its evolutionary relationships and validate structural insights using affinity assays and live cell imaging.
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.
Understanding a drug candidate’s mechanism of action is crucial for its further development. However, kinetic schemes are often complex and multi-parametric, especially for proteins in ...oligomerization equilibria. Here, we demonstrate the use of particle swarm optimization (PSO) as a method to select between different sets of parameters that are too far apart in the parameter space to be found by conventional approaches. PSO is based upon the swarming of birds: each bird in the flock assesses multiple landing spots while at the same time sharing that information with its neighbors. We applied this approach to the kinetics of HSD17β13 enzyme inhibitors, which displayed unusually large thermal shifts. Thermal shift data for HSD17β13 indicated that the inhibitor shifted the oligomerization equilibrium toward the dimeric state. Validation of the PSO approach was provided by experimental mass photometry data. These results encourage further exploration of multi-parameter optimization algorithms as tools in drug discovery.
•Remove bias when interpreting mechanistic data for complex biological systems•Characterize the oligomeric state of a protein dependent on inhibitor concentration•Increase robustness for global analysis of thermal shift data•Detect global minimum in the presence of several local minima
Biological systems contain numerous components and points of regulation. These factors complicate mathematical modeling approaches; however, methods have been developed that explore the entire solution space before an optimal solution is selected. One such approach, particle swarm optimization, is based upon the swarming of birds. Each bird in the flock assesses multiple landing spots while at the same time sharing that information with its neighbors. Eventually, the entire flock chooses an optimal location based on food availability and the avoidance of predators. When transformed into multi-variable equations, this behavior can remove bias when interpreting complex datasets. This can be a powerful tool in the pharmaceutical industry for the study of complex equilibria. Enhancements to these modeling approaches may allow for the analysis of higher-order kinetic and thermodynamic problems in biochemistry.
Remove bias when interpreting mechanistic data for complex biological systems that characterize the oligomeric state of a protein dependent on inhibitor concentration. This method increases robustness for global analysis of thermal shift data with the potential to be applied to other data-rich biophysical techniques.
PDF
