Architecture of the major component of the type III secretion system export apparatus

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

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

Timing is everything: the regulation of type III secretion

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

Domain swapping and different oligomeric states for the complex between calmodulin and the calmodulin-binding domain of calcineurin A

The contraction–relaxation cycle of muscle cellstranslates into large movements of several filamentsystems in sarcomeres, requiring special molecularmechanisms to maintain their structural integrity.Recent structural and functional data from three fila-ments harboring extensive arrays of immunoglobulin-like domains–titin, filamin and myomesin–have, for thefirst time, unraveled a common function of their terminaldomains: assembly and anchoring of the respective fila-ments. In each case, the protein–protein interactions aremediated by antiparallel dimerization modules via inter-molecularb-sheets. These observations on terminal fila-ment assembly indicate an attractive model for severalother filament proteins that require structural character-izatio

Visualization and characterization of the pore formed by SpaP and SpaR.

<p>(A) Six selected class averages (4, 23, 29, 36, 55, 82) of negative-stained isolated SpaPR complexes imaged by electron microscopy. The length of the scale bar represents 50 Å. The two class averages at the top represent the SpaP₅ complex. Arrowheads in the class averages in the middle and at the bottom represent the anticipated position of SpaR on the SpaP₅ ring. The complete picture of all class averages can be seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006071#ppat.1006071.s009" target="_blank">S4 Fig</a>. (B) Fluorescent streptavidin detection of SDS PAGE-separated biotin maleimide-labeled proteins of whole cell lysates, cell culture supernatant, periplasmic fraction, or cytoplasmic fraction of <i>S</i>. Typhimurium Δ<i>prgHIJK</i>, <i>flhD</i>::<i>tet</i> moderately overexpressing indicated proteins from a medium copy number plasmid (pT12). (C) Blue native PAGE and immunodetection of a high molecular weight complex formed by EPEA-tagged SpaP alone.</p

Models of SpaP, SpaR, SpaQ, SpaS, and PrgJ in the T3SS needle complex and its assembly.

<p>(A) Model of the central SpaP complex with surrounding export apparatus components SpaQ, SpaR, and SpaS, and direct connection to the inner rod formed by PrgJ. These results suggest that SpaP, SpaR, and PrgJ form the socket structure on the periplasmic side of the inner membrane patch of the base. (B) Model of a view of the membrane patch of the needle complex from the cytoplasmic side highlighting SpaP, SpaQ, SpaR, and SpaS. (C) Model of needle complex assembly. The unified Sct nomenclature [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006071#ppat.1006071.ref023" target="_blank">23</a>] is shown in parenthesis.</p