Aberrant folding of proteins in the endoplasmic reticulum activates the bifunctional transmembrane kinase/endoribonuclease Ire1. Ire1 excises an intron from HAC1 messenger RNA in yeasts and Xbp1 ...messenger RNA in metozoans encoding homologous transcription factors. This non-conventional mRNA splicing event initiates the unfolded protein response, a transcriptional program that relieves the endoplasmic reticulum stress. Here we show that oligomerization is central to Ire1 function and is an intrinsic attribute of its cytosolic domains. We obtained the 3.2-A crystal structure of the oligomer of the Ire1 cytosolic domains in complex with a kinase inhibitor that acts as a potent activator of the Ire1 RNase. The structure reveals a rod-shaped assembly that has no known precedence among kinases. This assembly positions the kinase domain for trans-autophosphorylation, orders the RNase domain, and creates an interaction surface for binding of the mRNA substrate. Activation of Ire1 through oligomerization expands the mechanistic repertoire of kinase-based signalling receptors.
In all three kingdoms of life, co-translational targeting of secretory and membrane proteins to the prokaryotic plasma membrane or eukaryotic endoplasmic reticulum is mediated by a ribonucleoprotein ...complex, the signal recognition particle (SRP), and its membrane-associated receptor (SR). SRP binds to signal sequences of nascent proteins as they emerge from the exit tunnel of the ribosome. The resulting targeting complex, composed of the SRP and the ribosome–nascent chain complex (RNC), then docks with the SR in a GTP-dependent manner. Passing through a complex series of conformational states, SRP and SR deliver the RNC to the translocon, which in turn mediates protein translocation across or integration into the membrane. The core structural and mechanistic principles of SRP-dependent protein targeting are universally conserved. Recent structural investigations combining X-ray crystallography and cryo-electron microscopy have provided new insights into three essentials steps of the SRP-dependent protein targeting cycle: the assembly and interaction of the SRP ribonucleoprotein core, the GTP-dependent SRP–SR association, and the interaction between SRP and the ribosome.
Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player ...in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC's multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.
The structure of lactose permease, stabilized in a periplasmic open conformation by two Gly to Trp replacements (LacYww) and complexed with a nanobody directed against this conformation, provides the ...highest resolution structure of the symporter. The nanobody binds in a different manner than two other nanobodies made against the same mutant, which also bind to the same general region on the periplasmic side. This region of the protein may represent an immune hotspot. The CDR3 loop of the nanobody is held by hydrogen bonds in a conformation that partially blocks access to the substrate-binding site. As a result, kon and koff for galactoside binding to either LacY or the double mutant complexed with the nanobody are lower than for the other two LacY/nanobody complexes though the Kd values are similar, reflecting the fact that the nanobodies rigidify structures along the pathway. While the wild-type LacY/nanobody complex clearly stabilizes a similar 'extracellular open' conformation in solution, judged by binding kinetics, the complex with wild-type LacY did not yet crystallize, suggesting the nanobody does not bind strongly enough to shift the equilibrium to stabilize a periplasmic side-open conformation suitable for crystallization. However, the similarity of the galactoside binding kinetics for the nanobody-bound complexes with wild type LacY and with LacYWW indicates that they have similar structures, showing that the reported co-structures reliably show nanobody interactions with LacY.
Objective:
Aquaporin 4 (AQP4)‐specific autoantibodies in neuromyelitis optica (NMO) are immunoglobulin (Ig)G1, a T cell‐dependent Ig subclass, indicating that AQP4‐specific T cells participate in NMO ...pathogenesis. Our goal was to identify and characterize AQP4‐specific T cells in NMO patients and healthy controls (HC).
Methods:
Peripheral blood T cells from NMO patients and HC were examined for recognition of AQP4 and production of proinflammatory cytokines. Monocytes were evaluated for production of T cell‐polarizing cytokines and expression of costimulatory molecules.
Results:
T cells from NMO patients and HC proliferated to intact AQP4 or AQP4 peptides (p11–30, p21–40, p61–80, p131–150, p156–170, p211–230, and p261–280). T cells from NMO patients demonstrated greater proliferation to AQP4 than those from HC, and responded most vigorously to p61–80, a naturally processed immunodominant determinant of intact AQP4. T cells were CD4+, and corresponding to association of NMO with human leukocyte antigen (HLA)‐DRB1*0301 and DRB3, AQP4 p61–80‐specific T cells were HLA‐DR restricted. The T‐cell epitope within AQP4 p61–80 was mapped to 63–76, which contains 10 residues with 90% homology to a sequence within Clostridium perfringens adenosine triphosphate‐binding cassette (ABC) transporter permease. T cells from NMO patients proliferated to this homologous bacterial sequence, and cross‐reactivity between it and self‐AQP4 was observed, supporting molecular mimicry. In NMO, AQP4 p61–80‐specific T cells exhibited Th17 polarization, and furthermore, monocytes produced more interleukin 6, a Th17‐polarizing cytokine, and expressed elevated CD40 and CD80 costimulatory molecules, suggesting innate immunologic dysfunction.
Interpretation:
AQP4‐specific T‐cell responses are amplified in NMO, exhibit a Th17 bias, and display cross‐reactivity to a protein of an indigenous intestinal bacterium, providing new perspectives for investigating NMO pathogenesis. ANN NEUROL 2012;
Aquaporins are transmembrane channels found in cell membranes of all life forms. We examine their apparently paradoxical property, facilitation of efficient permeation of water while excluding ...protons, which is of critical importance to preserving the electrochemical potential across the cell membrane. We have determined the structure of the Escherichia coli aquaglyceroporin GlpF with bound water, in native (2.7 angstroms) and in W48F/F200T mutant (2.1 angstroms) forms, and carried out 12-nanosecond molecular dynamics simulations that define the spatial and temporal probability distribution and orientation of a single file of seven to nine water molecules inside the channel. Two conserved asparagines force a central water molecule to serve strictly as a hydrogen bond donor to its neighboring water molecules. Assisted by the electrostatic potential generated by two half-membrane spanning loops, this dictates opposite orientations of water molecules in the two halves of the channel, and thus prevents the formation of a "proton wire," while permitting rapid water diffusion. Both simulations and observations revealed a more regular distribution of channel water and an increased water permeability for the W48F/F200T mutant.
Aquaporin (AQP) 4 is the predominant water channel in the mammalian brain, abundantly expressed in the blood-brain and brain-cerebrospinal fluid interfaces of glial cells. Its function in cerebral ...water balance has implications in neuropathological disorders, including brain edema, stroke, and head injuries. The 1.8-Å crystal structure reveals the molecular basis for the water selectivity of the channel. Unlike the case in the structures of water-selective AQPs AqpZ and AQP1, the asparagines of the 2 Asn-Pro-Ala motifs do not hydrogen bond to the same water molecule; instead, they bond to 2 different water molecules in the center of the channel. Molecular dynamics simulations were performed to ask how this observation bears on the proposed mechanisms for how AQPs remain totally insulating to any proton conductance while maintaining a single file of hydrogen bonded water molecules throughout the channel.
The unfolded protein response (UPR) detects and restores deficits in the endoplasmic reticulum (ER) protein folding capacity. Ceapins specifically inhibit the UPR sensor ATF6α, an ER-tethered ...transcription factor, by retaining it at the ER through an unknown mechanism. Our genome-wide CRISPR interference (CRISPRi) screen reveals that Ceapins function is completely dependent on the ABCD3 peroxisomal transporter. Proteomics studies establish that ABCD3 physically associates with ER-resident ATF6α in cells and in vitro in a Ceapin-dependent manner. Ceapins induce the neomorphic association of ER and peroxisomes by directly tethering the cytosolic domain of ATF6α to ABCD3's transmembrane regions without inhibiting or depending on ABCD3 transporter activity. Thus, our studies reveal that Ceapins function by chemical-induced misdirection which explains their remarkable specificity and opens up new mechanistic routes for drug development and synthetic biology.
Structure of LacY with an α-substituted galactoside Kumar, Hemant; Finer-Moore, Janet S.; Kaback, H. Ronald ...
Proceedings of the National Academy of Sciences - PNAS,
07/2015, Letnik:
112, Številka:
29
Journal Article
Recenzirano
Odprti dostop
The X-ray crystal structure of a conformationally constrained mutant of theEscherichia colilactose permease (the LacY double-Trp mutant Gly-46→Trp/Gly-262→Trp) with ...boundp-nitrophenyl-α-D-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, Phe-20, Met-23, and Phe-27 (helix I) are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substitue.
Eukaryotic Ca(2+) regulation involves sequestration into intracellular organelles, and expeditious Ca(2+) release into the cytosol is a hallmark of key signalling transduction pathways. Bulk removal ...of Ca(2+) after such signalling events is accomplished by members of the Ca(2+):cation (CaCA) superfamily. The CaCA superfamily includes the Na(+)/Ca(2+) (NCX) and Ca(2+)/H(+) (CAX) antiporters, and in mammals the NCX and related proteins constitute families SLC8 and SLC24, and are responsible for the re-establishment of Ca(2+) resting potential in muscle cells, neuronal signalling and Ca(2+) reabsorption in the kidney. The CAX family members maintain cytosolic Ca(2+) homeostasis in plants and fungi during steep rises in intracellular Ca(2+) due to environmental changes, or following signal transduction caused by events such as hyperosmotic shock, hormone response and response to mating pheromones. The cytosol-facing conformations within the CaCA superfamily are unknown, and the transport mechanism remains speculative. Here we determine a crystal structure of the Saccharomyces cerevisiae vacuolar Ca(2+)/H(+) exchanger (Vcx1) at 2.3 Å resolution in a cytosol-facing, substrate-bound conformation. Vcx1 is the first structure, to our knowledge, within the CAX family, and it describes the key cytosol-facing conformation of the CaCA superfamily, providing the structural basis for a novel alternating access mechanism by which the CaCA superfamily performs high-throughput Ca(2+) transport across membranes.
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