Ligand-gated ion channels transduce chemical signals into electrical impulses by opening a transmembrane pore in response to binding one or more neurotransmitter molecules. After activation, many ligand-gated ion channels enter a desensitized state in which the neurotransmitter remains bound but the ion channel is closed. Although receptor desensitization is crucial to the functioning of many ligand-gated ion channels in vivo, the molecular basis of this important process has until now defied analysis. Using the GluR2 AMPA-sensitive glutamate receptor, we show here that the ligand-binding cores form dimers and that stabilization of the intradimer interface by either mutations or allosteric modulators reduces desensitization. Perturbations that destabilize the interface enhance desensitization. Receptor activation involves conformational changes within each subunit that result in an increase in the separation of portions of the receptor that are linked to the ion channel. Our analysis defines the dimer interface in the resting and activated state, indicates how ligand binding is coupled to gating, and suggests modes of dimer dimer interaction in the assembled tetramer. Desensitization occurs through rearrangement of the dimer interface, which disengages the agonist-induced conformational change in the ligand-binding core from the ion channel gate.

The family of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels are crucial for a range of electrical signalling, including cardiac and neuronal pacemaker activity, setting resting membrane electrical properties and dendritic integration. These nonselective cation channels, underlying the I(f), I(h) and I(q) currents of heart and nerve cells, are activated by membrane hyperpolarization and modulated by the binding of cyclic nucleotides such as cAMP and cGMP. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by beta-adrenergic agonists. Here we have investigated the mechanism underlying this modulation by studying a carboxy-terminal fragment of HCN2 containing the cyclic nucleotide-binding domain (CNBD) and the C-linker region that connects the CNBD to the pore. X-ray crystallographic structures of this C-terminal fragment bound to cAMP or cGMP, together with equilibrium sedimentation analysis, identify a tetramerization domain and the mechanism for cyclic nucleotide specificity, and suggest a model for ligand-dependent channel modulation. On the basis of amino acid sequence similarity to HCN channels, the cyclic nucleotide-gated, and eag- and KAT1-related families of channels are probably related to HCN channels in structure and mechanism.

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Pore-forming toxins (PFTs) are potent cytolytic agents secreted by pathogenic bacteria that protect microbes against the cellmediated immune system (by targeting phagocytic cells), disrupt epithelial barriers, and liberate materials necessary to sustain growth and colonization. Produced by gram-positive and gramnegative bacteria alike, PFTs are released as water-soluble monomeric or dimeric species, bind specifically to target membranes, and assemble transmembrane channels leading to cell damage and/or lysis. Structural and biophysical analyses of individual steps in the assembly pathway are essential to fully understanding the dynamic process of channel formation. To work toward this goal, we solved by X-ray diffraction the 2.9-Å structure of the 450-kDa heptameric Vibrio cholerae cytolysin (VCC) toxin purified and crystallized in the presence of detergent. This structure, together with our previously determined 2.3-Å structure of the VCC water-soluble monomer, reveals in detail the architectural changes that occur within the channel region and accessory lectin domains during pore formation including substantial rearrangements of hydrogen-bonding networks in the pore-forming amphipathic loops. Interestingly, a ring of tryptophan residues forms the narrowest constriction in the transmembrane channel reminiscent of the phenylalanine clamp identified in anthrax protective antigen [Krantz BA, et al. (2005) Science 309:777-781]. Our work provides an example of a β-barrel PFT (β-PFT) for which soluble and assembled structures are available at high-resolution, providing a template for investigating intermediate steps in assembly.hemolysin | membrane protein | X-ray crystallography | virulence factor

Upon activation by tyrosine kinases, members of the STAT family of transcription factors form stable dimers that are able to rapidly translocate to the nucleus and bind DNA. Although crystal structures of activated, near full-length, Stat1 and Stat3 illustrate how STATs bind to DNA, they provide little insight into the dynamic regulation of STAT activity. To explore the unique structural changes Stat1 and Stat3 undergo when they become activated, full-length inactive recombinant proteins were prepared. To our surprise, even though these proteins are unable to bind DNA, our studies demonstrate that they exist as stable homodimers. Similarly, the Stat1 and Stat3 found in the cytoplasm of unstimulated cells also exhibit a dimeric structure. These observations indicate that Stat1 and Stat3 exist as stable homodimers prior to activation.Cytokines are important regulators of intercellular communication. They mediate pleiotropic cellular responses by binding to specific transmembrane spanning receptors (reviewed in Refs. 1 and 2). These receptors in turn activate intracellular signaling pathways, resulting in the induction of gene expression. The STAT 1 (signal transducers and activators of transcription) family of transcription factors transmits signals in response to cytokines. This family consists of seven members, Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6. Structural and functional studies have led to the identification of six conserved STAT domains (3-5): 1) the amino-terminal domain (ϳamino acids 1-125), which has been implicated in cooperativity of DNA binding to tandem gamma activation site (GAS) elements, nuclear translocation, and in receptor association (2); 2) the coiled-coil domain (ϳamino acids 125-325), which has been shown to mediate interactions with several other proteins (2); 3) the DNA binding domain (ϳamino acids 325-475); 4) the linker domain (ϳamino acids 475-575); 5) the SH2 domain ϩ tyrosine activation motif (ϳamino acids 575-710), which is essential for STAT recruitment to the receptor, STAT activation, and dimerization (2); and 6) the carboxyl-terminal transcriptional activation domain.The general outline for the STAT signaling paradigm elucidated a decade ago is now widely accepted (1, 2, 6). In this model, a specific interaction between a cytokine and its cognate receptor brings the cytoplasmic domains of this receptor into apposition, thereby promoting the transphosphorylation of receptor associated tyrosine kinases from the Janus kinase family. These activated Janus kinases in turn phosphorylate specific tyrosine motifs found in receptor endodomains, which then serve to mediate recruitment of the specific monomeric STATs to the receptor complex. Once at the receptor, the STATs become activated by a single tyrosine phosphorylation event. The activated STATs are then released, where upon they dimerize through the interaction between the SH2 domain of one STAT and the tyrosine-phosphorylated tail segment of the other STAT. This renders them competent for both rapid translocation ...