During neurotransmitter release, the neuronal SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 form a four-helix bundle, the SNARE complex, that pulls the synaptic vesicle and plasma membranes together possibly causing membrane fusion. Complexin binds tightly to the SNARE complex and is essential for efficient Ca(2+)-evoked neurotransmitter release. A combined X-ray and TROSY-based NMR study now reveals the atomic structure of the complexin/SNARE complex. Complexin binds in an antiparallel alpha-helical conformation to the groove between the synaptobrevin and syntaxin helices. This interaction stabilizes the interface between these two helices, which bears the repulsive forces between the apposed membranes. These results suggest that complexin stabilizes the fully assembled SNARE complex as a key step that enables the exquisitely high speed of Ca(2+)-evoked neurotransmitter release.

The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament. Here we report the crystal structure of the yeast Bni1p FH2 domain in complex with tetramethylrhodamine-actin. Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus. Biochemical properties of heterodimeric FH2 mutants suggest that the wild-type protein equilibrates between two bound states at the barbed end: one permitting monomer binding and the other permitting monomer dissociation. Interconversion between these states allows processive barbed-end polymerization and depolymerization in the presence of bound FH2 domain. Kinetic and/or thermodynamic differences in the conformational and binding equilibria can explain the variable activity of different FH2 domains as well as the effects of the actin-binding protein profilin on FH2 function.

Cytochrome P450s constitute a superfamily of enzymes that catalyze the oxidation of a vast number of structurally and chemically diverse hydrophobic substrates. Herein, we describe the crystal structure of a complex between the bacterial P450BM-3 and the novel substrate N-palmitoylglycine at a resolution of 1.65 A, which reveals previously unrecognizable features of active site reorganization upon substrate binding. N-palmitoylglycine binds with higher affinity than any other known substrate and reacts with a higher turnover number than palmitic acid but with unaltered regiospecificity along the fatty acid moiety. Substrate binding induces conformational changes in distinct regions of the enzyme including part of the I-helix adjacent to the active site. These changes cause the displacement by about 1 A of the pivotal water molecule that ligands the heme iron, resulting in the low-spin to high-spin conversion of the iron. The water molecule is trapped close to the heme group, which allows it to partition between the iron and the new binding site. This partitioning explains the existence of a high-spin-low-spin equilibrium after substrate binding. The close proximity of the water molecule to the heme iron indicates that it may also participate in the proton-transfer cascade that leads to heterolytic bond scission of oxygen in P450BM-3.

No abstract

Histone methylation regulates diverse chromatin-templated processes, including transcription. Many transcriptional corepressor complexes contain lysine-specific demethylase 1 (LSD1) and CoREST that collaborate to demethylate mono- and dimethylated H3-K4 of nucleosomes. Here, we report the crystal structure of the LSD1-CoREST complex. LSD1-CoREST forms an elongated structure with a long stalk connecting the catalytic domain of LSD1 and the CoREST SANT2 domain. LSD1 recognizes a large segment of the H3 tail through a deep, negatively charged pocket at the active site and possibly a shallow groove on its surface. CoREST SANT2 interacts with DNA. Disruption of the SANT2-DNA interaction diminishes CoREST-dependent demethylation of nucleosomes by LSD1. The shape and dimension of LSD1-CoREST suggest its bivalent binding to nucleosomes, allowing efficient H3-K4 demethylation. This spatially separated, multivalent nucleosome binding mode may apply to other chromatin-modifying enzymes that generally contain multiple nucleosome binding modules.

The hypoxia-inducible factor (HIF) basic helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim (bHLH-PAS) transcription factors are master regulators of the conserved molecular mechanism by which metazoans sense and respond to reductions in local oxygen concentrations. In humans, HIF is critically important for the sustained growth and metastasis of solid tumors. Here, we describe crystal structures of the heterodimer formed by the C-terminal PAS domains from the HIF2␣ and ARNT subunits of the HIF2 transcription factor, both in the absence and presence of an artificial ligand. Unexpectedly, the HIF2␣ PAS-B domain contains a large internal cavity that accommodates ligands identified from a small-molecule screen. Binding one of these ligands to HIF2␣ PAS-B modulates the affinity of the HIF2␣:ARNT PAS-B heterodimer in vitro. Given the essential role of PAS domains in forming active HIF heterodimers, these results suggest a presently uncharacterized ligand-mediated mechanism for regulating HIF2 activity in endogenous and clinical settings.internal cavity ͉ NMR ͉ X-ray crystallography ͉ hypoxia ͉ protein-ligand interactions T he hypoxia-inducible factor (HIF) transcription factors are present in multicellular organisms and adopt conserved roles in maintaining cellular oxygen homeostasis. In humans, HIF misregulation correlates with aggressive solid tumor growth and poor clinical outcomes (1, 2). Transcriptionally active HIF proteins are heterodimers of the HIF␣ and aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF␤) subunits (3, 4), each containing an N-terminal basic helix-loophelix (bHLH) domain for specific DNA binding, two tandem Per-ARNT-Sim (PAS) domains to facilitate heterodimerization and C-terminal regulatory regions (5-7). Three known human HIF␣ subunit isoforms share ARNT as their bHLH-PAS protein binding partner. HIF1␣ and HIF2␣ are similarly regulated, but show cell line-specific differences in expression and gene regulation patterns (8). HIF3␣ and its splicing isoforms (9, 10) lack C-terminal sequences that recruit transcriptional coactivator proteins, suggesting that these proteins act as dominant negative pathway regulators by forming regulatory-incompetent heterodimers with ARNT.Regulation of this pathway is governed in large part by posttranslational modifications that down-regulate HIF activity under adequate cellular oxygenation levels (normoxia). The best characterized of these modifications are hydroxylations of key proline and asparagine residues in the HIF␣ C-terminal region (11,12). These hydroxylated prolines recruit the von Hippel Lindau (pVHL) E3 ubiquitin ligase, which ultimately downregulates HIF␣ protein levels through proteasomal degradation, whereas the hydroxylated asparagines block HIF␣-coactivator interactions. Oxygen-insufficient conditions (hypoxia) inactivate the hydroxylases, allowing HIF␣ subunits to escape degradation, heterodimerize with ARNT, and ultimately control the levels of Ͼ100 proteins (13). Misregulation of the HIF path...

Munc13 is a multidomain protein of presynaptic active zones that mediates the priming and plasticity of synaptic vesicle exocytosis, but the mechanisms involved remain unclear. Here, we use biophysical, biochemical, and electrophysiological approaches to demonstrate that the central C2B-domain of Munc13 functions as a Ca2+-regulator of short-term synaptic plasticity. The crystal structure of the C2B-domain revealed an unusual Ca2+-binding site with an amphipathic α-helix. This configuration confers onto the C2B-domain unique Ca2+-dependent phospholipid-binding properties favoring phosphatidylinositolphosphates. A mutation that inactivated Ca2+-dependent phospholipid binding to the C2B-domain did not alter neurotransmitter release evoked by isolated action potentials, but depressed release evoked by action potential trains. In contrast, a mutation that increased Ca2+-dependent phosphatidylinositolbisphosphate binding to the C2B-domain enhanced release evoked by isolated action potentials and by action potential trains. Our data suggest that during repeated action potentials, Ca2+- and phosphatidylinositolphosphate-binding to the Munc13 C2B-domain potentiate synaptic vesicle exocytosis, thereby offsetting synaptic depression induced by vesicle depletion.

Diaphanous-related formins (DRFs) regulate dynamics of unbranched actin filaments during cell contraction and cytokinesis. DRFs are autoinhibited through intramolecular binding of a Diaphanous autoinhibitory domain (DAD) to a conserved N-terminal regulatory element. Autoinhibition is relieved through binding of the GTPase RhoA to the N-terminal element. We report the crystal structure of the dimeric regulatory domain of the DRF, mDia1. Dimerization is mediated by an intertwined six-helix bundle, from which extend two Diaphanous inhibitory domains (DIDs) composed of five armadillo repeats. NMR and biochemical mapping indicate the RhoA and DAD binding sites on the DID partially overlap, explaining activation of mDia1 by the GTPase. RhoA binding also requires an additional structurally independent segment adjacent to the DID. This regulatory construction, involving a GTPase binding site spanning a flexibly tethered arm and the inhibitory module, is observed in many autoinhibited effectors of Ras superfamily GTPases, suggesting evolutionary pressure for this design.