Diana R. Tomchick

Three-dimensional structure of the complexin/SNARE complex.

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.

Crystal structure of a 12 ANK repeat stack from human ankyrinR

Ankyrins are multifunctional adaptors that link specific proteins to the membrane‐associated, spectrin–actin cytoskeleton. The N‐terminal, ‘membrane‐binding’ domain of ankyrins contains 24 ANK repeats and mediates most binding activities. Repeats 13–24 are especially active, with known sites of interaction for the Na/K ATPase, Cl/HCO3 anion exchanger, voltage‐gated sodium channel, clathrin heavy chain and L1 family cell adhesion molecules. Here we report the crystal structure of a human ankyrinR construct containing ANK repeats 13–24 and a portion of the spectrin‐binding domain. The ANK repeats are observed to form a contiguous spiral stack with which the spectrin‐binding domain fragment associates as an extended strand. The structural information has been used to construct models of all 24 repeats of the membrane‐binding domain as well as the interactions of the repeats with the Cl/HCO3 anion exchanger and clathrin. These models, together with available binding studies, suggest that ion transporters such as the anion exchanger associate in a large central cavity formed by the ANK repeat spiral, while clathrin and cell adhesion molecules associate with specific regions outside this cavity.

Structural basis of histone demethylation by LSD1 revealed by suicide inactivation

Histone methylation regulates diverse chromatin-templated processes, including transcription. The recent discovery of the first histone lysine–specific demethylase (LSD1) has changed the long-held view that histone methylation is a permanent epigenetic mark. LSD1 is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that demethylates histone H3 Lys4 (H3-K4). However, the mechanism by which LSD1 achieves its substrate specificity is unclear. We report the crystal structure of human LSD1 with a propargylamine-derivatized H3 peptide covalently tethered to FAD. H3 adopts three consecutive γ-turns, enabling an ideal side chain spacing that places its N terminus into an anionic pocket and positions methyl-Lys4 near FAD for catalysis. The LSD1 active site cannot productively accommodate more than three residues on the N-terminal side of the methyllysine, explaining its H3-K4 specificity. The unusual backbone conformation of LSD1-bound H3 suggests a strategy for designing potent LSD1 inhibitors with therapeutic potential.

Structure of Cdc42 in a complex with the GTPase-binding domain of the cell polarity protein, Par6.

Cdc42 is a small GTPase that is required for cell polarity establishment in eukaryotes as diverse as budding yeast and mammals. Par6 is also implicated in metazoan cell polarity establishment and asymmetric cell divisions. Cdc42·GTP interacts with proteins that contain a conserved sequence called a CRIB motif. Uniquely, Par6 possesses a semi‐CRIB motif that is not sufficient for binding to Cdc42. An adjacent PDZ domain is also necessary and is required for biological effects of Par6. Here we report the crystal structure of a complex between Cdc42 and the Par6 GTPase‐binding domain. The semi‐CRIB motif forms a β‐strand that inserts between the four strands of Cdc42 and the three strands of the PDZ domain to form a continuous eight‐stranded sheet. Cdc42 induces a conformational change in Par6, detectable by fluorescence resonance energy transfer spectroscopy. Nuclear magnetic resonance studies indicate that the semi‐CRIB motif of Par6 is at least partially structured by the PDZ domain. The structure highlights a novel role for a PDZ domain as a structural scaffold.

Artificial ligand binding within the HIF2α PAS-B domain of the HIF2 transcription factor

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 HIF2α and ARNT subunits of the HIF2 transcription factor, both in the absence and presence of an artificial ligand. Unexpectedly, the HIF2α PAS-B domain contains a large internal cavity that accommodates ligands identified from a small-molecule screen. Binding one of these ligands to HIF2α PAS-B modulates the affinity of the HIF2α: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.

Munc13 C2B domain is an activity-dependent Ca2+ regulator of synaptic exocytosis

Munc13 is a multidomain protein present in 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 show 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 that favor phosphatidylinositolphosphates. A mutation that inactivated Ca2+-dependent phospholipid binding to the C2B domain did not alter neurotransmitter release evoked by isolated action potentials, but it did depress 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.

A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria

The antimalarial drug DSM265 displays activity against blood and liver stages of Plasmodium falciparum and has a long predicted half-life in humans. Long-acting new treatment for drug-resistant malaria Malaria kills 0.6 million people annually, yet current malaria drugs are no longer fully effective because the parasite that causes malaria is becoming resistant to these agents. Phillips et al. have identified a new drug that kills both drug-sensitive and drug-resistant malaria parasites by targeting the ability of the parasite to synthesize the nucleotide precursors required for synthesis of DNA and RNA. This drug kills parasites in both the blood and liver and is sufficiently long-acting that it is expected to cure malaria after a single dose or to be effective if dosed weekly for chemoprevention. Malaria is one of the most significant causes of childhood mortality, but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in combating malaria requires development of new, easy to administer drug combinations with broad-ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective toward DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200 to 400 mg. DSM265 was well tolerated in repeat-dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood- and liver-stage activity, and predicted long half-life in humans position DSM265 as a new potential drug combination partner for either single-dose treatment or once-weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive against the parasite liver stage.

Munc13 C[subscript 2]B domain is an activity-dependent Ca[superscript 2+] regulator of synaptic exocytosis

Munc13 is a multidomain protein present in 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 show 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 that favor phosphatidylinositolphosphates. A mutation that inactivated Ca2+-dependent phospholipid binding to the C2B domain did not alter neurotransmitter release evoked by isolated action potentials, but it did depress 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.

Structural and Energetic Mechanisms of Cooperative Autoinhibition and Activation of Vav1

Vav proteins are guanine nucleotide exchange factors (GEFs) for Rho family GTPases. They control processes including T cell activation, phagocytosis, and migration of normal and transformed cells. We report the structure and biophysical and cellular analyses of the five-domain autoinhibitory element of Vav1. The catalytic Dbl homology (DH) domain of Vav1 is controlled by two energetically coupled processes. The DH active site is directly, but weakly, inhibited by a helix from the adjacent Acidic domain. This core interaction is strengthened 10-fold by contacts of the calponin homology (CH) domain with the Acidic, pleckstrin homology, and DH domains. This construction enables efficient, stepwise relief of autoinhibition: initial phosphorylation events disrupt the modulatory CH contacts, facilitating phosphorylation of the inhibitory helix and consequent GEF activation. Our findings illustrate how the opposing requirements of strong suppression of activity and rapid kinetics of activation can be achieved in multidomain systems.

The assembly of a GTPase-kinase signalling complex by a bacterial catalytic scaffold.

The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding, and the identity of the effectors and their mechanisms of action are poorly understood. Here we identify the enterohaemorrhagic Escherichia coli O157:H7 type III effector EspG as a regulator of endomembrane trafficking using a functional screen, and report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAKs) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GTPase-activating-protein-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signalling inhibition at organelle membranes. In addition, the 2.8 Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAKs are organized on adjacent surfaces of EspG, indicating its role as a ‘catalytic scaffold’ that effectively reprograms cellular events through the functional assembly of GTPase-kinase signalling complex.