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Although the pathogenesis of schizophrenia (SCZ) is proposed to involve alterations of neural circuits via synaptic dysfunction, the underlying molecular mechanisms remain poorly understood. Recent exome sequencing studies of SCZ have uncovered numerous single-nucleotide variants (SNVs); however, the majority of these SNVs have unknown functional consequences, leaving their disease relevance uncertain. Filling this knowledge gap requires systematic application of quantitative and scalable assays to assess known and novel biological functions of genes. Here we demonstrate loss-of-function effects of multiple rare coding SNVs found in SCZ subjects in the GIT1 ( G protein-coupled receptor kinase interacting ArfGAP 1 ) gene using functional cell-based assays involving coexpression of GIT1 and PAK3 (p21 protein (Cdc42/Rac)-activated kinase 3). Most notably, a GIT1-R283W variant reported in four independent SCZ cases was defective in activating PAK3 as well as MAPK (mitogen-activated protein kinase). Similar functional deficits were found for a de novo SCZ variant GIT1-S601N. Additional assays revealed deficits in the capacity of GIT1-R283W to stimulate PAK phosphorylation in cultured hippocampal neurons. In addition, GIT1-R283W showed deficits in the induction of GAD1 (glutamate decarboxylase 1) protein expression. Extending these functional assays to 10 additional rare GIT1 variants revealed the existence of an allelic series with the majority of the SCZ case variants exhibiting loss of function toward MAPK activation in a manner correlated with loss of PAK3 activation. Taken together, we propose that rare variants in GIT1, along with other genetic and environmental factors, cause dysregulation of PAK3 leading to synaptic deficits in SCZ.

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.

Signals generated by cryptochrome (CRY) blue-light photoreceptors are responsible for a variety of developmental and circadian responses in plants. The CRYs are also identified as circadian blue-light photoreceptors in Drosophila and components of the mammalian circadian clock. These flavoproteins all have an N-terminal domain that is similar to photolyase, and most have an additional C-terminal domain of variable length. We present here the crystal structure of the photolyase-like domain of CRY-1 from Arabidopsis thaliana. The structure reveals a fold that is very similar to photolyase, with a single molecule of FAD noncovalently bound to the protein. The surface features of the protein and the dissimilarity of a surface cavity to that of photolyase account for its lack of DNA-repair activity. Previous in vitro experiments established that the photolyase-like domain of CRY-1 can bind Mg·ATP, and we observe a single molecule of an ATP analog bound in the aforementioned surface cavity, near the bound FAD cofactor. The structure has implications for the signaling mechanism of CRY blue-light photoreceptors.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro . In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

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/HCO 3 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/HCO 3 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.

The neuronal protein synaptotagmin 1 functions as a Ca 2+ sensor in exocytosis via two Ca 2+ -binding C 2 domains. The very similar synaptotagmin 4, which includes all the predicted Ca 2+ -binding residues in the C 2 B domain but not in the C 2 A domain, is also thought to function as a neuronal Ca 2+ sensor. Here we show that, unexpectedly, both C 2 domains of fly synaptotagmin 4 exhibit Ca 2+ -dependent phospholipid binding, whereas neither C 2 domain of rat synaptotagmin 4 binds Ca 2+ or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca 2+ ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C 2 B domain unable to form full Ca 2+ -binding sites. These results indicate that synaptotagmin 4 is a Ca 2+ sensor in the fly but not in the rat, that the Ca 2+ -binding properties of C 2 domains cannot be reliably predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties.

Nucleotide excision repair (NER) is the most important DNA-repair mechanism in living organisms. In prokaryotes, three enzymes forming the UvrABC system initiate NER of a variety of structurally different DNA lesions. UvrB, the central component of this system, is responsible for the ultimate DNA damage recognition and participates in the incision of the damaged DNA strand. The crystal structure of Thermus thermophilus UvrB reveals a core that is structurally similar to core regions found in helicases, where they constitute molecular motors. Additional domains implicated in binding to DNA and various components of the NER system are attached to this central core. The architecture and distribution of DNA binding sites suggest a possible model for the DNA damage recognition process.

Mitochondrial protein kinases (mPKs) are molecular switches that down-regulate the oxidation of branched-chain α-ketoacids and pyruvate. Elevated levels of these metabolites are implicated in disease states such as insulin-resistant Type II diabetes, branchedchain ketoaciduria, and primary lactic acidosis. We report a three-dimensional structure of a member of the mPK family, rat branched-chain α-ketoacid dehydrogenase kinase (BCK). BCK features a characteristic nucleotide-binding domain and a four-helix bundle domain. These two domains are reminiscent of modules found in protein histidine kinases (PHKs), which are involved in two-component signal transduction systems. Unlike PHKs, BCK dimerizes through direct interaction of two opposing nucleotide-binding domains. Nucleotide binding to BCK is uniquely mediated by both potassium and magnesium. Binding of ATP induces disorder-order transitions in a loop region at the nucleotide-binding site. These structural changes lead to the formation of a quadruple aromatic stack in the interface between the nucleotide-binding domain and the four-helix bundle domain, where they induce a movement of the top portion of two helices. Phosphotransfer induces further ordering of the loop region, effectively trapping the reaction product ADP, which explains product inhibition in mPKs. The BCK structure is a prototype for all mPKs and will provide a framework for structure-assisted inhibitor design for this family of kinases.

Munc13 primes synaptic vesicles for exocytosis. Studies now reveal that the central C2B domain of Munc13 contains a structurally unusual Ca 2+ binding site that confers specific phospholipid-binding properties to Munc13 and is essential for regulating exocytosis upon trains of action potentials. 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 C 2 B domain of Munc13 functions as a Ca 2+ regulator of short-term synaptic plasticity. The crystal structure of the C 2 B domain revealed an unusual Ca 2+ -binding site with an amphipathic α-helix. This configuration confers onto the C 2 B domain unique Ca 2+ -dependent phospholipid-binding properties that favor phosphatidylinositolphosphates. A mutation that inactivated Ca 2+ -dependent phospholipid binding to the C 2 B 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 Ca 2+ -dependent phosphatidylinositolbisphosphate binding to the C 2 B domain enhanced release evoked by isolated action potentials and by action-potential trains. Our data suggest that, during repeated action potentials, Ca 2+ and phosphatidylinositolphosphate binding to the Munc13 C 2 B domain potentiate synaptic vesicle exocytosis, thereby offsetting synaptic depression induced by vesicle depletion.

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.