Members of the Bin/amphiphysin/Rvs (BAR) domain protein superfamily are involved in membrane remodeling in various cellular pathways ranging from endocytic vesicle and T-tubule formation to cell migration and neuromorphogenesis. Membrane curvature induction and stabilization are encoded within the BAR or Fer-CIP4 homology-BAR (F-BAR) domains, α-helical coiled coils that dimerize into membrane-binding modules. BAR/F-BAR domain proteins often contain an SH3 domain, which recruits binding partners such as the oligomeric membrane-fissioning GTPase dynamin. How precisely BAR/F-BAR domain-mediated membrane deformation is regulated at the cellular level is unknown. Here we present the crystal structures of full-length syndapin 1 and its F-BAR domain. Our data show that syndapin 1 F-BAR-mediated membrane deformation is subject to autoinhibition by its SH3 domain. Release from the clamped conformation is driven by association of syndapin 1 SH3 with the proline-rich domain of dynamin 1, thereby unlocking its potent membrane-bending activity. We hypothesize that this mechanism might be commonly used to regulate BAR/F-BAR domain-induced membrane deformation and to potentially couple this process to dynamin-mediated fission. Our data thus suggest a structure-based model for SH3-mediated regulation of BAR/F-BAR domain function.

The regulation of the number of ␥2-subunit-containing GABAA receptors (GABAARs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface ␥2-subunit-containing GABAARs is regulated. Here, we identify a ␥2-subunit-specific Yxx-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for ␥2-subunit tyrosine phosphorylation. Blocking GABAAR-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxx motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that ␥2-subunit-containing heteromeric GABAARs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABAAR surface levels and synaptic inhibition.endocytosis ͉ phosphorylation ͉ structure ͉ synaptic transmission ͉ tyrosine kinase T he GABA A receptor (GABA A R), a ligand-gated ion channel, mediates the majority of fast inhibitory synaptic transmission in the mammalian CNS. Identifying the molecular mechanisms important for regulating these receptors is essential for our understanding of how synaptic inhibition and neuronal excitability are controlled. GABA A Rs are pentameric heterooligomers assembled from seven subunit classes (␣1-6, ␤1-3, ␥1-3, ␦, , , and ). It is generally assumed that the majority of GABA A Rs in the brain are assembled from at least 2 ␣-, 2 ␤-, and 1 ␥2-subunits (1). The GABA A R ␥2-subunit confers important pharmacological, functional, and membrane-trafficking properties to GABA A Rs, including benzodiazepine sensitivity, the selective targeting of GABA A Rs to inhibitory postsynaptic domains, and correct animal behavior (2, 3). The phosphorylation of tyrosine (Y) residues within the ␥2-subunit intracellular domain (ICD) at Y 365 and Y 367 increases GABA A R function. However, the mechanisms that underlie this regulation remain unclear (4, 5). Furthermore, it has recently been demonstrated that altered membrane trafficking of ␥2-subunit-containing GABA A Rs may underlie certain pathological conditions, such as the generation of pharmacoresistance and self-sustaining seizures in status epilepticus and the increased excitotoxicity in ischemia (6-8). Currently, little is known regarding the molecular mechanisms and protein interactions that underlie ␥2-subunit-dependent regulation of receptor membrane trafficking under normal or pathological conditions.A potential mechanism to regulate synaptic inhibition is to alter the number of surface and synaptic GABA A Rs. This surface receptor number can be determined, in part, by receptor endocytosis and the interaction with the clathrin adaptor protein (AP2) complex (9, 10). The AP2 complex...

The active site metal ion of superoxide dismutase (SOD) is reduced and reoxidized as it disproportionates superoxide to dioxygen and hydrogen peroxide. Thus, the reduction midpoint potential (Em) is a critical determinant of catalytic activity. In E. coli Fe-containing SOD (FeSOD), reduction of Fe3+ is accompanied by protonation of a coordinated OH-, to produce Fe2+ coordinated by H2O. The coordinated solvent's only contact with the protein beyond the active site is a conserved Gln residue. Mutation of this Gln to His or Glu resulted in elevation of the Em by 220 mV and more than 660 mV, respectively [Yikilmaz et al., Biochemistry 2006, 45, 1151-1161], despite the fact that overall protein structure was preserved, His is a chemically conservative replacement for Gln, and neutral Glu is isostructural and isoelectronic with Gln. Therefore, we have investigated several possible bases for the elevated Em's, including altered Fe electronic structure, altered active site electrostatics, altered H-bonding and altered redox-coupled proton transfer. Using EPR, MCD, and NMR spectroscopies, we find that the active site electronic structures of the two mutants resemble that of the WT enzyme, for both oxidation states, and Q69E-FeSOD's apparent deviation from WT-like Fe3+ coordination in the oxidized state can be explained by increased affinity for a small anion. Spontaneous coordination of an exogenous anion can only stabilize oxidized Q69E-Fe3+SOD and, therefore, cannot account for the increased Em of Q69E FeSOD. WT-like anion binding affinities and active site pK's indicate that His69 of Q69H-FeSOD is neutral in both oxidation states, like Gln69 of WT-FeSOD, whereas Glu69 appears to be neutral in the oxidized state but ionized in the reduced state of Q69E-FeSOD. A 1.1 A resolution crystal structure of Q69E-Fe2+SOD indicates that Glu69 accepts a strong H-bond from coordinated solvent in the reduced state, in contrast to the case in WT-FeSOD where Gln69 donates an H-bond. These data and DFT calculations lead to the proposal that the elevated Em of Q69E-FeSOD can be substantially explained by (1) relief from enforced H-bond donation in the reduced state, (2) Glu69's capacity to provide a proton for proton-coupled Fe3+ reduction, and (3) strong hydrogen bond acceptance in the reduced state, which stabilizes coordinated H2O. Our results thus support the hypothesis that the protein matrix can apply significant redox tuning via its influence over redox-coupled proton transfer and the energy associated with it.

Clathrin-mediated synaptic vesicle (SV) recycling involves the spatiotemporally controlled assembly of clathrin coat components at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P 2 ]-enriched membrane sites within the periactive zone. Such spatiotemporal control is needed to coordinate SV cargo sorting with clathrin/AP2 recruitment and to restrain membrane fission and synaptojanin-mediated uncoating until membrane deformation and clathrin coat assembly are completed. The molecular events underlying these control mechanisms are unknown. Here we show that the endocytic SH3 domaincontaining accessory protein intersectin 1 scaffolds the endocytic process by directly associating with the clathrin adaptor AP2. Acute perturbation of the intersectin 1-AP2 interaction in lamprey synapses in situ inhibits the onset of SV recycling. Structurally, complex formation can be attributed to the direct association of hydrophobic peptides within the intersectin 1 SH3A-B linker region with the "side sites" of the AP2 α-and β-appendage domains. AP2 appendage association of the SH3A-B linker region inhibits binding of the inositol phosphatase synaptojanin 1 to intersectin 1. These data identify the intersectin-AP2 complex as an important regulator of clathrinmediated SV recycling in synapses.endocytosis | synapse | scaffolding proteins | appendage | synaptojanin S ynaptic vesicles (SVs), following their activity-dependent exocytic fusion with the presynaptic plasma membrane, are recycled by compensatory endocytosis at the periactive zone (1-3), largely via clathrin-mediated reinternalization of fully fused SV membrane (4). Clathrin-coated pit (CCP) formation (5) proceeds through the assembly of endocytic proteins at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P 2 ]-enriched membrane sites (6, 7). A key factor in the assembly pathway is the heterotetrameric adaptor complex AP2, whose α-and β2-appendage domains act as major recruitment platforms for accessory proteins (6, 7), regulating distinct steps within the pathway. Despite our extensive knowledge regarding the endocytic interactome, we know comparably little about the structural components within the periactive zone that scaffold the endocytic process, thereby allowing the high fidelity of SV recycling. Such spatiotemporal control is needed to coordinate SV cargo protein sorting with coat recruitment (8) and to restrain membrane fission and uncoating until membrane deformation and CCP assembly are completed. Moreover, stabilizing scaffolds may aid coupling of SV exo-and endocytosis (1, 3). The Drosophila multidomain protein Dap160, an ortholog of mammalian intersectin, has been postulated to act as an endocytic scaffold of the periactive zone (9-11), although its precise role in SV recycling in mammalian nerve terminals remains largely unclear (12).Here we show that intersectin 1 scaffolds the endocytic process by directly associating with AP2. Acute perturbation of intersectin-AP2 complex formation blocks the onset of SV recycling. Moreover, association of the SH3A-B l...

Crystals of insulin grown in microgravity on Space Shuttle Mission STS-95 were extremely well ordered and unusually large (many >2 mm). The physical characteristics of six microgravity and six earthgrown crystals were examined by X-ray analysis employing super®ne 9 slicing and unfocused synchrotron radiation. This experimental setup allowed hundreds of re¯ections to be precisely examined from each crystal in a short period of time. The microgravity crystals were on average 34 times larger, had sevenfold lower mosaicity, had 54-fold higher re¯ection peak heights and diffracted to signi®cantly higher resolution than their earth-grown counterparts. A single mosaic domain model could account for the observed re¯ection pro®les in microgravity crystals, whereas data from earth crystals required a model with multiple mosaic domains. This statistically signi®cant and unbiased characterization indicates that the microgravity environment was useful for the improvement of crystal growth and the resultant diffraction quality in insulin crystals and may be similarly useful for macromolecular crystals in general.

No abstract

We have designed and constructed fusion genes of C-terminal (Ct) or N-terminal (Nt) bmrA with EGFP vectors and successfully expressed them in ΔBmrA (BmrA deletion strain of Bacillus subtilis), generating two new strains of B. subtilis (Ct-BmrA-EGFP and Nt-BmrA-EGFP). The fusion genes were characterized using gel electrophoresis and DNA sequencing. Their expression in live cells was determined by measuring the fluorescence of EGFP in single live cells using fluorescence microscopy and spectroscopy. The efflux function of the new strains was studied by measuring their accumulation kinetics of intracellular Hoechst dye molecules (a pump substrate) using fluorescence spectroscopy, which were compared with wild-type (WT-BmrA) and ΔBmrA strains. Both new strains show lower accumulation rates than ΔBmrA, and their efflux kinetics are inhibited by a pump inhibitor (orthovanadate). The results suggest that both strains extrude the dye molecules and the fusion proteins retain the efflux function of BmrA (ATP-binding cassette, ABC, transporter). Notably, Nt-BmrA-EGFP strain shows lower accumulation rates (higher efflux rates) than Ct-BmrA-EGFP. Modeled structures of the fusion proteins illustrate a highly flexible linker region connecting EGFP with BmrA, suggesting a minimal obstruction of EGFP to the BmrA. A closer distance of two C termini (~14 Å) than two N termini (47.9 Å) of the "closed" BmrA dimer depicts the larger steric effect of C-terminal fusion. This study also shows that glucose affects the fluorescence study of efflux function of BmrA, suggesting that efflux kinetics of ABC membrane transporters in live cells must be characterized in the absence of glucose.

Rhodopsin, a light-activated G protein coupled receptor (GPCR), has been the subject of numerous biochemical and structural investigations, serving as a model receptor for GPCRs and their activation. Herein we present the 2.3 Å resolution structure of native-source rhodopsin stabilized in a conformation competent for G protein binding. An extensive water-mediated hydrogen bond network linking the chromophore binding site to the site of G protein binding is observed, providing connections to conserved motifs essential for GPCR activation. Comparison of this extensive solvent mediated hydrogen-bonding network to the positions of ordered solvent in earlier crystallographic structures of rhodopsin photointermediates reveals both static structural and dynamic functional water-protein interactions present during the activation process. When taken with observations that solvent occupies similar positions in the structures of other GPCRs, these analyses strongly support an integral role for this dynamic ordered water network in both rhodopsin and GPCR activation.