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.