Hermann Schindelin

A left-hand beta-helix revealed by the crystal structure of a carbonic anhydrase from the archaeon Methanosarcina thermophila.

A carbonic anhydrase from the thermophilic archaeon Methanosarcina thermophila that exhibits no significant sequence similarity to known carbonic anhydrases has recently been characterized. Here we present the structure of this enzyme, which adopts a left‐handed parallel beta‐helix fold. This fold is of particular interest since it contains only left‐handed crossover connections between the parallel beta‐strands, which so far have been observed very infrequently. The active form of the enzyme is a trimer with three zinc‐containing active sites, each located at the interface between two monomers. While the arrangement of active site groups differs between this enzyme and the carbonic anhydrases from higher vertebrates, there are structural similarities in the zinc coordination environment, suggestive of convergent evolution dictated by the chemical requirements for catalysis of the same reaction. Based on sequence similarities, the structure of this enzyme is the prototype of a new class of carbonic anhydrases with representatives in all three phylogenetic domains of life.

Mechanism of Ubiquitin Activation Revealed by the Structure of a Bacterial MoeB-MoaD Complex

The activation of ubiquitin and related protein modifiers is catalysed by members of the E1 enzyme family that use ATP for the covalent self-attachment of the modifiers to a conserved cysteine. The Escherichia coli proteins MoeB and MoaD are involved in molybdenum cofactor (Moco) biosynthesis, an evolutionarily conserved pathway. The MoeB- and E1-catalysed reactions are mechanistically similar, and despite a lack of sequence similarity, MoaD and ubiquitin display the same fold including a conserved carboxy-terminal Gly-Gly motif. Similar to the E1 enzymes, MoeB activates the C terminus of MoaD to form an acyl-adenylate. Subsequently, a sulphurtransferase converts the MoaD acyl-adenylate to a thiocarboxylate that acts as the sulphur donor during Moco biosynthesis. These findings suggest that ubiquitin and E1 are derived from two ancestral genes closely related to moaD and moeB. Here we present the crystal structures of the MoeB–MoaD complex in its apo, ATP-bound, and MoaD-adenylate forms, and highlight the functional similarities between the MoeB– and E1–substrate complexes. These structures provide a molecular framework for understanding the activation of ubiquitin, Rub, SUMO and the sulphur incorporation step during Moco and thiamine biosynthesis.

Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation.

Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea and eukaryotes, including humans. Genetic deficiencies of enzymes involved in Moco biosynthesis in humans lead to a severe and usually fatal disease. Moco contains a tricyclic pyranopterin, termed molybdopterin (MPT), that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of MPT is generated by MPT synthase, which consists of a large and small subunits. The 1.45 Å resolution crystal structure of MPT synthase reveals a heterotetrameric protein in which the C-terminus of each small subunit is inserted into a large subunit to form the active site. In the activated form of the enzyme this C-terminus is present as a thiocarboxylate. In the structure of a covalent complex of MPT synthase, an isopeptide bond is present between the C-terminus of the small subunit and a Lys side chain in the large subunit. The strong structural similarity between the small subunit of MPT synthase and ubiquitin provides evidence for the evolutionary antecedence of the Moco biosynthetic pathway to the ubiquitin dependent protein degradation pathway.

Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes.

Ubiquitin (Ub) and ubiquitin-like proteins (Ubls) are conjugated to their targets by specific cascades involving three classes of enzymes, E1, E2, and E3. Each E1 adenylates the C terminus of its cognate Ubl, forms a E1 approximately Ubl thioester intermediate, and ultimately generates a thioester-linked E2 approximately Ubl product. We have determined the crystal structure of yeast Uba1, revealing a modular architecture with individual domains primarily mediating these specific activities. The negatively charged C-terminal ubiquitin-fold domain (UFD) is primed for binding of E2s and recognizes their positively charged first alpha helix via electrostatic interactions. In addition, a mobile loop from the domain harboring the E1 catalytic cysteine contributes to E2 binding. Significant, experimentally observed motions in the UFD around a hinge in the linker connecting this domain to the rest of the enzyme suggest a conformation-dependent mechanism for the transthioesterification function of Uba1; however, this mechanism clearly differs from that of other E1 enzymes.

The Residence Time of GABAARs at Inhibitory Synapses Is Determined by Direct Binding of the Receptor α1 Subunit to Gephyrin

The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABAARs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABAARs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ∼20 μm, mediated by residues 360–375 within the intracellular domain of this receptor subunit. Mutating residues 360–375 decreases both the accumulation of α1-containing GABAARs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit-containing GABAARs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360–375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABAARs at synaptic sites, thereby modulating the strength of synaptic inhibition.

The crystal structure of Escherichia coli MoeA and its relationship to the multifunctional protein gephyrin.

BACKGROUND Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in archaea, eubacteria, and eukaryotes. In humans, genetic abnormalities in the biosynthetic pathway result in Moco deficiency, which is accompanied by severe neurological symptoms and death shortly after birth. The Escherichia coli MoeA and MogA proteins are involved in the final step of Moco biosynthesis: the incorporation of molybdenum into molybdopterin (MPT), the organic pyranopterin moiety of Moco. RESULTS The crystal structure of E. coli MoeA has been refined at 2 A resolution and reveals that the highly elongated MoeA monomer consists of four clearly separated domains, one of which is structurally related to MogA, indicating a divergent evolutionary relationship between both proteins. The active form of MoeA is a dimer, and a putative active site appears to be localized to a cleft formed between domain II of the first monomer and domains III and IV of the second monomer. CONCLUSIONS In eukaryotes, MogA and MoeA are fused into a single polypeptide chain. The corresponding mammalian protein gephyrin has also been implicated in the anchoring of glycinergic receptors to the cytoskeleton at inhibitory synapses. Based on the structures of MoeA and MogA, gephyrin is surmised to be a highly organized molecule containing at least five domains. This multidomain arrangement could provide a structural basis for its functional diversity. The oligomeric states of MoeA and MogA suggest how gephyrin could assemble into a hexagonal scaffold at inhibitory synapses.

Deciphering the structural framework of glycine receptor anchoring by gephyrin

Glycine is the major inhibitory neurotransmitter in the spinal cord and brain stem. Gephyrin is required to achieve a high concentration of glycine receptors (GlyRs) in the postsynaptic membrane, which is crucial for efficient glycinergic signal transduction. The interaction between gephyrin and the GlyR involves the E‐domain of gephyrin and a cytoplasmic loop located between transmembrane segments three and four of the GlyR β subunit. Here, we present crystal structures of the gephyrin E‐domain with and without the GlyR β‐loop at 2.4 and 2.7 Å resolutions, respectively. The GlyR β‐loop is bound in a symmetric ‘key and lock’ fashion to each E‐domain monomer in a pocket adjacent to the dimer interface. Structure‐guided mutagenesis followed by in vitro binding and in vivo colocalization assays demonstrate that a hydrophobic interaction formed by Phe 330 of gephyrin and Phe 398 and Ile 400 of the GlyR β‐loop is crucial for binding.

The Catalytic Activity of Protein-disulfide Isomerase Requires a Conformationally Flexible Molecule

Protein-disulfide isomerase (PDI) catalyzes the formation of the correct pattern of disulfide bonds in secretory proteins. A low resolution crystal structure of yeast PDI described here reveals large scale conformational changes compared with the initially reported structure, indicating that PDI is a highly flexible molecule with its catalytic domains, a and a′, representing two mobile arms connected to a more rigid core composed of the b and b′ domains. Limited proteolysis revealed that the linker between the a domain and the core is more susceptible to degradation than that connecting the a′ domain to the core. By restricting the two arms with inter-domain disulfide bonds, the molecular flexibility of PDI, especially that of its a domain, was demonstrated to be essential for the enzymatic activity in vitro and in vivo. The crystal structure also featured a PDI dimer, and a propensity to dimerize in solution and in the ER was confirmed by cross-linking experiments and the split green fluorescent protein system. Although sedimentation studies suggested that the self-association of PDI is weak, we hypothesize that PDI exists as an interconvertible mixture of monomers and dimers in the endoplasmic reticulum due to its high abundance in this compartment.

Gephyrin, the enigmatic organizer at GABAergic synapses

GABAA receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows for an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABAA receptors have also been shown to directly interact with gephyrin and interaction sites have been identified and mapped within the intracellular loops of the GABAA receptor α1, α2, and α3 subunits. Gephyrin-binding to GABAA receptors seems to be at least one order of magnitude weaker than to glycine receptors (GlyRs) and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but also plays a crucial role in synaptic dynamics and is a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signaling proteins into close spatial proximity.

Candidate Amino Acids Involved in H+ Gating of Acid-sensing Ion Channel 1a

Acid-sensing ion channels are ligand-gated cation channels, gated by extracellular H+. H+ is the simplest ligand possible, and whereas for larger ligands that gate ion channels complex binding sites in the three-dimensional structure of the proteins have to be assumed, H+ could in principle gate a channel by titration of a single amino acid. Experimental evidence suggests a more complex situation, however. For example, it has been shown that extracellular Ca2+ ions compete with H+; probably Ca2+ ions bound to the extracellular loop of ASICs stabilize the closed state of the channel and have to be displaced before the channel can open. In such a scheme, amino acids contributing to Ca2+ binding would also be candidates contributing to H+ gating. In this study we systematically screened more than 40 conserved, charged amino acids in the extracellular region of ASIC1a for a possible contribution to H+ gating. We identified four amino acids where substitution strongly affects H+ gating: Glu63, His72/His73, and Asp78. These amino acids are highly conserved among H+-sensitive ASICs and are candidates for the “H+ sensor” of ASICs.