Structural characterization of gephyrin by AFM and SAXS reveals a mixture of compact and extended states

Sander, Bodo; Tria, Giancarlo; Shkumatov, Alexander V.; Kim, Eun Young; Grossmann, J. Günter; Teßmer, Ingrid; Svergun, Dmitri I.; Schindelin, Hermann

doi:10.1107/s0907444913018714

Cited by 48 publications

(49 citation statements)

References 68 publications

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“…S9), compared to the shallow minimum observed for GTF180-DN, may indicate that the full-length protein is slightly more flexible. Studies [22,23] have found a functional role for protein flexibility, although whether this also applies to glucansucrases such as GTF180 remains to be established. In this light, we considered performing SAXS measurements of GTF180 (or GTF180-DN) in the presence of its substrate sucrose.…”

Section: Discussionmentioning

confidence: 99%

Flexibility of truncated and full‐length glucansucrase GTF180 enzymes from Lactobacillus reuteri 180

et al. 2014

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Glucansucrase enzymes synthesize high-molecular-mass extracellular aglucan polysaccharides from sucrose. Previously, the crystal structure of truncated glucansucrase glucosyltransferase (GTF)180-DN from Lactobacillus reuteri 180 (lacking the N-terminal domain) revealed an elongated overall structure with two remote domains (IV and V) extending away from the core. By contrast, a new crystal form of the a-1,6/a-1,3 specific glucansucrase GTF180-DN shows an approximate 120 o rotation of domain V about a hinge located between domains IV and V, giving a much more compact structure than before. Positional variability of domain V in solution is confirmed by small angle X-ray scattering experiments and rigid-body ensemble calculations. In addition, small angle Xray scattering measurements of full-length GTF180 also provide the first structural data for a full-length glucansucrase, showing that the enzyme has an almost symmetric boomerang-like molecular shape, with a bend likely located between domains IV and V. The~700-residue N-terminal domain, which is not present in the crystal structures, extends away from domain V and the catalytic core of the enzyme. We conclude that, as a result of the hinge region, in solution, GTF180-DN (and likely also the full-length GTF180) shows conformational flexibility; this may be a general feature of GH70 glucansucrases.

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Section: Discussionmentioning

confidence: 99%

Flexibility of truncated and full‐length glucansucrase GTF180 enzymes from Lactobacillus reuteri 180

et al. 2014

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“…The generated models may include folded regions or domains or even nucleic acids if needed; for oligomeric structures, asymmetric or symmetric multimers can be generated. The latter approach was employed in the study of a flexible protein gephyrin which consists of two folded domains (of which one forms a trimer) connected by a 150 amino acid residues linker [48]. The possibility of combining multiple independent pools further expands the range of applicability of EOM: in a recent study of a full-length mitochondrial glutaminase C [49], pools consisting of dimers, trimers and octamers were combined to explain how disordered regions influence enzymatic activity and the oligomeric state.…”

Section: Ensemble Approachmentioning

confidence: 99%

A practical guide to small angle X‐ray scattering (SAXS) of flexible and intrinsically disordered proteins

Kikhney¹,

2015

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Edited by Wilhelm Just Keywords:Small angle scattering Small-angle X-ray scattering Flexibility Disorder Intrinsically disordered protein a b s t r a c t Small-angle X-ray scattering (SAXS) is a biophysical method to study the overall shape and structural transitions of biological macromolecules in solution. SAXS provides low resolution information on the shape, conformation and assembly state of proteins, nucleic acids and various macromolecular complexes. The technique also offers powerful means for the quantitative analysis of flexible systems, including intrinsically disordered proteins (IDPs). Here, the basic principles of SAXS are presented, and profits and pitfalls of the characterization of multidomain flexible proteins and IDPs using SAXS are discussed from the practical point of view. Examples of the synergistic use of SAXS with high resolution methods like X-ray crystallography and nuclear magnetic resonance (NMR), as well as other experimental and in silico techniques to characterize completely, or partially unstructured proteins, are presented.

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“…However, a recent structural analysis of gephyrin (expressed in E. coli) using atomic force microscopy and synchrotron X-ray scattering revealed that gephyrin forms trimers with multiple conformations, owing to partial flexibility of the C domain, and confirmed that the linker domain prevents E domain dimerization 174 , as was shown previously 164 . Visualization of gephyrin clusters in glycinergic synapses using super-resolution microscopy revealed a 1:1 stoichiometry with GlyRs 110 .…”

Section: Box 1: Gephyrin Structurementioning

confidence: 99%

Gephyrin: a master regulator of neuronal function?

2014

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The neurotransmitters GABA and glycine mediate fast synaptic inhibition by activating ligandgated chloride channels-namely, type A GABA (GABA(A)) and glycine receptors. Both types of receptors are anchored postsynaptically by gephyrin, which self-assembles into a scaffold and interacts with the cytoskeleton. Current research indicates that postsynaptic gephyrin clusters are dynamic assemblies that are held together and regulated by multiple protein-protein interactions. Moreover, post-translational modifications of gephyrin regulate the formation and plasticity of GABAergic synapses by altering the clustering properties of postsynaptic scaffolds and thereby the availability and function of receptors and other signalling molecules. Here, we discuss the formation and regulation of the gephyrin scaffold, its role in GABAergic and glycinergic synaptic function and the implications for the pathophysiology of brain disorders caused by abnormal inhibitory neurotransmission. Shiva Tyagarajan graduated as a molecular virologist at the Pennsylvania State University (USA) and later trained as a neurobiologist during his post-doctoral studies at the University of Zurich. He is currently a group leader and the research focus of his group is to elucidate molecular and cellular mechanisms that couple transcriptional and post-transcriptional programs to regulate GABAergic synaptic plasticity. 3 AbstractThe neurotransmitters GABA and glycine mediate fast synaptic inhibition by activating ligand-gated Cl  channels, namely the GABA A and glycine receptors. Both types of receptors are anchored postsynaptically by gephyrin, which self-assembles into a scaffold and interacts with the cytoskeleton. Current research indicates that gephyrin postsynaptic clusters are dynamic assemblies that are held together and regulated by multiple protein-protein interactions. Moreover, posttranslational modifications of gephyrin regulate the formation and plasticity of GABAergic synapses, by altering the clustering properties of postsynaptic scaffolds and thereby the availability and function of receptors and other signalling molecules.Here, we discuss the formation and regulation of the gephyrin scaffold, its role in GABAergic and glycinergic synaptic function and the implications for the pathophysiology of brain disorders caused by abnormal inhibitory neurotransmission.On-line summary  Gephyrin is a multi-functional protein, responsible for Moco biosynthesis in all organisms, and for postsynaptic clustering of glycine receptors and GABAA receptors in vertebrate CNS  Gephyrin forms a protein scaffold by self-assembly from trimeric complexes, which interacts with numerous, structurally different proteins, in order to form a high ordered signalling complex in glycinergic and GABAergic synapses  Gephyrin function as a scaffolding protein is regulated by alternate mRNA splicing and by multiple post-transcriptional and posttranslational modifications, which only begin to be understood.  Regulation of gephyrin scaffold by multiple signallin...

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Structural characterization of gephyrin by AFM and SAXS reveals a mixture of compact and extended states

Cited by 48 publications

References 68 publications

Flexibility of truncated and full‐length glucansucrase GTF180 enzymes from Lactobacillus reuteri 180

Flexibility of truncated and full‐length glucansucrase GTF180 enzymes from Lactobacillus reuteri 180

A practical guide to small angle X‐ray scattering (SAXS) of flexible and intrinsically disordered proteins

Gephyrin: a master regulator of neuronal function?

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