I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13440]
Open Access
Abstract: The activity-regulated cytoskeleton-associated protein (Arc) is important for synaptic plasticity and the normal function of the brain. Arc interacts with neuronal postsynaptic proteins, but the mechanistic details of its function have not been fully established. The C-terminal domain of Arc consists of tandem domains, termed the N- and C-lobe. The N-lobe harbours a peptide binding site, able to bind multiple targets. By measuring the affinity of human Arc towards various peptides from stargazin and guanylate kinase-associated protein (GKAP), we have refined its specificity determinants. We found two sites in the GKAP repeat region that bind to Arc and confirmed these interactions by X-ray crystallography. Phosphorylation of the stargazin peptide did not affect binding affinity but caused changes in thermodynamic parameters. Comparison of the crystal structures of three high-resolution human Arc-peptide complexes identifies three conserved C–H…π interactions at the binding cavity, explaining the sequence specificity of short linear motif binding by Arc. We further characterise central residues of the Arc lobe fold, show the effects of peptide binding on protein dynamics, and identify acyl carrier proteins as structures similar to the Arc lobes. We hypothesise that Arc may affect protein-protein interactions and phase separation at the postsynaptic density, affecting protein turnover and re-modelling of the synapse. The present data on Arc structure and ligand binding will help in further deciphering these processes.
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Jul 2021
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19951]
Open Access
Abstract: Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders. Despite the common involvement of ganglioside-induced differentiation-associated protein 1 (GDAP1) in CMT, the protein structure and function, as well as the pathogenic mechanisms, remain unclear. We determined the crystal structure of the complete human GDAP1 core domain, which shows a novel mode of dimerization within the glutathione S-transferase (GST) family. The long GDAP1-specific insertion forms an extended helix and a flexible loop. GDAP1 is catalytically inactive toward classical GST substrates. Through metabolite screening, we identified a ligand for GDAP1, the fatty acid hexadecanedioic acid, which is relevant for mitochondrial membrane permeability and Ca2+ homeostasis. The fatty acid binds to a pocket next to a CMT-linked residue cluster, increases protein stability, and induces changes in protein conformation and oligomerization. The closest homologue of GDAP1, GDAP1L1, is monomeric in its full-length form. Our results highlight the uniqueness of GDAP1 within the GST family and point toward allosteric mechanisms in regulating GDAP1 oligomeric state and function.
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Jan 2021
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B21-High Throughput SAXS
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Open Access
Abstract: N‐myc downstream‐regulated gene 1 (NDRG1) is a tumour suppressor involved in vesicular trafficking and stress response. NDRG1 participates in peripheral nerve myelination, and mutations in the NDRG1 gene lead to Charcot‐Marie‐Tooth neuropathy. The 43‐kDa NDRG1 is considered as an inactive member of the α/β hydrolase superfamily. In addition to a central α/β hydrolase fold domain, NDRG1 consists of a short N terminus and a C‐terminal region with three 10‐residue repeats. We determined the crystal structure of the α/β hydrolase domain of human NDRG1 and characterised the structure and dynamics of full‐length NDRG1. The structure of the α/β hydrolase domain resembles the canonical α/β hydrolase fold with a central β sheet surrounded by α helices. Small‐angle X‐ray scattering and CD spectroscopy indicated a variable conformation for the N‐ and C‐terminal regions. NDRG1 binds to various types of lipid vesicles, and the conformation of the C‐terminal region is modulated upon lipid interaction. Intriguingly, NDRG1 interacts with metal ions, such as nickel, but is prone to aggregation in their presence. Our results uncover the structural and dynamic features of NDRG1, as well as elucidate its interactions with metals and lipids, and encourage studies to identify a putative hydrolase activity of NDRG1.
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Jan 2021
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B21-High Throughput SAXS
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Maria S.
Eriksen
,
Oleksii
Nikolaienko
,
Erik I.
Hallin
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Sverre
Grødem
,
Helene J.
Bustad
,
Marte I.
Flydal
,
Ian
Merski
,
Tomohisa
Hosokawa
,
Daniela
Lascu
,
Shreeram
Akerkar
,
Jorge
Cuellar
,
James J.
Chambers
,
Rory
O’connell
,
Gopinath
Muruganandam
,
Remy
Loris
,
Christine
Touma
,
Tambudzai
Kanhema
,
Yasunori
Hayashi
,
Margaret M.
Stratton
,
José M.
Valpuesta
,
Petri
Kursula
,
Aurora
Martinez
,
Clive R.
Bramham
Abstract: Activity‐regulated cytoskeleton‐associated protein (Arc) is a protein interaction hub with diverse roles in intracellular neuronal signaling, and important functions in neuronal synaptic plasticity, memory, and postnatal cortical development. Arc has homology to retroviral Gag protein and is capable of self‐assembly into virus‐like capsids implicated in the intercellular transfer of RNA. However, the molecular basis of Arc self‐association and capsid formation is largely unknown. Here, we identified a 28‐amino‐acid stretch in the mammalian Arc N‐terminal (NT) domain that is necessary and sufficient for self‐association. Within this region, we identified a 7‐residue oligomerization motif, critical for the formation of virus‐like capsids. Purified wild‐type Arc formed capsids as shown by transmission and cryo‐electron microscopy, whereas mutant Arc with disruption of the oligomerization motif formed homogenous dimers. An atomic‐resolution crystal structure of the oligomerization region peptide demonstrated an antiparallel coiled‐coil interface, strongly supporting NT‐NT domain interactions in Arc oligomerization. The NT coil–coil interaction was also validated in live neurons using fluorescence lifetime FRET imaging, and mutation of the oligomerization motif disrupted Arc‐facilitated endocytosis. Furthermore, using single‐molecule photobleaching, we show that Arc mRNA greatly enhances higher‐order oligomerization in a manner dependent on the oligomerization motif. In conclusion, a helical coil in the Arc NT domain supports self‐association above the dimer stage, mRNA‐induced oligomerization, and formation of virus‐like capsids.
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Nov 2020
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B21-High Throughput SAXS
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Abstract: Actin capping proteins belong to the core set of proteins minimally required for actin-based motility and are present in virtually all eukaryotic cells. They bind to the fast-growing barbed end of an actin filament, preventing addition and loss of monomers, thus restricting growth to the slow-growing pointed end. Actin capping proteins are usually heterodimers of two subunits. The Plasmodium orthologs are an exception, as their α subunits are able to form homodimers. We show here that, while the β subunit alone is unstable, the α subunit of the Plasmodium actin capping protein forms functional homo- and heterodimers. This implies independent functions for the αα homo- and αβ heterodimers in certain stages of the parasite life cycle. Structurally, the homodimers resemble canonical αβ heterodimers, although certain rearrangements at the interface must be required. Both homo- and heterodimers bind to actin filaments in a roughly equimolar ratio, indicating they may also bind other sites than barbed ends.
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Mar 2020
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B21-High Throughput SAXS
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Open Access
Abstract: The process of myelination in the nervous system requires a coordinated formation of both transient and stable supramolecular complexes. Myelin-specific proteins play key roles in these assemblies, which may link membranes to each other or connect the myelinating cell cytoskeleton to the extracellular matrix. The myelin protein periaxin is known to play an important role in linking the Schwann cell cytoskeleton to the basal lamina through membrane receptors, such as the dystroglycan complex. Mutations that truncate periaxin from the C terminus cause demyelinating peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease type 4F, indicating a function for the periaxin C-terminal region in myelination. We identified the cytoplasmic domain of β4 integrin as a specific high-affinity binding partner for periaxin. The C-terminal region of periaxin remains unfolded and flexible when bound to the third fibronectin type III domain of β4 integrin. Our data suggest that periaxin is able to link the Schwann cell cytoplasm to the basal lamina through a two-pronged interaction via different membrane protein complexes, which bind close to the N and C terminus of this elongated, flexible molecule.
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Apr 2019
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B21-High Throughput SAXS
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Abstract: The activity‐regulated cytoskeleton‐associated protein (ARC) is critical for long‐term synaptic plasticity and memory formation. Acting as a protein interaction hub, ARC regulates diverse signalling events in postsynaptic neurons. A protein interaction site is present in the ARC C‐terminal domain (CTD), a bilobar structure homologous to the retroviral Gag capsid domain. We hypothesized that detailed knowledge of the three‐dimensional molecular structure of monomeric full‐length ARC is crucial to understand its function; therefore, we set out to determine the structure of ARC to understand its various functional modalities. We purified recombinant ARC and analyzed its structure using small‐angle X‐ray scattering and synchrotron radiation circular dichroism spectroscopy. Monomeric full‐length ARC has a compact, closed structure, in which the oppositely charged N‐terminal domain (NTD) and CTD are juxtaposed, and the flexible linker between them is not extended. The modeled structure of ARC is supported by intramolecular live‐cell Förster resonance energy transfer imaging in rat hippocampal slices. Peptides from several postsynaptic proteins, including stargazin, bind to the N‐lobe, but not to the C‐lobe, of the bilobar CTD. This interaction does not induce large‐scale conformational changes in the CTD or flanking unfolded regions. The ARC NTD contains long helices, predicted to form an anti‐parallel coiled coil; binding of ARC to phospholipid membranes requires the NTD. Our data support a role for the ARC NTD in oligomerization as well as lipid membrane binding. The findings have important implications for the structural organization of ARC with respect to distinct functions, such as postsynaptic signal transduction and virus‐like capsid formation.
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Nov 2018
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B21-High Throughput SAXS
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Abstract: The close association of myelinated axons and their myelin sheaths involves numerous intercellular molecular interactions. For example, myelin‐associated glycoprotein (MAG) mediates myelin‐to‐axon adhesion and signalling via molecules on the axonal surface. However, knowledge about intracellular binding partners of myelin proteins, including MAG, has remained limited. The two splice isoforms of MAG, S‐ and L‐MAG, display distinct cytoplasmic domains and spatiotemporal expression profiles. We used yeast 2‐hybrid screening to identify interaction partners of L‐MAG and found the dynein light chain DYNLL1 (also termed DLC8). DYNLL1 homodimers are known to facilitate dimerization of target proteins. L‐MAG and DYNLL1 associate with high affinity, as confirmed with recombinant proteins in vitro. Structural analyses of the purified complex indicate that the DYNLL1‐binding segment is localized close to the L‐MAG C terminus, next to the Fyn kinase Tyr phosphorylation site. The crystal structure of the complex between DYNLL1 and its binding segment on L‐MAG shows 2:2 binding in a parallel arrangement, indicating a heterotetrameric complex. The homology between L‐MAG and previously characterized DYNLL1‐ligands is limited, and some details of binding‐site interactions are unique for L‐MAG. The structure of the complex between the entire L‐MAG cytoplasmic domain and DYNLL1, as well as that of the the extracellular domain of MAG, were modeled based on small‐angle X‐ray scattering data, allowing structural insights into L‐MAG interactions on both membrane surfaces. Our data imply that DYNLL1 dimerizes L‐MAG, but not S‐MAG, through the formation of a specific 2:2 heterotetramer. This arrangement is likely to affect, in an isoform‐specific manner, the functions of MAG in adhesion and myelin‐to‐axon signalling.
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Sep 2018
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I04-Macromolecular Crystallography
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Abstract: The Shank proteins are crucial scaffolding elements of the post-synaptic density (PSD). One of the best-characterized domains in Shank is the PDZ domain, which binds to C-terminal segments of several other PSD proteins. We carried out a detailed structural analysis of Shank3 PDZ domain-peptide complexes, in order to understand determinants of binding affinity towards different ligand proteins. Ligand peptides from four different proteins were cocrystallized with the Shank3 PDZ domain, and binding affinities were determined calorimetrically. In addition, to conserve class I interactions between the first and third C-terminal peptide residue and Shank3, side chain interactions of other residues in the peptide with the PDZ domain are important factors in defining affinity. Structural conservation suggests that the binding specificities of the PDZ domains from different Shanks are similar. Two conserved buried water molecules in PDZ domains may affect correct local folding of ligand recognition determinants. The solution structure of a tandem Shank3 construct containing the SH3 and PDZ domains showed that the two domains are close to each other, which could be of relevance, when recognizing and binding full target proteins. The SH3 domain did not affect the affinity of the PDZ domain towards short target peptides, and the schizophrenia-linked Shank3 mutation R536W in the linker between the domains had no effect on the structure or peptide interactions of the Shank3 SH3-PDZ unit. Our data show the spatial arrangement of two adjacent Shank domains and pinpoint affinity determinants for short PDZ domain ligands with limited sequence homology.
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Feb 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Salla
Ruskamo
,
Tuomo
Nieminen
,
Cecilie K.
Kristiansen
,
Guro H.
Vatne
,
Anne
Baumann
,
Erik I.
Hallin
,
Arne
Raasakka
,
Päivi
Joensuu
,
Ulrich
Bergmann
,
Ilpo
Vattulainen
,
Petri
Kursula
Diamond Proposal Number(s):
[14794]
Open Access
Abstract: Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neuropathies. Recently, three CMT1-associated point mutations (I43N, T51P, and I52T) were discovered in the abundant peripheral myelin protein P2. These mutations trigger abnormal myelin structure, leading to reduced nerve conduction velocity, muscle weakness, and distal limb atrophy. P2 is a myelin-specific protein expressed by Schwann cells that binds to fatty acids and membranes, contributing to peripheral myelin lipid homeostasis. We studied the molecular basis of the P2 patient mutations. None of the CMT1-associated mutations alter the overall folding of P2 in the crystal state. P2 disease variants show increased aggregation tendency and remarkably reduced stability, T51P being most severe. In addition, P2 disease mutations affect protein dynamics. Both fatty acid binding by P2 and the kinetics of its membrane interactions are affected by the mutations. Experiments and simulations suggest opening of the β barrel in T51P, possibly representing a general mechanism in fatty acid-binding proteins. Our findings demonstrate that altered biophysical properties and functional dynamics of P2 may cause myelin defects in CMT1 patients. At the molecular level, a few malformed hydrogen bonds lead to structural instability and misregulation of conformational changes related to ligand exchange and membrane binding.
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Jul 2017
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