I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18666]
Open Access
Abstract: Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is poorly understood. Arc has an N-terminal domain (NTD) involved in membrane binding and a C-terminal domain (CTD) that binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerisation, including assembly of retrovirus-like capsids involved in intercellular signalling. To obtain new tools for studies on Arc structure and function, we produced and characterised six high-affinity anti-Arc nanobodies (Nb). The CTD of rat and human Arc were both crystallised in ternary complexes with two Nbs. One Nb bound deep into the stargazin-binding pocket of Arc CTD and suggested competitive binding with Arc ligand peptides. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. The collapsed conformation closely resembles Drosophila Arc in capsids, suggesting that we have trapped a capsid-like conformation of the human Arc CTD. Our data obtained with the help of anti-Arc Nbs suggest that structural dynamics of the CTD and dimerisation of the NTD may promote the formation of capsids. Taken together, the recombinant high-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function, as well as mechanisms of Arc capsid formation, both in vitro and in vivo. For example, the Nbs could serve as a genetically encoded tools for inhibition of endogenous Arc interactions in the study of neuronal function and plasticity.
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Jun 2022
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Apicomplexan parasites, such as Plasmodium spp., rely on an unusual actomyosin motor, termed glideosome, for motility and host cell invasion. The actin filaments are maintained by a small set of essential regulators, which provide control over actin dynamics in the different stages of the parasite life cycle. Actin filament capping proteins (CPs) are indispensable heterodimeric regulators of actin dynamics. CPs have been extensively characterized in higher eukaryotes, but their role and functional mechanism in Apicomplexa remain enigmatic. Here, we present the first crystal structure of a homodimeric CP from the malaria parasite and compare the homo- and heterodimeric CP structures in detail. Despite retaining several characteristics of a canonical CP, the homodimeric Plasmodium berghei (Pb)CP exhibits crucial differences to the canonical heterodimers. Both homo- and heterodimeric PbCPs regulate actin dynamics in an atypical manner, facilitating rapid turnover of parasite actin, without affecting its critical concentration. Homo- and heterodimeric PbCPs show partially redundant activities, possibly to rescue actin filament capping in life cycle stages where the β-subunit is downregulated. Our data suggest that the homodimeric PbCP also influences actin kinetics by recruiting lateral actin dimers. This unusual function could arise from the absence of a β-subunit, as the asymmetric PbCP homodimer lacks structural elements essential for canonical barbed end interactions suggesting a novel CP binding mode. These findings will facilitate further studies aimed at elucidating the precise actin filament capping mechanism in Plasmodium.
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Feb 2022
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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
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Diamond Proposal Number(s):
[27081]
Abstract: Peripheral myelin protein 2 (P2) is a fatty acid-binding protein expressed in vertebrate peripheral nervous system myelin, as well as in human astrocytes. Suggested functions of P2 include membrane stacking and lipid transport. Mutations in the PMP2 gene, encoding P2, are associated with Charcot-Marie-Tooth disease (CMT). Recent studies have revealed three novel PMP2 mutations in CMT patients. To shed light on the structure and function of these P2 variants, we used X-ray and neutron crystallography, small-angle X-ray scattering, circular dichroism spectroscopy, computer simulations and lipid binding assays. The crystal and solution structures of the I50del, M114T and V115A variants of P2 showed minor differences to the wild-type protein, whereas their thermal stability was reduced. Vesicle aggregation assays revealed no change in membrane stacking characteristics, while the variants showed altered fatty acid binding. Time-lapse imaging of lipid bilayers indicated formation of double membrane structures induced by P2, which could be related to its function in stacking of two myelin membrane surfaces in vivo. In order to better understand the links between structure, dynamics and function, the crystal structure of perdeuterated P2 was refined from room temperature data using neutrons and X-rays and the results were compared to simulations and cryocooled crystal structures. Our data indicate similar properties for all known human P2 CMT variants; while crystal structures are nearly identical, thermal stability and function of CMT variants are impaired. Our data provide new insights into the structure-function relationships and dynamics of P2 in health and disease.
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Jun 2021
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18666]
Open Access
Abstract: Synaptic plasticity is vital for brain function and memory formation. One of the key proteins in long-term synaptic plasticity and memory is the activity-regulated cytoskeleton-associated protein (Arc). Mammalian Arc forms virus-like capsid structures in a process requiring the N-terminal domain and contains two C-terminal lobes that are structural homologues to retroviral capsids. Drosophila has two isoforms of Arc, dArc1 and dArc2, with low sequence similarity to mammalian Arc, but lacking a large N-terminal domain. Both dArc isoforms are related to the Ty3/gypsy retrotransposon capsid, consisting of N- and C-terminal lobes. Structures of dArc1, as well as capsids formed by both dArc isoforms, have been recently determined. We carried out structural characterization of the four individual dArc lobe domains. As opposed to the corresponding mammalian Arc lobe domains, which are monomeric, the dArc lobes were all oligomeric in solution, indicating a strong propensity for homophilic interactions. A truncated N-lobe from dArc2 formed a domain-swapped dimer in the crystal structure, resulting in a novel dimer interaction that could be relevant for capsid assembly or other dArc functions. This domain-swapped structure resembles the dimeric protein C of flavivirus capsids, as well as the structure of histones dimers, domain-swapped transcription factors, and membrane-interacting BAK domains. The strong oligomerization properties of the isolated dArc lobe domains explain the ability of dArc to form capsids in the absence of any large N-terminal domain, in contrast to the mammalian protein.
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May 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
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Oleksii
Nikolaienko
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Erik I.
Hallin
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Sverre
Grødem
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Helene J.
Bustad
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Marte I.
Flydal
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Ian
Merski
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Tomohisa
Hosokawa
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Daniela
Lascu
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Shreeram
Akerkar
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Jorge
Cuellar
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James J.
Chambers
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Rory
O’connell
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Gopinath
Muruganandam
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Remy
Loris
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Christine
Touma
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Tambudzai
Kanhema
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Yasunori
Hayashi
,
Margaret M.
Stratton
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José M.
Valpuesta
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Petri
Kursula
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Aurora
Martinez
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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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