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• W2943929497 abstract "•The Arc capsid domain has a rigid bilobar structure•The N-terminal subdomain exists in an alternative conformation•Arc CA binds both the GluN2A and GluN2B NMDA receptor subunits•Ligand binding inhibits temperature-induced oligomerization of the Arc CA domain The activity-regulated cytoskeleton-associated protein, Arc, is highly expressed in neuronal dendrites and is involved in synaptic scaling and plasticity. Arc exhibits homology to the capsid-forming Gag proteins from retroviruses and can encapsulate its own mRNA and transport it to neighboring neurons. However, the molecular events that lead to the assembly of Arc capsids and how the capsid formation is regulated are not known. Here we show that the capsid domain of Arc may transiently form homogeneous oligomers of similar size as capsids formed by full-length Arc. We determined a high-resolution structure of the monomeric Arc capsid domain and mapped the initial structural change in the oligomerization process to the N-terminal part of the capsid domain. Peptide ligands from the NMDA receptor subunits inhibit oligomerization, which suggests that Arc's ability to transfer mRNA between cells may be regulated by protein-protein interactions at the synapse. The activity-regulated cytoskeleton-associated protein, Arc, is highly expressed in neuronal dendrites and is involved in synaptic scaling and plasticity. Arc exhibits homology to the capsid-forming Gag proteins from retroviruses and can encapsulate its own mRNA and transport it to neighboring neurons. However, the molecular events that lead to the assembly of Arc capsids and how the capsid formation is regulated are not known. Here we show that the capsid domain of Arc may transiently form homogeneous oligomers of similar size as capsids formed by full-length Arc. We determined a high-resolution structure of the monomeric Arc capsid domain and mapped the initial structural change in the oligomerization process to the N-terminal part of the capsid domain. Peptide ligands from the NMDA receptor subunits inhibit oligomerization, which suggests that Arc's ability to transfer mRNA between cells may be regulated by protein-protein interactions at the synapse. Storage of information in the human brain is a highly dynamic process requiring both rapid scaling of synaptic responses and reorganization of the synaptic connections between networks of neurons. The neuronal Activity-Regulated Cytoskeleton-Associated protein (Arc) is a regulator of both synaptic scaling (Plath et al., 2006Plath N. Ohana O. Dammermann B. Errington M.L. Schmitz D. Gross C. Mao X. Engelsberg A. Mahlke C. Welzl H. et al.Arc/Arg3.1 is essential for the consolidation of synaptic plasticity and memories.Neuron. 2006; 52: 437-444Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar, Shepherd et al., 2006Shepherd J.D. Rumbaugh G. Wu J. Chowdhury S. Plath N. Kuhl D. Huganir R.L. Worley P.F. Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors.Neuron. 2006; 52: 475-484Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar) and dendritic remodeling (Fujimoto et al., 2004Fujimoto T. Tanaka H. Kumamaru E. Okamura K. Miki N. Arc interacts with microtubules/microtubule-associated protein 2 and attenuates microtubule-associated protein 2 immunoreactivity in the dendrites.J. Neurosci. Res. 2004; 76: 51-63Crossref PubMed Scopus (54) Google Scholar, Newpher et al., 2018Newpher T.M. Harris S. Pringle J. Hamilton C. Soderling S. Regulation of spine structural plasticity by Arc/Arg3.1.Semin. Cell Dev. Biol. 2018; 77: 25-32Crossref PubMed Scopus (22) Google Scholar). Arc is essential for consolidation of memory in vivo (Plath et al., 2006Plath N. Ohana O. Dammermann B. Errington M.L. Schmitz D. Gross C. Mao X. Engelsberg A. Mahlke C. Welzl H. et al.Arc/Arg3.1 is essential for the consolidation of synaptic plasticity and memories.Neuron. 2006; 52: 437-444Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar) and is implicated in variety of neurological and neurobehavioral disorders including Alzheimer's disease (Wu et al., 2011Wu J. Petralia R.S. Kurushima H. Patel H. Jung M.-y. Volk L. Chowdhury S. Shepherd J.D. Dehoff M. Li Y. et al.Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent β-amyloid generation.Cell. 2011; 147: 615-628Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), Fragile X syndrome, Angelman's syndrome (Greer et al., 2010Greer P.L. Hanayama R. Bloodgood B.L. Mardinly A.R. Lipton D.M. Flavell S.W. Kim T.K. Griffith E.C. Waldon Z. Maehr R. et al.The Angelman syndrome protein Ube3A regulates synapse development by ubiquitinating arc.Cell. 2010; 140: 704-716Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, Park et al., 2008Park S. Park J.M. Kim S. Kim J.A. Shepherd J.D. Smith-Hicks C.L. Chowdhury S. Kaufmann W. Kuhl D. Ryazanov A.G. et al.Elongation factor 2 and fragile X mental retardation protein control the dynamic translation of Arc/Arg3.1 essential for mGluR-LTD.Neuron. 2008; 59: 70-83Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar), and schizophrenia (Fromer et al., 2014Fromer M. Pocklington A.J. Kavanagh D.H. Williams H.J. Dwyer S. Gormley P. Georgieva L. Rees E. Palta P. Ruderfer D.M. et al.De novo mutations in schizophrenia implicate synaptic networks.Nature. 2014; 506: 179-184Crossref PubMed Scopus (1127) Google Scholar, Manago et al., 2016Manago F. Mereu M. Mastwal S. Mastrogiacomo R. Scheggia D. Emanuele M. De Luca M.A. Weinberger D.R. Wang K.H. Papaleo F. Genetic disruption of Arc/Arg3.1 in mice causes alterations in dopamine and neurobehavioral phenotypes related to schizophrenia.Cell Rep. 2016; 16: 2116-2128Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Purcell et al., 2014Purcell S.M. Moran J.L. Fromer M. Ruderfer D. Solovieff N. Roussos P. O'Dushlaine C. Chambert K. Bergen S.E. Kahler A. et al.A polygenic burden of rare disruptive mutations in schizophrenia.Nature. 2014; 506: 185-190Crossref PubMed Scopus (1001) Google Scholar) suggesting Arc as a key player in the dynamic regulation of synaptic homeostasis within several different neuronal networks. Arc belongs to the immediate-early genes, which are dynamically regulated in the brain by neural activity (Guzowski, 2002Guzowski J.F. Insights into immediate-early gene function in hippocampal memory consolidation using antisense oligonucleotide and fluorescent imaging approaches.Hippocampus. 2002; 12: 86-104Crossref PubMed Scopus (319) Google Scholar, Guzowski et al., 2005Guzowski J.F. Timlin J.A. Roysam B. McNaughton B.L. Worley P.F. Barnes C.A. Mapping behaviorally relevant neural circuits with immediate-early gene expression.Curr. Opin. Neurobiol. 2005; 15: 599-606Crossref PubMed Scopus (299) Google Scholar). Upon transcription, Arc mRNA is transported to the dendritic compartments where it is translated by polyribosomal units (Ninomiya et al., 2016Ninomiya K. Ohno M. Kataoka N. Dendritic transport element of human arc mRNA confers RNA degradation activity in a translation-dependent manner.Genes Cells. 2016; 21: 1263-1269Crossref PubMed Scopus (9) Google Scholar, Steward et al., 1998Steward O. Wallace C.S. Lyford G.L. Worley P.F. Synaptic activation causes the mRNA for the IEG Arc to localize selectively near activated postsynaptic sites on dendrites.Neuron. 1998; 21: 741-751Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, Steward and Worley, 2001Steward O. Worley P.F. A cellular mechanism for targeting newly synthesized mRNAs to synaptic sites on dendrites.Proc. Natl. Acad. Sci. U S A. 2001; 98: 7062-7068Crossref PubMed Scopus (193) Google Scholar). Arc expression is particularly enriched in hippocampal and cortical neurons responsible for learning and memory formation (Guzowski et al., 1999Guzowski J.F. McNaughton B.L. Barnes C.A. Worley P.F. Environment-specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles.Nat. Neurosci. 1999; 2: 1120-1124Crossref PubMed Scopus (807) Google Scholar, Link et al., 1995Link W. Konietzko U. Kauselmann G. Krug M. Schwanke B. Frey U. Kuhl D. Somatodendritic expression of an immediate early gene is regulated by synaptic activity.Proc. Natl. Acad. Sci. U S A. 1995; 92: 5734-5738Crossref PubMed Scopus (576) Google Scholar), and the function of Arc has mostly been studied in glutamatergic neurons. Here, Arc is necessary for group I metabotropic glutamate receptor (mGluR)-induced long-term depression (LTD) by promoting endocytosis of ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors (AMPAR) (Park et al., 2008Park S. Park J.M. Kim S. Kim J.A. Shepherd J.D. Smith-Hicks C.L. Chowdhury S. Kaufmann W. Kuhl D. Ryazanov A.G. et al.Elongation factor 2 and fragile X mental retardation protein control the dynamic translation of Arc/Arg3.1 essential for mGluR-LTD.Neuron. 2008; 59: 70-83Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, Rial Verde et al., 2006Rial Verde E.M. Lee-Osbourne J. Worley P.F. Malinow R. Cline H.T. Increased expression of the immediate-early gene arc/arg3.1 reduces AMPA receptor-mediated synaptic transmission.Neuron. 2006; 52: 461-474Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, Shepherd et al., 2006Shepherd J.D. Rumbaugh G. Wu J. Chowdhury S. Plath N. Kuhl D. Huganir R.L. Worley P.F. Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors.Neuron. 2006; 52: 475-484Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, Wang et al., 2016Wang H. Ardiles A.O. Yang S. Tran T. Posada-Duque R. Valdivia G. Baek M. Chuang Y.A. Palacios A.G. Gallagher M. et al.Metabotropic glutamate receptors induce a form of LTP controlled by translation and Arc signaling in the hippocampus.J. Neurosci. 2016; 36: 1723-1729Crossref PubMed Scopus (50) Google Scholar, Waung et al., 2008Waung M.W. Pfeiffer B.E. Nosyreva E.D. Ronesi J.A. Huber K.M. Rapid translation of Arc/Arg3.1 selectively mediates mGluR-dependent LTD through persistent increases in AMPAR endocytosis rate.Neuron. 2008; 59: 84-97Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, Wilkerson et al., 2018Wilkerson J.R. Albanesi J.P. Huber K.M. Roles for Arc in metabotropic glutamate receptor-dependent LTD and synapse elimination: implications in health and disease.Semin. Cell Dev. Biol. 2018; 77: 51-62Crossref PubMed Scopus (28) Google Scholar) that mediate the vast majority of fast excitatory transmission in the brain. Arc interacts with several proteins associated with AMPAR including the transmembrane AMPAR regulatory protein γ2 (TARPγ2) (Jackson and Nicoll, 2011Jackson A.C. Nicoll R.A. The expanding social network of ionotropic glutamate receptors: TARPs and other transmembrane auxiliary subunits.Neuron. 2011; 70: 178-199Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar) and Ca2+/calmodulin dependent kinase (CaMKII) (Okuno et al., 2012Okuno H. Akashi K. Ishii Y. Yagishita-Kyo N. Suzuki K. Nonaka M. Kawashima T. Fujii H. Takemoto-Kimura S. Abe M. et al.Inverse synaptic tagging of inactive synapses via dynamic interaction of Arc/Arg3.1 with CaMKIIbeta.Cell. 2012; 149: 886-898Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar) as well as multiple components belonging to the clathrin-dependent endocytosis machinery, including the clathrin adaptor protein 2 (AP-2) (DaSilva et al., 2016DaSilva L.L. Wall M.J. P de Almeida L. Wauters S.C. Januario Y.C. Muller J. Correa S.A. Activity-regulated cytoskeleton-associated protein controls AMPAR endocytosis through a direct interaction with clathrin-adaptor protein 2.eNeuro. 2016; 3https://doi.org/10.1523/ENEURO.0144-15.2016Crossref PubMed Scopus (47) Google Scholar), dynamin-2, and endophilin-2/3 (Chowdhury et al., 2006Chowdhury S. Shepherd J.D. Okuno H. Lyford G. Petralia R.S. Plath N. Kuhl D. Huganir R.L. Worley P.F. Arc/Arg3.1 interacts with the endocytic machinery to regulate AMPA receptor trafficking.Neuron. 2006; 52: 445-459Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). LTD and LTP (long-term potentiation) involving endocytosis or reinsertion of specific subsets of AMPARs can also be induced by activation of ionotropic N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDAR), and it has been shown that Arc colocalizes also with NMDA receptors in neuronal postsynaptic supercomplexes (Fernandez et al., 2017Fernandez E. Collins M.O. Frank R.A.W. Zhu F. Kopanitsa M.V. Nithianantharajah J. Lempriere S.A. Fricker D. Elsegood K.A. McLaughlin C.L. et al.Arc requires PSD95 for assembly into postsynaptic complexes involved with neural dysfunction and intelligence.Cell Rep. 2017; 21: 679-691Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). While colocalization seems dependent on the highly abundant synaptic scaffolding protein PSD-95, the predominant GluN2A and GluN2B subunits of the NMDAR contain putative binding sites for Arc (Zhang et al., 2015Zhang W. Wu J. Ward M.D. Yang S. Chuang Y.-A. Xiao M. Li R. Leahy D.J. Worley P.F. Structural basis of arc binding to synaptic proteins: implications for cognitive disease.Neuron. 2015; 86: 490-500Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). How Arc influences NMDAR function or whether Arc serves as an integrator between mGluR and NMDA-induced LTD/LTP is not yet clear. The Arc protein contains structural elements similar to those found within the viral group-specific antigen (Gag). A bioinformatics analysis of the human genome by Campillos et al., 2006Campillos M. Doerks T. Shah P.K. Bork P. Computational characterization of multiple Gag-like human proteins.Trends Genet. 2006; 22: 585-589Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar identified at least 85 human proteins that have evolved from Gag proteins of the Ty3/Gypsy retrotransposon lineage and have developed new functions in human (Brandt et al., 2005Brandt J. Veith A.M. Volff J.N. A family of neofunctionalized Ty3/gypsy retrotransposon genes in mammalian genomes.Cytogenet. Genome Res. 2005; 110: 307-317Crossref PubMed Scopus (50) Google Scholar, Haig, 2012Haig D. Retroviruses and the placenta.Curr. Biol. 2012; 22: R609-R613Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Arc is by far the most studied mammalian Gag-like protein and has retained the topology of a retroviral Gag protein, including a predicted N-terminal matrix (MA) domain and a two-lobe capsid (CA) domain (Bi et al., 2018Bi R. Kong L.L. Xu M. Li G.D. Zhang D.F. Li T. Fang Y. Zhang C. Zhang B. et al.Alzheimer's Disease Neuroimaging InitiativeThe Arc gene confers genetic susceptibility to Alzheimer's disease in Han Chinese.Mol. Neurobiol. 2018; 55: 1217-1226Crossref PubMed Scopus (19) Google Scholar, Zhang et al., 2015Zhang W. Wu J. Ward M.D. Yang S. Chuang Y.-A. Xiao M. Li R. Leahy D.J. Worley P.F. Structural basis of arc binding to synaptic proteins: implications for cognitive disease.Neuron. 2015; 86: 490-500Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Moreover, the Arc protein is still fully capable of forming viral-like capsids and facilitating intercellular mRNA transfer between adjacent cells much like functional retroviruses such as human immunodeficiency virus (HIV) and human T-lymphotropic viruses (Ashley et al., 2018Ashley J. Cordy B. Lucia D. Fradkin L.G. Budnik V. Thomson T. Retrovirus-like Gag protein Arc1 binds RNA and traffics across synaptic boutons.Cell. 2018; 172: 262-274Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, Pastuzyn et al., 2018Pastuzyn E.D. Day C.E. Kearns R.B. Kyrke-Smith M. Taibi A.V. McCormick J. Yoder N. Belnap D.M. Erlendsson S. Morado D.R. et al.The neuronal gene arc encodes a repurposed retrotransposon Gag protein that mediates intercellular RNA transfer.Cell. 2018; 172: 275-288Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Neither the physiological significance of the retroviral-like properties nor the detailed molecular architecture of Arc or any other Ty3/Gypsy-related capsid assemblies are currently known, although the structures of the separate N- and C-lobes of Arc CA have been determined by X-ray crystallography (Zhang et al., 2015Zhang W. Wu J. Ward M.D. Yang S. Chuang Y.-A. Xiao M. Li R. Leahy D.J. Worley P.F. Structural basis of arc binding to synaptic proteins: implications for cognitive disease.Neuron. 2015; 86: 490-500Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Moreover, the structural arrangement of the two lobes of Arc CA relative to each other is not known. Furthermore, the structure of the N lobe was solved in the presence of ligand. Here, we show that the ligand-free Arc CA domain retains a well-defined bilobar structure with low flexibility that directly binds to polypeptide sequences near the C-termini of the GluN2A and GluN2B subunits of the NMDA receptor. The binding of ligands stabilizes the monomeric state of Arc CA and inhibits the formation of higher order Arc CA oligomers. This suggests that intracellular functions of Arc are governed by CA-mediated oligomerization or that capsid formation may be influenced by ligand binding. Such a mechanism could decouple the ability of Arc to regulate synaptic function by modulating the surface expression of receptors from its ability to facilitate intercellular RNA transfer in a viral-like manner. The CA domain of Arc comprises 160 amino acid residues from positions 206 to 364 in full-length Arc (Figure 1A). In solution Arc CA is stable, monomeric, and homogeneous as gauged from size-exclusion chromatography and the 1H-15N heteronuclear single-quantum coherence (HSQC) spectrum (Figures S1A and S1B). The final structure of Arc CA calculated from nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS) restraints (Figures S1C and S1D; Table 1) is well defined from residue 217 to residue 357, with a pairwise root-mean-square deviation (RMSD) of 0.9 ± 0.2 Å for the 20 models representing the structure (Figure 1B). Arc CA has two globular subdomains, the N-lobe and the C-lobe, consisting of four and five α helices, respectively. Each of the lobes has slightly lower pairwise backbone RMSD when aligned individually; 0.57 Å for the N-lobe and 0.64 Å for the C-lobe. The two lobes are connected by a short non-helical segment (residues 278–280) between H4 and H5 (Figure 1C, inset ii). Except for the lack of an N-terminal helix in the N-lobe, the two lobes share the same fold with an interdomain RMSD of 2.74 Å. The core of the N-lobe (Figure 1C, inset i) is stabilized primarily by stacking bulky hydrophobic residues (221L, 224L, 228L, 242I, 254W, 264W, 267F, 271F), and a highly hydrophobic groove between H1, H2, and H4 is exposed in our structure (Figure 1D). Zhang et al., 2015Zhang W. Wu J. Ward M.D. Yang S. Chuang Y.-A. Xiao M. Li R. Leahy D.J. Worley P.F. Structural basis of arc binding to synaptic proteins: implications for cognitive disease.Neuron. 2015; 86: 490-500Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar found that peptides from TARPγ2 and CaMKII bind in this groove and form a two-stranded β sheet with the N-terminus of Arc CA, which is disordered in our ligand-free structure. The hydrophobic core of the C-lobe is primarily constituted by aliphatic residues (Figure 1C, inset iv), which are not solvent exposed. The surface of the C-lobe is positively charged around H8 (Figure 1E). H7 in Arc, corresponding to H9 in the HIV-1 capsid protein, is negatively charged. In the HIV capsid protein, H9 is key in forming the initial intramolecular interactions during oligomerization and capsid formation, and therefore capsid formation of Arc could involve electrostatic interactions between C-lobes. The backbone of the helical parts of our NMR structure align with the previous X-ray structures of the individual lobes with RMSDs of 1.08 Å and 2.02 Å for the N-lobe (PDB: 4x3i) and the C-lobe (PDB: 4x3x), respectively. The most notable differences to the two structures of the individual lobes are (except for the presence for the ligand in the N-lobe) that 236W in the N-lobe is flipped 90° and that the orientations of helices 5 and 9 in the C-lobe are a slightly different. Particularly, the orientation of H5 in the X-ray structure appears to be affected by the missing N-lobe.Table 1Statistics for the Structural Ensemble of the Arc CA Domain (PDB: 6GSE)RestraintsNOE-based restraints Intraresidual (|i – j| = 0)361 Sequential (|i – j| = 1)617 Medium range (2 ≤ |i – j| ≤ 4)830 Long (|i – j| > 5)398Total2206Dihedral angle restraints264Residual dipolar couplings140Restraint StatisticsMean RMSD from experimental restraints NOE-based distances (Å)0.023 ± 0.001 Dihedrals (°)0.57 ± 0.09 Residual dipolar coupling (Q factor)1.65 ± 0.12 SAXS6.0 ± 0.3Structure StatisticsaPROCHECK. Most favored regions94.6% Allowed regions5.2% Generously allowed regions0.2% Disallowed regions0.0%Coordinate Precision RMSD (Å)bMOLMOL. Arc (217–356) Backbone heavy atoms (N, Cα, and C′)0.9 ± 0.2 Heavy atoms1.79 ± 0.17 NTD (217–277) Backbone heavy atoms (N, Cα, and C′)0.57 ± 0.14 Heavy atoms1.51 ± 0.14 CTD (281–356) Backbone heavy atoms (N, Cα, and C′)0.6 ± 0.2 Heavy atoms1.7 ± 0.2None of the structures exhibits distance violations >0.3 Å or dihedral angle violations >5°. Values for restraints statistics and coordinate precision are reported as the mean ± SD for the 20 structures in the ensemble.a PROCHECK.b MOLMOL. Open table in a new tab None of the structures exhibits distance violations >0.3 Å or dihedral angle violations >5°. Values for restraints statistics and coordinate precision are reported as the mean ± SD for the 20 structures in the ensemble. In the HIV-1 capsid protein, the two lobes are connected by a flexible linker, and the interdomain orientation changes substantially between the immature and mature structures that the HIV-1 capsid protein can form (Bush and Vogt, 2014Bush D.L. Vogt V.M. In vitro assembly of retroviruses.Annu. Rev. Virol. 2014; 1: 561-580Crossref PubMed Scopus (36) Google Scholar, Deshmukh et al., 2013Deshmukh L. Schwieters C.D. Grishaev A. Ghirlando R. Baber J.L. Clore G.M. Structure and dynamics of full-length HIV-1 capsid protein in solution.J. Am. Chem. Soc. 2013; 135: 16133-16147Crossref PubMed Scopus (93) Google Scholar, Perilla and Gronenborn, 2016Perilla J.R. Gronenborn A.M. Molecular architecture of the retroviral capsid.Trends Biochem. Sci. 2016; 41: 410-420Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Therefore, we characterized the dynamics of Arc CA by NMR spectroscopy. Fast conformational dynamics on the picosecond-to-nanosecond time scale was gauged by measuring R1, R2, and the heteronuclear Overhauser effect (NOE) (Figure S1E) and calculating the generalized order parameter S2 of the backbone amide 15N nuclei (Figure 2A). Overall the protein is well ordered. The helices have S2 above 0.7 and the connecting loops have S2 between 0.6 and 0.7. Only the N- and C-termini are disordered with S2 below 0.5. The three non-helical residues between the two lobes are not more flexible on the fast time scales than the short loops connecting the helices in the two lobes. The helical segments of the C-lobe are the most ordered parts of the structure with S2 approaching 1 for many residues, which suggest that the C-lobe is more rigid than the N-lobe. Conformational exchange processes on the microsecond-to-millisecond time scales were identified by 15N Carr-Purcell-Meiboom-Gill (CPMG) spin relaxation dispersion. Six of the backbone amides (239L, 243Q, 254W, 256F, 263N, 271F) primarily in H2 and H3 near the ligand binding site in the N-lobe show clear dispersions and thus experience conformational exchange (Figures 2B and 2C). The dispersions can be fit to a single two-state process with a rate-constant, kex = (6.9 ± 1.1) × 102 s−1 and a population of the minor state, pminor = 0.6% ± 0.1% (discussed below). This process is independent of protein concentration as dispersion curves recorded on a 2-fold diluted sample follow the same two-state kinetics (Figure S1F). Thus, the slow process is intramolecular and not a result of oligomerization. For the rest of the protein, including the linker between the two lobes, the dispersions are flat and no slow conformational exchange processes are seen (Figure S1G). Taken together, the relaxation analysis demonstrates that the relative orientation of the two subdomains is well defined with little interdomain flexibility on the picosecond-to-nanosecond and microsecond-to-millisecond time scales. The low interdomain flexibility is supported by the residual dipolar coupling and SAXS data (Figures S1C and S1D), which are sensitive to dynamics on time scales from nanoseconds to seconds. We only needed a single conformation of the Arc structure to fit these data. The low interdomain flexibility separates Arc from Gag proteins from active retroviruses where high interdomain flexibility is important for capsid assembly and maturation (Byeon et al., 2012Byeon I.-J.L. Hou G. Han Y. Suiter C.L. Ahn J. Jung J. Byeon C.-H. Gronenborn A.M. Polenova T. Motions on the millisecond time scale and multiple conformations of HIV-1 capsid protein: implications for structural polymorphism of CA assemblies.J. Am. Chem. Soc. 2012; 134: 6455-6466Crossref PubMed Scopus (74) Google Scholar, Campos-Olivas et al., 2000Campos-Olivas R. Newman J.L. Summers M.F. Solution structure and dynamics of the Rous sarcoma virus capsid protein and comparison with capsid proteins of other retroviruses.J. Mol. Biol. 2000; 296: 633-649Crossref PubMed Scopus (103) Google Scholar). A subset of the 85 other domesticated Gag proteins of Ty3/Gypsy origin appears to have the same bilobar CA structure as Arc. We made a multiple sequence alignment with PSI-Coffee (Di Tommaso et al., 2011Di Tommaso P. Moretti S. Xenarios I. Orobitg M. Montanyola A. Chang J.M. Taly J.F. Notredame C. T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension.Nucleic Acids Res. 2011; 39: W13-W17Crossref PubMed Scopus (731) Google Scholar) of nine of the human sequences with Arc from Rattus norvegicus to substantiate this assumption (Figure 3A). In the alignment we also included the Ty3 Gag protein from Saccharomyces cerevisiae and the Gypsy Gag protein from the fish Latimeria chalumnae, which are archetypical Gag proteins of the Ty3/Gypsy family (Pastuzyn et al., 2018Pastuzyn E.D. Day C.E. Kearns R.B. Kyrke-Smith M. Taibi A.V. McCormick J. Yoder N. Belnap D.M. Erlendsson S. Morado D.R. et al.The neuronal gene arc encodes a repurposed retrotransposon Gag protein that mediates intercellular RNA transfer.Cell. 2018; 172: 275-288Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Although the sequence identity to Arc is low (12%–20%), several positions in the sequences have high conservation scores and coincide with the hydrophobic cores of the two lobes in Arc (Figure 3). Between H6 and H7, between H7 and H8, and between H8 and H9, additional amino acid residues are inserted in Pnma-1, MOAP-1, Pnma-3, and Zcchc12 that all belong to the Paraneoplastic subfamily. These insertions all localize to the C-lobe, which in Arc appears rigid, but is known to undergo structural changes in the HIV Gag protein and be important for capsid stability (Deshmukh et al., 2013Deshmukh L. Schwieters C.D. Grishaev A. Ghirlando R. Baber J.L. Clore G.M. Structure and dynamics of full-length HIV-1 capsid protein in solution.J. Am. Chem. Soc. 2013; 135: 16133-16147Crossref PubMed Scopus (93) Google Scholar). In the proteins of the Sushi family (the RTLs and PEG10), additional residues are found between H1 and H2, and just before the start of H4, suggesting that this region, which binds ligands in Arc, may have developed different properties in the different Gag proteins. For the Paraneoplastic subfamily and Arc we also observe a high frequency of fast-evolving residues (Ser and Thr) in the linker region connecting the two lobes, suggesting that this region has been subject to strong selection pressure (Creixell et al., 2012Creixell P. Schoof E.M. Tan C.S. Linding R. Mutational properties of amino acid residues: implications for evolvability of phosphorylatable residues.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2012; 367: 2584-2593Crossref PubMed Scopus (31) Google Scholar). Arc is able to regulate LTD by removal of AMPARs from the plasma membrane independently of NMDAR stimulation (Wang et al., 2016Wang H. Ardiles A.O. Yang S. Tran T. Posada-Duque R. Valdivia G. Baek M. Chuang Y.A. Palacios A.G. Gallagher M. et al.Metabotropic glutamate receptors induce a form of LTP controlled by translation and Arc signaling in the hippocampus.J. Neurosci. 2016; 36: 1723-1729Crossref PubMed Scopus (50) Google Scholar). However, Arc is found in NMDAR containing postsynaptic MDa complexes where its presence depends on PSD-95 (Fernandez et al., 2017Fernandez E. Collins M.O. Frank R.A.W. Zhu F. Kopanitsa M.V. Nithianantharajah J. Lempriere S.A. Fricker D. Elsegood K.A. McLaughlin C.L. et al.Arc requires PSD95 for assembly into postsynaptic complexes involved with neural dysfunction and intelligence.Cell Rep. 2017; 21: 679-691Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This observation is intriguing, as previous work by Zhang et al., 2015Zhang W. Wu J. Ward M.D. Yang S. Chuang Y.-A. Xiao M. Li R. Leahy D.J. Worley P.F. Structural basis of arc binding to synaptic proteins: implications for cognitive disease.Neuron. 2015; 86: 490-500Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar showed that CaMKII and TARPγ2 bind to Arc with a consensus binding motif (X-P-X-ψ, where X is any residue and ψ is either" @default.
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• W2943929497 date "2019-07-01" @default.
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• W2943929497 title "The Capsid Domain of Arc Changes Its Oligomerization Propensity through Direct Interaction with the NMDA Receptor" @default.
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