Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cold Spring Harbor Laboratory  

Abstract

Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including E. coli RseP. Our observations are consistent with a rearrangement of the RseP domains surrounding the active center to expose the substrate-binding site where a conserved electrostatic linkage between the transmembrane and membrane-associated domains mediates the conformational changes, suggesting that RseP has a gating mechanism to regulate substrate entry. Mutational analysis also supports that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet and is clamped at the active center for efficient cleavage. Furthermore, this substrate accommodation mechanism appears to be common across distinct intramembrane proteases.

DOI： 10.1126/sciadv.abp9011

DOI： 10.1101/2022.01.31.478169

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Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.

DOI： 10.1107/S2059798321002527

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The large, secreted glycoprotein reelin regulates embryonic brain development as well as adult brain functions. Although reelin binds to its receptors via its central part, the N-terminal region directs multimer formation and is critical for efficient signal transduction. In fact, the inhibitory antibody CR-50 interacts with the N-terminal region and prevents higher-order multimerization and signalling. Reelin is a multidomain protein in which the central part is composed of eight characteristic repeats, named reelin repeats, each of which is further divided by insertion of a epidermal growth factor (EGF) module into two subrepeats. In contrast, the N-terminal region shows unique 'irregular' domain architecture since it comprises three consecutive subrepeats without the intervening EGF module. Here, we determined the crystal structure of the murine reelin fragment named RX-R1 including the irregular region and the first reelin repeat at 2.0-Å resolution. The overall structure of RX-R1 has a branched Y-shaped form. Interestingly, two incomplete subrepeats cooperatively form one entire subrepeat structure, though an additional subrepeat is inserted between them. We further reveal that Arg335 of RX-R1 is crucial for binding CR-50. A possible self-association mechanism via the N-terminal region is proposed based on our results.

DOI： 10.1093/jb/mvaa144

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An antibody fragment that recognizes the tertiary structure of a target protein with high affinity can be utilized as a crystallization chaperone. Difficulties in establishing conformation-specific antibodies, however, limit the applicability of antibody fragment-assisted crystallization. Here, we attempted to establish an alternative method to promote the crystallization of target proteins using an already established anti-tag antibody. The monoclonal antibody NZ-1 recognizes the PA tag with an extremely high affinity. It was also established that the PA tag is accommodated in the antigen-binding pocket in a bent conformation, compatible with an insertion into loop regions on the target. We, therefore, explored the application of NZ-1 Fab as a crystallization chaperone that complexes with a target protein displaying a PA tag. Specifically, we inserted the PA tag into the β-hairpins of the PDZ tandem fragment of a bacterial Site-2 protease. We crystallized the PA-inserted PDZ tandem mutants with the NZ-1 Fab and solved the co-crystal structure to analyze their interaction modes. Although the initial insertion designs produced only moderate-resolution structures, eliminating the solvent-accessible space between the NZ-1 Fab and target PDZ tandem improved the diffraction qualities remarkably. Our results demonstrate that the NZ-1-PA system efficiently promotes crystallization of the target protein. The present work also suggests that β-hairpins are suitable sites for the PA insertion because the PA tag contains a Pro-Gly sequence with a propensity for a β-turn conformation.

DOI： 10.1002/pro.3580

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Fragile X mental retardation protein (FMRP), a causative gene (FMR1) product of Fragile X syndrome (FXS), is an RNA-binding protein to regulate local protein synthesis in dendrites for postsynaptic functions. However, involvement of FMRP in local protein synthesis in axons for presynaptic functions remains unclear. Here we investigated role of FMRP in local translation of the active zone protein Munc18-1 during presynapse formation. We found that leucine-rich repeat transmembrane neuronal 2 (LRRTM2)-conjugated beads, which promotes synchronized presynapse formation, induced simultaneous accumulation of FMRP and Munc18-1 in presynapses of axons of mouse cortical neurons in neuronal cell aggregate culture. The LRRTM2-induced accumulation of Munc18-1 in presynapses was observed in axons protein-synthesis-dependently, even physically separated from cell bodies. The accumulation of Munc18-1 was enhanced in Fmr1-knockout (KO) axons as compared to wild type (WT), suggesting FMRP-regulated suppression for local translation of Munc18-1 in axons during presynapse formation. Using naturally formed synapses of dissociated culture, structured illumination microscope revealed that accumulation of Munc18-1 puncta in Fmr1-KO neurons increased significantly at 19 days in vitro, as compared to WT. Our findings lead the possibility that excessive accumulation of Munc18-1 in presynapses at early stage of synaptic development in Fmr1-KO neurons may have a critical role in impaired presynaptic functions in FXS.

DOI： 10.1016/j.neures.2018.09.013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Verlag  

The low-density lipoprotein receptor (LDLR) and its homologs capture and internalize lipoproteins into the cell. Due to the fact that LDLR family members possess a modular ectodomain that undergoes dynamic conformational changes, multi-scale structural analysis has been performed so as to understand the ligand capture and release mechanism. For example, crystallographic analyses have provided models for both the entire ectodomain and high-resolution structures of individual modules. In addition, nuclear magnetic resonance spectroscopic analyses have shown the rigidity and flexibility of inter-module linkers to restrict the mobility of ectodomain. Accumulated structural data suggest that the ectodomains of LDLR family members are flexible at the cell surface and switch between two metastable conformations, that is, the extended and contracted conformations. Recent structural analysis of ApoER2, a close homolog of LDLR, raised the possibility that the receptor binds with the ligand in the contracted conformation. After transport to an endosome by endocytosis, the receptor undergoes a conformational change to the closed conformation for completion of ligand release. In contrast, LDLR has been reported to adopt the extended conformation when it binds with a inhibitory regulator that recruits LDLR toward the degradation pathway. These findings support a mechanism of different ectodomain conformations for binding the ligand versus binding the regulatory protein. In this review, I provide an overview of studies that analyze the structural and biophysical properties of the ectodomains of LDLR family members and discuss a hypothetical model for ligand uptake and receptor recycling that integrates the known ectodomain conformational variability.

DOI： 10.1007/s12551-017-0362-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Apolipoprotein E receptor 2 (ApoER2) is a close homologue of low-density lipoprotein receptor (LDLR) that mediates the endocytosis of ligands, including LDL particles. LDLR family members have been presumed to explore a large conformational space to capture ligands in the extended conformation at the cell surface. Ligands are subsequently released through a pH-titrated structural transition to a self-docked, contracted-closed conformation. In addition to lipoprotein uptake, ApoER2 is implicated in signal transduction during brain development through capture of the extracellular protein reelin. From crystallographic analysis, we determine that the full-length ApoER2 ectodomain adopts an intermediate contracted-open conformation when complexed with the signaling-competent reelin fragment, and we identify a previously unappreciated auxiliary low-affinity binding interface. Based on mutational analyses, we propose that the pH shift during endocytosis weakens the affinity of the auxiliary interface and destabilizes the ligand-receptor complex. Furthermore, this study elucidates that the contracted-open conformation of ligand-bound ApoER2 at neutral pH resembles the contracted-closed conformation of ligand-unbound LDLR at acidic pH in a manner suggestive of being primed for ligand release even prior to internalization.

DOI： 10.15252/embr.201643521

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Orchestration of the multiple enzymes engaged in O-mannose glycan synthesis provides a matriglycan on α-dystroglycan (α-DG) which attracts extracellular matrix (ECM) proteins such as laminin. Aberrant O-mannosylation of α-DG leads to severe congenital muscular dystrophies due to detachment of ECM proteins from the basal membrane. Phosphorylation at C6-position of O-mannose catalyzed by protein O-mannosyl kinase (POMK) is a crucial step in the biosynthetic pathway of O-mannose glycan. Several mis-sense mutations of the POMK catalytic domain are known to cause a severe congenital muscular dystrophy, Walker-Warburg syndrome. Due to the low sequence similarity with other typical kinases, structure-activity relationships of this enzyme remain unclear. Here, we report the crystal structures of the POMK catalytic domain in the absence and presence of an ATP analogue and O-mannosylated glycopeptide. The POMK catalytic domain shows a typical protein kinase fold consisting of N- and C-lobes. Mannose residue binds to POMK mainly via the hydroxyl group at C2-position, differentiating from other monosaccharide residues. Intriguingly, the two amino acid residues K92 and D228, interacting with the triphosphate group of ATP, are donated from atypical positions in the primary structure. Mutations in this protein causing muscular dystrophies can now be rationalized.

DOI： 10.1111/gtc.12480

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Humana Press Inc.  

Plexins are type I membrane proteins that function as receptors for semaphorins. All of the known plexins contain a large globular domain, termed the sema domain, in the N-terminal extracellular region, which interacts with semaphorins during signal transduction. Here, we describe procedures for protein production and purification that we utilized in the crystallographic study of the mouse Plexin A2 (mPlxnA2) extracellular fragment, including the sema domain. A mutant mammalian cell line, HEK293S GnTI−, was used as an expression host for the production of a crystallizable-quality mPlxnA2 fragment, which contains several N-glycosylation sites and disulfide bonds.

DOI： 10.1007/978-1-4939-6448-2_4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELIFE SCIENCES PUBLICATIONS LTD  

Molecular mechanisms underlying substrate recognition and cleavage by Escherichia coli RseP, which belongs to S2P family of intramembrane-cleaving proteases, remain unclear. We examined the function of a conserved region looped into the membrane domain of RseP to form a beta-hairpin-like structure near its active site in substrate recognition and cleavage. We observed that mutations disturbing the possible beta-strand conformation of the loop impaired RseP proteolytic activity and that some of these mutations resulted in the differential cleavage of different substrates. Coimmunoprecipitation and crosslinking experiments suggest that the loop directly interacts with the transmembrane segments of substrates. Helix-destabilising mutations in the transmembrane segments of substrates suppressed the effect of loop mutations in an allele-specific manner. These results suggest that the loop promotes substrate cleavage by selectively recognising the transmembrane segments of substrates in an extended conformation and by presenting them to the proteolytic active site, which contributes to substrate discrimination.

DOI： 10.7554/eLife.08928

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

SorLA is a neuronal sorting receptor considered to be a major risk factor for Alzheimer's disease. We have recently reported that it directs lysosomal targeting of nascent neurotoxic amyloid-beta (All) peptides by directly binding A beta. Here, we determined the crystal structure of the human sorLA domain responsible for A beta capture, Vps10p, in an unbound state and in complex with two ligands. Vps10p assumes a ten-bladed beta-propeller fold with a large tunnel at the center. An internal ligand derived from the sorLA propeptide bound inside the tunnel to extend the beta-sheet of one of the propeller blades. The structure of the sorLA Vps10p-A beta complex revealed that the same site is used. Peptides are recognized by sorLA Vps10p in redundant modes without strict dependence on a particular amino acid sequence, thus suggesting a broad specificity toward peptides with a propensity for beta-sheet formation.

DOI： 10.1038/nsmb.2954

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Peptide-based epitope tagging technology is universally used in nearly all kind of research projects that involve biochemical characterization of a target protein, but not many systems are fully compatible with purification purpose. By utilizing an anti-human podoplanin antibody NZ-I, we constructed a novel epitope tag system. NZ-1 possesses exceptionally high affinity toward a dodecapeptide dubbed "PA tag", with a characteristic slow dissociation kinetics. Because of its high affinity, PA-tagged proteins in a dilute sample can be captured by immobilized NZ-1 resin in a near complete fashion and eluted by a solution of free PA peptide. This enabled efficient one-step purification of various proteins including soluble (an ectodomain fragment of neuropilin-1) and membrane (epidermal growth factor receptor) proteins expressed in mammalian cells. Mild regeneration condition of the peptide-bound antibody ensures repeated use of the antibody resin, indicating a cost-efficient nature of the system. Together with its outstanding performance in the immunodetection experiments (i.e., Western blotting and flow cytometry), PA tag/NZ-1 system will offer a great chance to facilitate protein production in many biomedical research projects. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.pep.2014.01.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

During the extracytoplasmic stress response in Escherichia coli, the intramembrane protease RseP cleaves the anti-sigma(E) protein RseA only after the membrane-anchored protease DegS truncates the perk plasmic part of RseA that suppresses the action of RseP. Here we analyzed the three-dimensional structure of the two tandemly arranged PSD-95/DIg/ZO-1 (PDZ) domains (PDZ tandem) present in the periplasmic region of RseP and revealed that the two putative ligand-binding grooves constitute a single pocket-like structure that would lie just above the active center sequestrated within the membrane. Complete removal of the PDZ tandem from RseP led to the intramembrane cleavage of RseA without prior truncation by DegS. Furthermore, mutations expected to destabilize the tertiary structure of the PDZ tandem also caused the deregulation of the sequential cleavage. These observations suggest that the PDZ tandem serves as a size-exclusion filter to accommodate the truncated form of RseA into the active center.

DOI： 10.1016/j.str.2013.12.003

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S217_2

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Polymorphic adhesion molecules neurexin and neuroligin (NL) mediate asymmetric trans-synaptic adhesion, which is crucial for synapse development and function. It is not known whether or how individual synapse function is controlled by the interactions between variants and isoforms of these molecules with differing ectodomain regions. At a physiological concentration of Ca2+, the ectodomain complex of neurexin-1 beta isoform (Nrx1 beta) and NL1 spontaneously assembled into crystals of a lateral sheet-like super-structure topologically compatible with transcellular adhesion. Correlative light-electron microscopy confirmed extracellular sheet formation at the junctions between Nrx1 beta- and NL1-expressing non-neuronal cells, mimicking the close, parallel synaptic membrane apposition. The same NL1-expressing cells, however, did not form this higher-order architecture with cells expressing the much longer neurexin-1 alpha isoform, suggesting a functional discrimination mechanism between synaptic contacts made by different isoforms of neurexin variants.

DOI： 10.1016/j.celrep.2012.06.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Integrin alpha 5 beta 1 is a major cellular receptor for the extracellular matrix protein fibronectin and plays a fundamental role during mammalian development. A crystal structure of the alpha 5 beta 1 integrin headpiece fragment bound by an allosteric inhibitory antibody was determined at a 2.9-angstrom resolution both in the absence and presence of a ligand peptide containing the Arg-Gly-Asp (RGD) sequence. The antibody-bound beta 1 chain accommodated the RGD ligand with very limited structural changes, which may represent the initial step of cell adhesion mediated by nonactivated integrins. Furthermore, a molecular dynamics simulation pointed to an important role for Ca2+ in the conformational coupling between the ligand-binding site and the rest of the molecule. The RGD-binding pocket is situated at the center of a trenchlike exposed surface on the top face of alpha 5 beta 1 devoid of glycosylation sites. The structure also enabled the precise prediction of the acceptor residue for the auxiliary synergy site of fibronectin on the alpha 5 subunit, which was experimentally confirmed by mutagenesis and kinetic binding assays.

DOI： 10.1083/jcb.201111077

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.270

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Other Link： http://search.jamas.or.jp/link/ui/2012089364

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Neurexins (Nrxs) are presynaptic membrane proteins with a single membrane-spanning domain that mediate asymmetric trans-synaptic cell adhesion by binding to their postsynaptic receptor neuroligins. alpha-Nrx has a large extracellular region comprised of multiple copies of laminin, neurexin, sex-hormone-binding globulin (LNS) domains and epidermal growth factor (EGF) modules, while that of beta-Nrx has but a single LNS domain. It has long been known that the larger alpha-Nrx and the shorter beta-Nrx show distinct binding behaviors toward different isoforms/variants of neuroligins, although the underlying mechanism has yet to be elucidated. Here, we describe the crystal structure of a fragment corresponding to the C-terminal one-third of the Nrx1 alpha ectodomain, consisting of LNS5-EGF3-LNS6. The 2.3 angstrom-resolution structure revealed the presence of a domain configuration that was rigidified by inter-domain contacts, as opposed to the more common flexible "beads-on-a-string" arrangement. Although the neuroligin-binding site on the LNS6 domain was completely exposed, the location of the alpha-Nrx specific LNS5-EGF3 segment proved incompatible with the loop segment inserted in the B+ neuroligin variant, which explains the variant-specific neuroligin recognition capability observed in alpha-Nrx. This, combined with a low-resolution molecular envelope obtained by a single particle reconstruction performed on negatively stained full-length Nrx1 alpha sample, allowed us to derive a structural model of the alpha-Nrx ectodomain. This model will help us understand not only how the large alpha-Nrx ectodomain is accommodated in the synaptic cleft, but also how the trans-synaptic adhesion mediated by alpha- and beta-Nrxs could differentially affect synaptic structure and function.

DOI： 10.1371/journal.pone.0019411

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Low-density lipoprotein receptor (LDLR) relative with 11 binding repeats (LR11; also known as sorLA) is genetically associated with late-onset Alzheimer's disease and is thought to be involved in neurodegenerative processes. LR11 contains a vacuolar protein-sorting 10 protein (Vps10p) domain. As this domain has been implicated in protein-protein interaction in other receptors, its structure and function are of great biological interest. Human LR11 Vps10p domain was expressed in mammalian cells and the purified protein was crystallized using the hanging-drop vapour-diffusion method. Enzymatic deglycosylation of the sample was critical to obtaining diffraction-quality crystals. Deglycosylated LR11 Vps10p-domain crystals belonged to the hexagonal space group P6(1)22. A diffraction data set was collected to 2.4 angstrom resolution and a clear molecular-replacement solution was obtained.

DOI： 10.1107/S1744309110048153

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Semaphorins and their receptor plexins constitute a pleiotropic cell-signalling system that is used in a wide variety of biological processes, and both protein families have been implicated in numerous human diseases(1-4). The binding of soluble or membrane-anchored semaphorins to the membrane-distal region of the plexin ecto-domain activates plexin's intrinsic GTPase-activating protein (GAP) at the cytoplasmic region, ultimately modulating cellular adhesion behaviour(5). However, the structural mechanism underlying the receptor activation remains largely unknown. Here we report the crystal structures of the semaphorin 6A (Sema6A) receptor-binding fragment and the plexin A2 (PlxnA2) ligand-binding fragment in both their pre-signalling (that is, before binding) and signalling (after complex formation) states. Before binding, the Sema6A ectodomain was in the expected 'face-to-face' homodimer arrangement, similar to that adopted by Sema3A and Sema4D, whereas PlxnA2 was in an unexpected 'head-on' homodimer arrangement. In contrast, the structure of the Sema6A-PlxnA2 signalling complex revealed a 2:2 heterotetramer in which the two PlxnA2 monomers dissociated from one another and docked onto the top face of the Sema6A homodimer using the same interface as the head-on homodimer, indicating that plexins undergo 'partner exchange'. Cell-based activity measurements using mutant ligands/receptors confirmed that the Sema6A face-to-face dimer arrangement is physiologically relevant and is maintained throughout signalling events. Thus, homodimer-to-heterodimer transitions of cell-surface plexin that result in a specific orientation of its molecular axis relative to the membrane may constitute the structural mechanism by which the ligand-binding 'signal' is transmitted to the cytoplasmic region, inducing GAP domain rearrangements and activation.

DOI： 10.1038/nature09473

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor, members of the low-density lipoprotein receptor (LDLR) protein family, function as neuronal receptors for a secreted glycoprotein reelin during brain development. In both receptors, the first LDLR class A (LA1) module is sufficient to bind reelin. Analysis of a 2.6 angstrom crystal structure of the reelin receptor-binding fragment in complex with the LA1 of ApoER2 revealed that Lys2467 of reelin is recognized by both a conserved Trp residue and calcium-coordinating acidic residues from LA1, which together with Lys2360 plays a critical role in the interaction. This "double-Lys" recognition mode is, in fact, shared among other LDLR family proteins in ligand binding. The interface between reelin and LA1 covers a small surface area of similar to 350 angstrom(2) on each side, which ensures a stable complex formation under physiological conditions. An examination of structure-guided mutagenesis on interface residues revealed key features of this interaction.

DOI： 10.1016/j.str.2010.01.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce a novel affinity peptide tagging system that uses a low affinity anti-peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18-residue affinity tag with ideal solution binding kinetics, providing a low-cost purification means when combined with nondenaturing elution by water-miscible organic solvents. Three-dimensional information provides a firm structural basis for the antibody-peptide interaction, opening opportunities for further improvements/modifications.

DOI： 10.1110/ps.038299.108

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F-spondin is a secreted and extracellular matrix-attached protein that has been implicated in axonal pathfinding during neural development as well as in vascular remodelling in adult tissues. F-spondin is composed of a reeler, a spondin and six thrombospondin type 1 repeat domains. The reeler domain shares homology with the amino-terminal domain of reelin, a large secreted glycoprotein that guides migrating neurons during cortical development. Crystal structures of the F-spondin reeler domain were determined at 1.45 and 2.70 A resolution. The structure revealed a nine-stranded antiparallel beta-sandwich fold similar to the immunoglobulin or fibronectin type III domains, but with a unique extra beta-hairpin. Moreover, an amino-terminal extension which is anchored at its beginning via a conserved disulfide bond loosely packs against one face of the beta-sandwich, making a major contribution to the surface features of the domain. Structural comparison among the different molecules contained in two different crystals reveals an unusual conformational plasticity of the amino-terminal loop, suggesting its role in molecular interactions.

DOI： 10.1107/S0907444908028308

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT UNION CRYSTALLOGRAPHY  

F-spondin is a secreted and extracellular matrix-attached protein that has been implicated in axonal pathfinding during neural development as well as in vascular remodelling in adult tissues. F-spondin is composed of a reeler, a spondin and six thrombospondin type 1 repeat domains. The reeler domain shares homology with the amino-terminal domain of reelin, a large secreted glycoprotein that guides migrating neurons during cortical development. Crystal structures of the F-spondin reeler domain were determined at 1.45 and 2.70 A resolution. The structure revealed a nine-stranded antiparallel beta-sandwich fold similar to the immunoglobulin or fibronectin type III domains, but with a unique extra beta-hairpin. Moreover, an amino-terminal extension which is anchored at its beginning via a conserved disulfide bond loosely packs against one face of the beta-sandwich, making a major contribution to the surface features of the domain. Structural comparison among the different molecules contained in two different crystals reveals an unusual conformational plasticity of the amino-terminal loop, suggesting its role in molecular interactions.

DOI： 10.1107/S0907444908028308

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC  

A monoclonal antibody that recognizes native G protein coupled receptors (GPCR) is generally difficult to obtain. Protease-activated receptor-4 (PAR4) is a GPCR that plays in important role in platelet activation as a low-affinity thrombin receptor. By immunizing peptide corresponding to the N-terminal segment of human PAR4, we obtained a monoclonal antibody that recognizes cell surface expressed PAR4. Epitope mapping using a series of artificial fusion proteins that carry PAR4-derived peptide revealed that the recognition motif is fully contained within the 6-residue portion adjacent to the thrombin cleavage site. The antibody blocked PAR4 peptide cleavage by thrombin, Suggesting its Utility in the functional study of PAR4 signaling.

DOI： 10.1089/hyb.2008.0027

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Language：Japanese   Publisher：The Crystallographic Society of Japan  

Membrane-associated proteins play pivotal roles in a wide variety of cellular events, such as bioenergetics, membrane transport and signal transduction. In eukaryotic cells, membrane traffic in the endomembrane system is also regulated by membrane-associated proteins. Here we report the structural analyses of membrane-associated proteins as follows ; the bacterial photosynthetic reaction center converting the light energy into chemical energy, the γ1-adaptin ear domain of the AP-1 complex in the membrane traffic and the signaling molecule reelin regulating the layer formation in the brain. We discuss their structural features from the viewpoint of protein-protein interactions.

DOI： 10.5940/jcrsj.49.151

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Reelin, a large secreted protein implicated in the cortical development of the mammalian brain, is composed of eight tandem concatenations of "reelin repeats" and binds to neuronal receptors belonging to the low-density lipoprotein receptor gene family. We found that both receptor-binding and subsequent Dab 1 phosphorylation occur solely in the segment spanning the fifth and sixth reelin repeats(R5-6). Monomeric fragment exhibited a suboptimal level of signaling activity and artificial oligomerization resulted in a 10-fold increase in activity, indicating the critical importance of higher-order multimerization in physiological reelin. A 2.0-angstrom crystal structure from the R5-6 fragment revealed not only a unique domain arrangement wherein two repeats were aligned side by side with the same orientation, but also the unexpected presence of bound Zn ions. Structure-guided alanine mutagenesis of R5-6 revealed that two Lys residues (Lys-2360 and Lys-2467) constitute a central binding site for the low-density lipoprotein receptor class A module in the receptor, indicating a strong similarity to the ligand recognition mode shared among the endocytic lipoprotein receptors.

DOI： 10.1073/pnas.0700438104

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The large extracellular glycoprotein reelin directs neuronal migration during brain development and plays a fundamental role in layer formation. It is composed of eight tandem repeats of an similar to 380-residue unit, termed the reelin repeat, which has a central epidermal growth factor (EGF) module flanked by two homologous subrepeats with no obvious sequence similarity to proteins of known structure. The 2.05 angstrom crystal structure of the mouse reelin repeat 3 reveals that the subrepeat assumes a beta-jelly-roll fold with unexpected structural similarity to carbohydrate-binding domains. Despite the interruption by the EGF module, the two subdomains make direct contact, resulting in a compact overall structure. Electron micrographs of a four-domain fragment encompassing repeats 3-6, which is capable of inducing Disabled-1 phosphorylation in neurons, show a rod-like shape. Furthermore, a three-dimensional molecular envelope of the fragment obtained by single-particle tomography can be fitted with four concatenated repeat 3 atomic structures, providing the first glimpse of the structural unit for this important signaling molecule.

DOI： 10.1038/sj.emboj.7601240

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The photosynthetic reaction centers (RCs) classified as the group II possess a peripheral cytochrome (Cyt) subunit, which serves as the electron mediator to the special-pair. In the cycle of the photosynthetic electron transfer reactions, the Cyt subunit accepts electrons from soluble electron carrier proteins, and re-reduces the photo-oxidized special-pair of the bacteriochlorophyll. Physiologically, high-potential cytochromes such as the cytochrome c(2) and the high-potential iron-sulfur protein (HiPIP) function as the electron donors to the Cyt subunit. Most of the Cyt subunits possess four heme c groups, and it was unclear which heme group first accepts the electron from the electron donor. The most distal heme to the special-pair, the heme-1, has a lower redox potential than the electron donors, which makes it difficult to understand the electron transfer mechanism mediated by the Cyt subunit. Extensive mutagenesis combined with kinetic studies has made a great contribution to our understanding of the molecular interaction mechanisms, and has demonstrated the importance of the region close to the heme-1 in the electron transfer. Moreover, crystallographic studies have elucidated two high-resolution three-dimensional structures for the RCs containing the Cyt subunit, the Blastochloris viridis and Thermochromatium tepidum RCs, as well as the structures of their electron donors. An examination of the structural data also suggested that the binding sites for both the cytochrome c(2) and the HiPIP are located adjacent to the solvent-accessible edge of the heme-1. In addition, it is also indicated by the structural and biochemical data that the cytochrome c(2) and the HiPIP dock with the Cyt subunit by different mechanisms although the two electron donors utilize the same region for the interactions; cytochrome c(2) is recognized through electrostatic interactions while hydrophobic interactions are important in the HiPIP docking.

DOI： 10.1007/s11120-004-2416-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

GGAs are critical for trafficking soluble proteins from the trans-Golgi network (TGN) to endosomes/lysosomes through interactions with TGN-sorting receptors, ADP-ribosylation factor (ARF) and clathrin. ARF-GTP bound to TGN membranes recruits its effector GGA by binding to the GAT domain, thus facilitating recognition of GGA for cargo-loaded receptors. Here we report the X-ray crystal structures of the human GGA1-GAT domain and the complex between ARF1-GTP and the N-terminal region of the GAT domain. When unbound, the GAT domain forms an elongated bundle of three a-helices with a hydrophobic core. Structurally, this domain, combined with the preceding VHS domain, resembles CALM, an AP180 homolog involved in endocytosis. In the complex with ARF1-GTP, a helix-loop-helix of the N-terminal part of GGA1-GAT interacts with the switches 1 and 2 of ARF1 predominantly in a hydrophobic manner. These data reveal a molecular mechanism underlying membrane recruitment of adaptor proteins by ARF-GTP.

DOI： 10.1038/nsb920

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE AMERICA INC  

The adaptor proteins AP-1 and GGA regulate membrane traffic between the trans-Golgi network (TGN) and endosomes/lysosomes through ARF-regulated membrane association, recognition of sorting signals, and recruitment of clathrin and accessory proteins. The gamma1-adaptin subunits of AP-1 and GGA possess homologous ear domains involved in the recruitment of accessory proteins, gamma-synergin and Rabaptin-5. The crystal structure of the human gamma1-adaptin ear domain consists solely of an immunoglobulin-like fold, unlike the alpha-adaptin ear domain. Structure-based mutational analyses reveal a binding site for the accessory proteins that is composed of conserved basic residues, indicating that the recruitment mechanism in gamma1-adaptin and GGA is distinct from that in gamma-adaptin.

DOI： 10.1038/nsb808

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

Crystals of the high-potential iron-sulfur protein (HiPIP) from Thermochromatium tepidum diffract X-rays to 0.80 Angstrom using synchrotron radiation at 100 K. The crystal structure of this HiPIP was refined at this ultrahigh resolution with anisotropic temperature factors for all atoms to conventional crystallographic R factors of 0.092 and 0.101 for F-o > 4sigma(F-o) and all reflections, respectively. The present structure provides a more precise picture than the previous 1.5 Angstrom structure and allows location of the positions of most H atoms. The structure revealed a partly hydrophobic cavity near the main hydrophobic area and a much larger inter-cluster approach distance (23.454 Angstrom, the c constant of the unit cell) in the crystal packing than other types of HiPIPs. The structural features involved in the electron-transfer reaction of HiPIP are discussed.

DOI： 10.1107/S0907444902006261

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

GGAs (Golgi-localizing, g-adaptin ear homology domain, ARF-interacting proteins) are critical for the transport of soluble proteins from the trans-Golgi network (TGN) to endosomes/lysosomes by means of interactions with TGN-sorting receptors, ADP-ribosylation factor (ARF), and clathrin(1,2). The amino-terminal VHS domains of GGAs form complexes with the cytoplasmic domains of sorting receptors by recognizing acidic-cluster dileucine (ACLL) sequences(1-6). Here we report the X-ray structure of the GGA1 VHS domain alone, and in complex with the carboxyterminal peptide of cation-independent mannose 6-phosphate receptor containing an ACLL sequence. The VHS domain forms a super helix with eight alpha-helices, similar to the VHS domains of TOM1 and Hrs. Unidirectional movements of helices alpha6 and alpha8, and some of their side chains, create a set of electrostatic and hydrophobic interactions for correct recognition of the ACLL peptide. This recognition mechanism provides the basis for regulation of protein transport from the TGN to endosomes/lysosomes, which is shared by sortilin and low-density lipoprotein receptor-related protein.

DOI： 10.1038/415937a

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DOI： 10.2210/pdb1jwf/pdb

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Other Link： http://orcid.org/0000-0001-7071-5093

Language：English   Publisher：JAPANESE BIOCHEMICAL SOC  

The photosynthetic reaction center (RC) is the first membrane protein whose three-dimensional structure was revealed at the atomic level by X-ray crystallograph more than fifteen years ago. Structural information about RC made a great contribution to the understanding of the reaction mechanism of the complicated membrane protein complex. High-resolution structures of RCs from three photosynthetic bacteria are now available, namely, those from two mesophilic purple non-sulfur bacteria, Blastochloris viridis and Rhodobacter sphaeroides, and that from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. In addition, a variety of structural studies, mainly by X-ray crystallography, are still being performed to give more detailed insight into the reaction mechanism of this membrane protein. This review deals with structural studies of bacterial RC complexes, and a discussion about the electron transfer reaction between RCs and electron donors is the main focus out of several topics addressed by these structural studies. The structural data from three RCs and their electron donors provided reliable models for molecular recognition in the primary step of bacterial photosynthesis.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

The photosynthetic reaction center (RC) is a transmembrane protein complex that catalyzes light-driven electron transport across the photosynthetic membrane. The complete amino-acid sequence of the H subunit of the RC from a thermophilic purple sulfur bacterium, Thermochromatium tepidum, has been determined for the first time among purple sulfur bacteria. The H subunit consists of 259 amino acids and has a molecular mass of 28 187. The deduced amino-acid sequences of this H subunit showed a significant (40%) degree of identity with those from mesophilic purple nonsulfur bacteria.
The determined primary structure of the H subunit was compared with the structures of mesophilic B. viridis and R. sphaeroides based on the three-dimensional structure of the H subunit from T. tepidum, which has been recently determined by X-ray crystallography. One lipid molecule was found in the crystal structure of the T. tepidum RC, and the head group of the lipid appears to be stabilized by the electrostatic interactions with the conserved basic residues in the H subunit. The above comparison has suggested the existence of a lipid-binding site on the molecular surface at which a lipid molecule can interact with the RC in a specific manner.

DOI： 10.1046/j.1432-1327.2001.02158.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The reaction center (RC) of photosynthetic bacteria is a membrane protein complex that promotes a light-induced charge separation during the primary process of photosynthesis. In the photosynthetic electron transfer chain, the soluble electron carrier proteins transport electrons to the RC and reduce the photo-oxidized special-pair of bacteriochlorophyll. The high-potential iron-sulfur protein (HiPIP) is known to serve as an electron donor to the RC in some species, where the c-type cytochrome subunit, the peripheral subunit of the RC, directly accepts electrons from the HiPIP, Here we report the crystal structures of the RC and the HiPIP from Thermochromatium (Tch,) tepidum, at 2.2-Angstrom and 1.5-Angstrom resolution, respectively, Tch, tepidum can grow at the highest temperature of all known purple bacteria, and the Tch, tepidum RC shows some degree of stability to high temperature. Comparison with the RCs of mesophiles. such as Blastochloris viridis, has shown that the Tch. tepidum RC possesses more Arg residues at the membrane surface. which might contribute to the stability of this membrane protein. The RC and the HiPIP both possess hydrophobic patches on their respective surfaces, and the HiPIP is expected to interact with the cytochrome subunit by hydrophobic interactions near the heme-1, the most distal heme to the special-pair.

DOI： 10.1073/pnas.240224997

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MUNKSGAARD INT PUBL LTD  

The high-potential iron-sulfur protein (HiPIP) is an electron carrier between the photosynthetic reaction centre and the cytochrome bc(1) complex in the electron-transfer chain of photosynthesis. The purified HiPIP from Thermochromatium tepidum (formerly Chromatium tepidum) was crystallized in a solution of 1.4 M ammonium sulfate and 0.1 M sodium citrate pH 3.5. The crystals diffract X-rays beyond 1.4 Angstrom resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 47.12 (6), b = 59.59 (10), c = 23.62 (3) Angstrom. The structure was preliminarily solved by the molecular-replacement method. The crystal structure of HiPIP from T. tepidum showed that the proteins exist as monomers, although HiPIPs from several other species can form dimers.

DOI： 10.1107/S0907444900003127

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.40.S126_4

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/S0022-0248(99)00902-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

The gene of V-1-ATPase B subunit from the thermophilic eubacterium Thermus thermophilus has been cloned and the protein overproduced in Escherichia coli. The purified protein, with a molecular weight of 53.2 kDa, was crystallized from 10% (w/v) polyethylene glycol 1000, 120 mM magnesium chloride, and 100 mM Na-tricine, pH 8.0, by the vapor diffusion method. The crystals diffracted X-rays beyond 3.5 Angstrom on a synchrotron radiation source. The crystals belong to the monoclinic space group C2, with unit cell dimensions of a = 153.1 Angstrom, b = 129.6 Angstrom, c = 92.7 Angstrom, and beta = 100.3 degrees. Assuming that three or four molecules are contained in an asymmetric unit, the V-M value is calculated as 2.8 or 2.1 Angstrom (3)/Da, respectively. (C) 1999 Academic Press.

DOI： 10.1006/jsbi.1999.4140

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.39.S105_3

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Language：Japanese   Publisher：The Crystallographic Society of Japan  

DOI： 10.5940/jcrsj.39.Supplement_138

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Language：Japanese   Publisher：第一三共生命科学研究振興財団  

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.218

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Other Link： https://jlc.jst.go.jp/DN/JALC/10021777126?from=CiNii

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.235

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

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Language：English   Publisher：Japanese Society of Plant Physiologists  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=183924

Language：Japanese   Presentation type：Oral presentation (invited, special)  

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