Trimeric Assembly Research Articles

2P-084 ハロロドプシンNpHRの三量体構造はF150残基の対称配置によって安定化される(膜蛋白質,第47回日本生物物理学会年会)

2P-084 ハロロドプシンNpHRの三量体構造はF150残基の対称配置によって安定化される(膜蛋白質,第47回日本生物物理学会年会)

Covalently linked trimer of the AcrB multidrug efflux pump provides support for the functional rotating mechanism.

Escherichia coli AcrB is a proton motive force-dependent multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export (functional rotation hypothesis). In this study, we devised a way to test this hypothesis by creating a giant gene in which three acrB sequences were connected together through short linker sequences. The "linked-trimer" AcrB was expressed well in the inner membrane fraction of DeltaacrB DeltarecA strains, as a large protein of approximately 300 kDa which migrated at the same rate as the wild-type AcrB trimer in native polyacrylamide gel electrophoresis. The strain expressing the linked-trimer AcrB showed resistance to some toxic compounds that was sometimes even higher than that of the cells expressing the monomeric AcrB, indicating that the linked trimer functions well in intact cells. When we inactivated only one of the three protomeric units in the linked trimer, either with mutations in the salt bridge/H-bonding network (proton relay network) in the transmembrane domain or by disulfide cross-linking of the external cleft in the periplasmic domain, the entire trimeric complex was inactivated. However, some residual activity was seen, presumably as a result of random recombination of monomeric fragments (produced by protease cleavage or by transcriptional/translational truncation). These observations provide strong biochemical evidence for the functionally rotating mechanism of AcrB pump action. The linked trimer will be useful for further biochemical studies of mechanisms of transport in the future.

NMR-based homology model for the solution structure of the C-terminal globular domain of EMILIN1

EMILIN1 is a glycoprotein of elastic tissues that has been recently linked to the pathogenesis of hypertension. The protein is formed by different independently folded structural domains whose role has been partially elucidated. In this paper the solution structure, inferred from NMR-based homology modelling of the C-terminal trimeric globular C1q domain (gC1q) of EMILIN1, is reported. The high molecular weight and the homotrimeric structure of the protein required the combined use of highly deuterated (15)N, (13)C-labelled samples and TROSY experiments. Starting from a homology model, the protein structure was refined using heteronuclear residual dipolar couplings, chemical shift patterns, NOEs and H-exchange data. Analysis of the gC1q domain structure of EMILIN1 shows that each protomer of the trimer adopts a nine-stranded beta sandwich folding topology which is related to the conformation observed for other proteins of the family. Distinguishing features, however, include a missing edge-strand and an unstructured 19-residue loop. Although the current data do not allow this loop to be precisely defined, the available evidence is consistent with a flexible segment that protrudes from each subunit of the globular trimeric assembly and plays a key role in inter-molecular interactions between the EMILIN1 gC1q homotrimer and its integrin receptor alpha4beta1.

Different Signaling Roles of Two Conserved Residues in the Cytoplasmic Hairpin Tip of Tsr, the Escherichia coli Serine Chemoreceptor

Bacterial chemoreceptors form ternary signaling complexes with the histidine kinase CheA through the coupling protein CheW. Receptor complexes in turn cluster into cellular arrays that produce highly sensitive responses to chemical stimuli. In Escherichia coli, receptors of different types form mixed trimer-of-dimers signaling teams through the tips of their highly conserved cytoplasmic domains. To explore the possibility that the hairpin loop at the tip of the trimer contact region might promote interactions with CheA or CheW, we constructed and characterized mutant receptors with amino acid replacements at the two nearly invariant hairpin charged residues of Tsr: R388, the most tip-proximal trimer contact residue, and E391, the apex residue of the hairpin turn. Mutant receptors were subjected to in vivo tests for the assembly and function of trimers, ternary complexes, and clusters. All R388 replacements impaired or destroyed Tsr function, apparently through changes in trimer stability or geometry. Large-residue replacements locked R388 mutant ternary complexes in the kinase-off (F, H) or kinase-on (W, Y) signaling state, suggesting that R388 contributes to signaling-related conformational changes in the trimer. In contrast, most E391 mutants retained function and all formed ternary signaling complexes efficiently. Hydrophobic replacements of any size (G, A, P, V, I, L, F, W) caused a novel phenotype in which the mutant receptors produced rapid switching between kinase-on and -off states, indicating that hairpin tip flexibility plays an important role in signal state transitions. These findings demonstrate that the receptor determinants for CheA and CheW binding probably lie outside the hairpin tip of the receptor signaling domain.

Biogenesis of Phycobiliproteins: III. CpcM IS THE ASPARAGINE METHYLTRANSFERASE FOR PHYCOBILIPROTEIN β-SUBUNITS IN CYANOBACTERIA

All phycobiliproteins contain a conserved, post-translational modification on asparagine 72 of their beta-subunits. Methylation of this Asn to produce gamma-N-methylasparagine has been shown to increase energy transfer efficiency within the phycobilisome and to prevent photoinhibition. We report here the biochemical characterization of the product of sll0487, which we have named cpcM, from the cyanobacterium Synechocystis sp. PCC 6803. Recombinant apo-phycocyanin and apo-allophycocyanin subunits were used as the substrates for assays with [methyl-3H]S-adenosylmethionine and recombinant CpcM. CpcM methylated the beta-subunits of phycobiliproteins (CpcB, ApcB, and ApcF) and did not methylate the corresponding alpha-subunits (CpcA, ApcA, and ApcD), although they are similar in primary and tertiary structure. CpcM preferentially methylated its CpcB substrate after chromophorylation had occurred at Cys82. CpcM exhibited lower activity on trimeric phycocyanin after complete chromophorylation and oligomerization had occurred. Based upon these in vitro studies, we conclude that this post-translational modification probably occurs after chromophorylation but before trimer assembly in vivo.

Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at approximately 14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.

Crystal structure of a PduO‐type ATP:cobalamin adenosyltransferase from Burkholderia thailandensis

Crystal structure of a PduO‐type ATP:cobalamin adenosyltransferase from <i>Burkholderia thailandensis</i>

Trimeric reassembly of the globular domain of human C1q

C1q is a versatile recognition protein which binds to a variety of targets and consequently triggers the classical pathway of complement. C1q is a hetero-trimer composed of three chains (A, B and C) arranged in three domains, a short N-terminal region, followed by a collagenous repeat domain that gives rise to the formation of (ABC) triple helices, each ending in a C-terminal hetero-trimeric globular domain, called gC1q, which is responsible for the recognition properties of C1q. The mechanism of the trimeric assembly of C1q and in particular the role of each domain in the process is unknown. Here, we have investigated if the gC1q domain was able to assemble into functional trimers, in vitro, in the absence of the collagenous domain, a motif known to promote obligatory trimers in other proteins. Acid-mediated gC1q protomers reassembled into functional trimers, once neutralized, indicating that it is the gC1q domain which possesses the information for trimerization. However, reassembly occurred after neutralization, only if the gC1q protomers had preserved a residual tertiary structure at the end of the acidic treatment. Thus, the collagenous domain of C1q might initialize the folding of the gC1q domain so that subsequent assembly of the entire molecule can occur.

Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli

Assembly of the adenovirus (Ad) homotrimeric fiber protein is nucleated by its C-terminal knob domain, which itself can trimerize when expressed as a recombinant protein fragment. The non-interlocked, globular structure of subunits in the knob trimer implies that trimers assemble from prefolded monomers through a dimer intermediate, but these intermediates have not been observed and the mechanism of assembly therefore remains uncharacterized. Here we report that expression of the Ad serotype 2 (Ad2) knob was toxic for thi- strains of Escherichia coli, which are defective in de novo synthesis of thiamine (vitamin B1). Ad2 knob trimers isolated from a thi+ strain copurified through multiple chromatography steps with a small molecule of mass equivalent to that of thiamine diphosphate (ThDP). Mutant analysis did not implicate any specific site for ThDP binding. Our results suggest that ThDP may associate with assembly intermediates and become trapped in assembled trimers, possibly within one of several large cavities that are partially solvent-accessible or buried completely within the trimer interior.

The Heteropolytungstate Core {BW13O46}11‐ Derived as Monomer, Dimer, and Trimer.

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Site-directed disulfide cross-linking shows that cleft flexibility in the periplasmic domain is needed for the multidrug efflux pump AcrB of Escherichia coli.

Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export. However, biochemical evidence for these conformational changes has not been provided previously. In this study, we took advantage of the observation that the external large cleft in the periplasmic domain of AcrB appears to become closed in the crystal structure of one of the three protomers, and we carried out in vivo cross-linking between cysteine residues introduced by site-directed mutagenesis on both sides of the cleft, as well as at the interface between the periplasmic domains of the AcrB trimer. Double-cysteine mutants with mutations in the cleft or the interface were inactive. The possibility that this was due to the formation of disulfide bonds was suggested by the restoration of transport activity of the cleft mutants in a dsbA strain, which had diminished activity to form disulfide bonds in the periplasm. Furthermore, rapidly reacting, sulfhydryl-specific chemical cross-linkers, methanethiosulfonates, inactivated the AcrB transporter with double-cysteine residues in the cleft expressed in dsbA cells, and this inactivation could be observed within a few seconds after the addition of a cross-linker in real time by increased ethidium influx into the cells. These observations indicate that conformational changes, including the closure of the external cleft in the periplasmic domain, are required for drug transport by AcrB.

Selection of Protein Phosphatase 2A Regulatory Subunits Is Mediated by the C Terminus of the Catalytic Subunit

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases all composed of a catalytic C, a structural A, and a regulatory B subunit. Assembly of the complex with the appropriate B subunit forms the key to the functional specificity and regulation of PP2A. Emerging evidence suggests a crucial role for methylation and phosphorylation of the PP2A C subunit in this process. In this study, we show that PP2A C subunit methylation was not absolutely required for binding the PR61/B' and PR72/B'' subunit families, whereas binding of the PR55/B subunit family was determined by methylation and the nature of the C-terminal amino acid side chain. Moreover mutation of the phosphorylatable Tyr(307) or Thr(304) residues differentially affected binding of distinct B subunit family members. Down-regulation of the PP2A methyltransferase LCMT1 by RNA interference gradually reduced the cellular amount of methylated C subunit and induced a dynamic redistribution of the remaining methylated PP2A(C) between different PP2A trimers consistent with their methylation requirements. Persistent knockdown of LCMT1 eventually resulted in specific degradation of the PR55/B subunit and apoptotic cell death. Together these results establish a crucial foundation for understanding PP2A regulatory subunit selection.

The Heteropolytungstate Core {BW13O46}11− Derived as Monomer, Dimer, and Trimer

A study of the borotungstate system has led to the characterization of new, original compounds based on the unconventional Keggin derivative [H(3)BW(13)O(46)](8-) ion (denoted as 1). [H(3)BW(14)O(48)](6-) (2) and the dimer [H(6)B(2)W(26)O(90)](12-) (3) crystallize as mixed cesium/ammonium salts and have been characterized by single-crystal X-ray diffraction analysis. Anion 2 reveals an unusual arrangement, consisting of an outer {W(3)O(9)} core grafted onto the monovacant [BW(11)O(39)](9-) Keggin moiety and exhibits an unprecedented distorted square-pyramidal arrangement for a cis-{WO(2)} core. Elemental analysis, supported by bond distance analysis, is consistent with the presence of three protons distributed over the terminal oxygens of the outer {W(3)O(7)} capping fragment. The [H(6)B(2)W(26)O(90)](12-) ion (3) is formally derived from the direct condensation of two [H(3)BW(13)O(46)](8-) subunits. The cisoid arrangement of the two [BW(11)O(39)](9-) subunits, coupled with the antiparallel arrangement of the two quasi-linear O=W...O=W-OH2 chains within the central {W(4)O(12)} connecting group, breaks any symmetry, thereby resulting in a chiral compound. Polarography and pH-metric titrations reveal the formation of the monomeric precursor [H(3)BW(13)O(46)](8-) (anion 1) under stoichiometric conditions. (183)W NMR analysis of 2 and 3 in solution gives complex spectra, consistent with the presence of equilibria between several species. In the frame of this study, we also report on a structural re-investigation of the [H(6)B(3)W(39)O(132)](15-) ion (4) based on reliable results obtained in the solid state by means of single-crystal X-ray diffraction analysis, and in solution by means of 1D and 2D COSY (183)W NMR. X-ray diffraction analysis revealed the presence of three attached aquo ligands on the central {W(6)O(15)} connecting core, generating three O=W...O=W-OH2 quasi-linear chains, which are responsible for the chirality of the trimeric assembly. This structural arrangement accounts for the 39-line (183)W solution spectrum. The 2D COSY spectrum permits reliable assignments of the six strongly shielded resonances (around -250 and -400 ppm) to the six central W atoms, as well as additional assignments. The origin of such strong shielding for these particular W atoms is discussed on the basis of previously published results. Infrared data for compounds 1, 3, and 4 are also presented.

Modification of supramolecular assemblies based on C4H7(CH3)Te heterocycle and cooperative participation of intermolecular I···I, Te···I, Te···O secondary bonds; C(sp3)–H···O and C(sp2)–H···O hydrogen bonds

2-Methyl-1,1-dicarboxylato-1-telluracyclopentanes C4H7(CH3)Te(OCOR)2 (R=OCO, C6H5, 4-NO2C6H4, 3,5-(NO2)2C6H3, C6H5CH=CH, 4-OCH3C6H4) (2–7) were synthesised from the reactions of 2-methyl-1,1-diiodo-1-telluracyclopentane (1) and corresponding silver salts and characterised by (IR &1HNMR) spectroscopy. The structures of C4H7(CH3)TeI2 (1), C4H7(CH3)Te(OCOC6H5)2 (3) and C4H7(CH3)Te(4-NO2C6H4OCO)2 (4) were established by single crystal X-ray diffraction studies. The structures of 1, 3 & 4 (the immediate environment about tellurium is that of distorted trigonal bipyramidal geometry with a stereochemically active electron lone pair) are described in the context of their ability to generate intermolecular I···I, Te···I, Te···O secondary bonds; C(sp3)–H···O and C(sp2)–H···O hydrogen bonds leading to the formation of polymeric, tetrameric and trimeric supramolecular assemblies. The modification of supramolecular assembly present in the precursor 1 is demonstrated and the cooperative participation of C(sp2)–H···O & C(sp3)–H···O hydrogen bonds, probably, helpful in strengthening the supramolecular assembly is discussed.

Crystal structure of the multidrug efflux transporter AcrB at 3.1 Å resolution reveals the N-terminal region with conserved amino acids

Crystal structures of the bacterial multidrug transporter AcrB in R32 and C2 space groups showing both symmetric and asymmetric trimeric assemblies, respectively, supplemented with biochemical investigations, have provided most of the structural basis for a molecular level understanding of the protein structure and mechanisms for substrate uptake and translocation carried out by this 114-kDa inner membrane protein. They suggest that AcrB captures ligands primarily from the periplasm. Substrates can also enter the inner cavity of the transporter from the cytoplasm, but the exact mechanism of this remains undefined. Analysis of the amino acid sequences of AcrB and its homologs revealed the presence of conserved residues at the N-terminus including two phenylalanines which may be exposed to the cytoplasm. Any potential role that these conserved residues may play in function has not been addressed by existing biochemical or structural studies. Since phenylalanine residues elsewhere in the protein have been implicated in ligand binding, we explored the structure of this N-terminal region to investigate structural determinants near the cytoplasmic opening that may mediate drug uptake. Our structure of AcrB in R32 space group reveals an N-terminus loop, reducing the diameter of the central opening to ∼15 Å as opposed to the previously reported value of ∼30 Å for crystal structures in this space group with disordered N-terminus. Recent structures of the AcrB in C2 space group have revealed a helical conformation of this N-terminus but have not discussed its possible implications. We present the crystal structure of AcrB that reveals the structure of the N-terminus containing the conserved residues. We hope that the structural information provides a structural basis for others to design further biochemical investigation of the role of this portion of AcrB in mediating cytoplasmic ligand discrimination and uptake.

Structural analysis of flexible proteins in solution by small angle X-ray scattering combined with crystallography

In the last few years, SAXS of biological materials has been rapidly evolving and promises to move structural analysis to a new level. Recent innovations in SAXS data analysis allow ab initio shape predictions of proteins in solution. Furthermore, experimental scattering data can be compared to calculated scattering curves from the growing data base of solved structures and also identify aggregation and unfolded proteins. Combining SAXS results with atomic resolution structures enables detailed characterizations in solution of mass, radius, conformations, assembly, and shape changes associated with protein folding and functions. SAXS can efficiently reveal the spatial organization of protein domains, including domains missing from or disordered in known crystal structures, and establish cofactor or substrate-induced conformational changes. For flexible domains or unstructured regions that are not amenable for study by many other structural techniques, SAXS provides a unique technology. Here, we present SAXS shape predictions for PCNA that accurately predict a trimeric ring assembly and for a full-length DNA repair glycosylase with a large unstructured region. These new results in combination with illustrative published data show how SAXS combined with high resolution crystal structures efficiently establishes architectures, assemblies, conformations, and unstructured regions for proteins and protein complexes in solution.

Conformational flexibility and strand arrangements of the membrane-associated HIV fusion peptide trimer probed by solid-state NMR spectroscopy.

The human immunodeficiency virus (HIV) fusion peptide (HFP) is the N-terminal apolar region of the HIV gp41 fusion protein and interacts with target cell membranes and promotes membrane fusion. The free peptide catalyzes vesicle fusion at least to the lipid mixing stage and serves as a useful model fusion system. For gp41 constructs which lack the HFP, high-resolution structures show trimeric protein and suggest that at least three HFPs interact with the membrane with their C-termini in close proximity. In addition, previous studies have demonstrated that HFPs which are cross-linked at their C-termini to form trimers (HFPtr) catalyze fusion at a rate which is 15-40 times greater than that of non-cross-linked HFP. In the present study, the structure of membrane-associated HFPtr was probed with solid-state nuclear magnetic resonance (NMR) methods. Chemical shift and intramolecular (13)CO-(15)N distance measurements show that the conformation of the Leu-7 to Phe-11 region of HFPtr has predominant helical conformation in membranes without cholesterol and beta strand conformation in membranes containing approximately 30 mol % cholesterol. Interstrand (13)CO-(13)CO and (13)CO-(15)N distance measurements were not consistent with an in-register parallel strand arrangement but were consistent with either (1) parallel arrangement with adjacent strands two residues out-of-register or (2) antiparallel arrangement with adjacent strand crossing between Phe-8 and Leu-9. Arrangement 1 could support the rapid fusion rate of HFPtr because of placement of the apolar N-terminal regions of all strands on the same side of the oligomer while arrangement 2 could support the assembly of multiple fusion protein trimers.

Characterization of the HIV N-terminal Fusion Peptide-containing Region in Context of Key gp41 Fusion Conformations

Central to our understanding of human immunodeficiency virus-induced fusion is the high resolution structure of fragments of the gp41 fusion protein folded in a low energy core conformation. However, regions fundamental to fusion, like the fusion peptide (FP), have yet to be characterized in the context of the cognate protein regardless of its conformation. Based on conformation-specific monoclonal antibody recognition, we identified the polar region consecutive to the N36 fragment as a stabilizer of trimeric coiled-coil assembly, thereby enhancing inhibitory potency. This tertiary organization is retained in the context of the hydrophobic FP (N70 fragment). Our data indicate that the N70 fragment recapitulates the expected organization of this region in the viral fusion intermediate (N-terminal half of the pre-hairpin intermediate (N-PHI)), which happens to be the prime target for fusion inhibitors. Regarding the low energy conformation, we show for the first time core formation in the context of the FP (N70 core). The alpha-helical and coiled-coil stabilizing polar region confers substantial thermal stability to the core, whereas the hydrophobic FP does not add further stability. For the two key fusion conformations, N-PHI and N70 core, we find that the FP adopts a nonhelical structure and directs higher order assembly (assembly of coiled coils in N-PHI and assembly of bundles in the N70 core). This supra-molecular organization of coiled coils or folded cores is seen only in the context of the FP. This study is the first to characterize the FP region in the context of the folded core and provides a basic understanding of the role of the elusive FP for key gp41 fusion conformations.

The Crystal Structure of the Costimulatory OX40-OX40L Complex

OX40 is a T cell costimulator activated by OX40L. Blockade of the OX40L-OX40 interaction has ameliorative effects in animal models of T cell pathologies. In order to better understand the interaction between OX40 and OX40L, we have determined the crystal structure of murine OX40L and of the human OX40-OX40L complex at 1.45 and 2.4 A, respectively. These structures show that OX40L is an unusually small member of the tumor necrosis factor superfamily (TNFSF). The arrangement of the OX40L protomers forming the functional trimer is atypical and differs from that of other members by a 15 degrees rotation of each protomer with respect to the trimer axis, resulting in an open assembly. Site-directed changes of the interfacial residues of OX40L suggest this interface lacks a single "hot spot" and that instead, binding energy is dispersed over at least two distinct areas. These structures demonstrate the structural plasticity of TNFSF members and their interactions with receptors.

Molecular insights into the self‐assembly mechanism of dystrophia myotonica kinase

Self-assembly via coiled-coil domains (CC) is crucial for the regulation of the dystrophia myotonica kinase (DMPK) -related family of kinases. These CC domains are thought to form dimeric arrangements and thus to mediate dimerization in these enzymes. Using size exclusion chromatography combined with multiangle static light scattering, we analyzed the oligomeric state of DMPK as well as that of a truncated variant lacking the CC fraction. Remarkably, both forms were found to assemble into robust dimers. In contrast, the CC domain in isolation yielded trimeric assemblies, indicating that the oligomerization properties of CC domains from this kinase family are more diversified than anticipated. The crystal structure of this CC has been elucidated to 1.6 angstroms resolution and its properties in solution established using sedimentation equilibrium and thermal denaturation. These data show that, contrary to expectations, the self-assembly of DMPK is not dictated by the association properties of its CC domain. Instead, it appears to be driven by sequence segments flanking both N and C termini of the catalytic kinase fraction, as suggested by models of head-to-head dimers based on small angle X-ray scattering data. Our findings support a shared pattern of assembly across DMPK, ROCKs, and MRCK members of this family.