Oligomeric Arrays Research Articles

Ribosomes hibernate on mitochondria during cellular stress.

Cell survival under nutrient-deprived conditions relies on cells' ability to adapt their organelles and rewire their metabolic pathways. In yeast, glucose depletion induces a stress response mediated by mitochondrial fragmentation and sequestration of cytosolic ribosomes on mitochondria. This cellular adaptation promotes survival under harsh environmental conditions; however, the underlying mechanism of this response remains unknown. Here, we demonstrate that upon glucose depletion protein synthesis is halted. Cryo-electron microscopy structure of the ribosomes show that they are devoid of both tRNA and mRNA, and a subset of the particles depicted a conformational change in rRNA H69 that could prevent tRNA binding. Our in situ structural analyses reveal that the hibernating ribosomes tether to fragmented mitochondria and establish eukaryotic-specific, higher-order storage structures by assembling into oligomeric arrays on the mitochondrial surface. Notably, we show that hibernating ribosomes exclusively bind to the outer mitochondrial membrane via the small ribosomal subunit during cellular stress. We identify the ribosomal protein Cpc2/RACK1 as the molecule mediating ribosomal tethering to mitochondria. This study unveils the molecular mechanism connecting mitochondrial stress with the shutdown of protein synthesis and broadens our understanding of cellular responses to nutrient scarcity and cell quiescence.

Controlled Dimerization of Mn12 Single-Molecule Magnets.

Controlled dimerization of Mn12 single-molecule magnets (SMMs) was achieved via a synthetic route involving a competition between bridging and terminal ligands, namely, diols and alcohols. The reaction using a 1:1 ratio of the competing ligands resulted in the isolation of a new family of covalently linked dimers of Mn12 SMMs. This is the first step toward the controlled growth of SMM oligomeric arrays.

The Great Nuclear Escape: Structure-Based Mechanism of Membrane Budding during Nuclear Egress of Herpesviruses

Herpesviruses are unusual among enveloped viruses because they bud twice yet acquire a single envelope. They are also the only known mammalian viruses that bud into the nuclear envelope. Recently, we discovered that the herpesvirus nuclear egress complex (NEC) could bud membranes without the help of other proteins and that it formed a coat-like hexagonal scaffold inside the budding membrane. This discovery established the NEC as the first virally encoded budding machine that operates at the nuclear, as opposed to cytoplasmic, membrane but left unknown the structure of the NEC coat and its role in the budding process. To bridge this gap in our knowledge, we determined the 2.8-A crystal structure of the NEC from Herpes Simplex virus (HSV). In crystals, NEC packs into a hexagonal lattice that mimics the hexagonal NEC coats within budded vesicles. The crystal structure of the NEC lattice thus reveals molecular interactions that generate the hexagonal coat. To determine the role of the hexagonal NEC lattice in budding, we mutated residues at the oligomeric interfaces observed in the crystals with mutagenesis. Perturbation of the oligomeric interfaces through mutagenesis blocked NEC-mediated budding in vitro confirming that formation of the hexagonal NEC lattice drives budding. The NEC structure provide a three-dimensional blueprint for further dissection of its unique budding mechanism. Moreover, the structure represents the first atomic-level view of an oligomeric array formed by a membrane-deforming protein.

A Tightly Stretched Ultralong Supramolecular Multiporphyrin Array Propagated by Double-Strand Formation.

A shape-programmed linearity through supramolecular polymerization is demonstrated by a step-growth double-strand formation of a telechelic oligomeric porphyrin array in which two alternating pyridyl-porphyrin sequenced units are held together by self-complementary ligand-to-metal coordination. The stiff rod-like structure and sufficiently large binding constant of the double-strand unit considerably extended a supramolecular array in the one dimension, which produced a tightly stretched string with a length that exceeded several micrometers.

Transformation and patterning of supermicelles using dynamic holographic assembly.

Although the solution self-assembly of block copolymers has enabled the fabrication of a broad range of complex, functional nanostructures, their precise manipulation and patterning remain a key challenge. Here we demonstrate that spherical and linear supermicelles, supramolecular structures held together by non-covalent solvophobic and coordination interactions and formed by the hierarchical self-assembly of block copolymer micelle and block comicelle precursors, can be manipulated, transformed and patterned with mediation by dynamic holographic assembly (optical tweezers). This allows the creation of new and stable soft-matter superstructures far from equilibrium. For example, individual spherical supermicelles can be optically held in close proximity and photocrosslinked through controlled coronal chemistry to generate linear oligomeric arrays. The use of optical tweezers also enables the directed deposition and immobilization of supermicelles on surfaces, allowing the precise creation of arrays of soft-matter nano-objects with potentially diverse functionality and a range of applications.

Structural basis of membrane budding by the nuclear egress complex of herpesviruses.

During nuclear egress, herpesvirus capsids bud at the inner nuclear membrane forming perinuclear viral particles that subsequently fuse with the outer nuclear membrane, releasing capsids into the cytoplasm. This unusual budding process is mediated by the nuclear egress complex (NEC) composed of two conserved viral proteins, UL31 and UL34. Earlier, we discovered that the herpesvirus nuclear egress complex (NEC) could bud synthetic membranes in vitro without the help of other proteins by forming a coat-like hexagonal scaffold inside the budding membrane. To understand the structural basis of NEC-mediated membrane budding, we determined the crystal structures of the NEC from two herpesviruses. The hexagonal lattice observed in the NEC crystals recapitulates the honeycomb coats within the budded vesicles. Perturbation of the oligomeric interfaces through mutagenesis blocks budding in vitro confirming that NEC oligomerization into a honeycomb lattice drives budding. The structure represents the first atomic-level view of an oligomeric array formed by a membrane-deforming protein, making possible the dissection of its unique budding mechanism and the design of inhibitors to block it.

Self-complementary double-stranded porphyrin arrays assembled from an alternating pyridyl-porphyrin sequence.

Oligomeric porphyrin arrays with an alternating pyridyl-porphyrin sequence were synthesized to explore double-strand formation through self-complementary pyridyl-to-zinc axial coordination bonds. Competitive titration experiments revealed the thermodynamic aspects involved in the zipper effect within double-strand formation. Multiple axial coordination bonds defined the stacked conformation, despite a marginal contribution to the stability of the double-strands. Thus, the zipper cooperativity was the dominant factor for the remarkable stability. Moreover, the dimeric and trimeric porphyrin arrays were independently assembled into double-strands by self-sorting from a binary mixture. Double-strand formation engineered discretely stacked π-systems. Successive slipped-cofacial stacks of the porphyrin rings progressively extended the π-system via exciton coupling over the double-strand while keeping a relatively high fluorescence quantum yield.

Novel Structural and Functional Insights into M3 Muscarinic Receptor Dimer/Oligomer Formation

Class A G protein-coupled receptors (GPCRs) are able to form homodimers and/or oligomeric arrays. We recently proposed, based on bioluminescence resonance energy transfer studies with the M3 muscarinic receptor (M3R), a prototypic class A GPCR, that the M3R is able to form multiple, structurally distinct dimers that are probably transient in nature (McMillin, S. M., Heusel, M., Liu, T., Costanzi, S., and Wess, J. (2011) J. Biol. Chem. 286, 28584-28598). To provide more direct experimental support for this concept, we employed a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (transfected COS-7 cells). Disulfide cross-linking studies were carried out with many mutant M3Rs containing single cysteine (Cys) substitutions within two distinct cytoplasmic M3R regions, the C-terminal portion of the second intracellular loop (i2) and helix H8 (H8). The pattern of cross-links that we obtained, in combination with molecular modeling studies, was consistent with the existence of two structurally distinct M3R dimer interfaces, one involving i2/i2 contacts (TM4-TM5-i2 interface) and the other one characterized by H8-H8 interactions (TM1-TM2-H8 interface). Specific H8-H8 disulfide cross-links led to significant impairments in M3R-mediated G protein activation, suggesting that changes in the structural orientation or mobility of H8 are critical for efficient receptor-G protein coupling. Our findings provide novel structural and functional insights into the mechanisms involved in M3R dimerization (oligomerization). Because the M3R shows a high degree of sequence similarity with many other class A GPCRs, our findings should be of considerable general interest.

Membrane Driven Spatial Organization of GPCRs

Spatial organization of G-protein coupled receptors (GPCRs) into dimers and higher order oligomers has been demonstrated in vitro and in vivo. The pharmacological readout was shown to depend on the specific interfaces, but why particular regions of the GPCR structure are involved, and how ligand-determined states change them remains unknown. Here we show why protein-membrane hydrophobic matching is attained upon oligomerization at specific interfaces from an analysis of coarse-grained molecular dynamics simulations of the spontaneous diffusion-interaction of the prototypical beta2-adrenergic (β2AR) receptors in a POPC lipid bilayer. The energy penalty from mismatch is significantly reduced in the spontaneously emerging oligomeric arrays, making the spatial organization of the GPCRs dependent on the pattern of mismatch in the monomer. This mismatch pattern is very different for β2AR compared to the highly homologous and structurally similar β1AR, consonant with experimentally observed oligomerization patterns of β2AR and β1AR. The results provide a mechanistic understanding of the structural context of oligomerization.

Structural basis for the MukB-topoisomerase IV interaction and its functional implications in vivo.

Chromosome partitioning in Escherichia coli is assisted by two interacting proteins, topoisomerase (topo) IV and MukB. MukB stimulates the relaxation of negative supercoils by topo IV; to understand the mechanism of their action and to define this functional interplay, we determined the crystal structure of a minimal MukB-topo IV complex to 2.3 Å resolution. The structure shows that the so-called 'hinge' region of MukB forms a heterotetrameric assembly with a C-terminal DNA binding domain (CTD) on topo IV's ParC subunit. Biochemical studies show that the hinge stimulates topo IV by competing for a site on the CTD that normally represses activity on negatively supercoiled DNA, while complementation tests using mutants implicated in the interaction reveal that the cellular dependency on topo IV derives from a joint need for both strand passage and MukB binding. Interestingly, the configuration of the MukB·topo IV complex sterically disfavours intradimeric interactions, indicating that the proteins may form oligomeric arrays with one another, and suggesting a framework by which MukB and topo IV may collaborate during daughter chromosome disentanglement.

Quantitative Modeling of Membrane Deformations by Multihelical Membrane Proteins: Application to G-Protein Coupled Receptors

The interpretation of experimental observations of the dependence of membrane protein function on the properties of the lipid membrane environment calls for a consideration of the energy cost of protein-bilayer interactions, including the protein-bilayer hydrophobic mismatch. We present a novel (to our knowledge) multiscale computational approach for quantifying the hydrophobic mismatch-driven remodeling of membrane bilayers by multihelical membrane proteins. The method accounts for both the membrane remodeling energy and the energy contribution from any partial (incomplete) alleviation of the hydrophobic mismatch by membrane remodeling. Overcoming previous limitations, it allows for radially asymmetric bilayer deformations produced by multihelical proteins, and takes into account the irregular membrane-protein boundaries. The approach is illustrated by application to two G-protein coupled receptors: rhodopsin in bilayers of different thickness, and the serotonin 5-HT2A receptor bound to pharmacologically different ligands. Analysis of the results identifies the residual exposure that is not alleviated by bilayer adaptation, and its quantification at specific transmembrane segments is shown to predict favorable contact interfaces in oligomeric arrays. In addition, our results suggest how distinct ligand-induced conformations of G-protein coupled receptors may elicit different functional responses through differential effects on the membrane environment.

Structural Basis of M3 Muscarinic Receptor Dimer/Oligomer Formation

Class A G protein-coupled receptors (GPCRs) are known to form dimers and/or oligomeric arrays in vitro and in vivo. These complexes are thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the "outer" (lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1-5 and -7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the bioluminescence resonance energy transfer data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Given the high structural homology found among all class A GPCRs, our results should be of considerable general relevance.

Disulfide-Mediated Oligomer Formation in Borrelia burgdorferi Outer Surface Protein C, a Critical Virulence Factor and Potential Lyme Disease Vaccine Candidate

Borrelia burgdorferi OspC is an outer membrane lipoprotein required for the establishment of infection in mammals. Due to its universal distribution among B. burgdorferi sensu lato strains and high antigenicity, it is being explored for the development of a next-generation Lyme disease vaccine. An understanding of the surface presentation of OspC will facilitate efforts to maximize its potential as a vaccine candidate. OspC forms homodimers at the cell surface, and it has been hypothesized that it may also form oligomeric arrays. Here, we employ site-directed mutagenesis to test the hypothesis that interdimeric disulfide bonds at cysteine 130 (C130) mediate oligomerization. B. burgdorferi B31 ospC was replaced with a C130A substitution mutant to yield strain B31::ospC(C130A). Recombinant protein was also generated. Disulfide-bond-dependent oligomer formation was demonstrated and determined to be dependent on C130. Oligomerization was not required for in vivo function, as B31::ospC(C130A) retained infectivity and disseminated normally. The total IgG response and the induced isotype pattern were similar between mice infected with untransformed B31 and those infected with the B31::ospC(C130A) strain. These data indicate that the immune response to OspC is not significantly altered by formation of OspC oligomers, a finding that has significant implications in Lyme disease vaccine design.

Quadruple hydrogen bonded cytosine modules: N-1 functionalised arrays

A ureidocytosine (UCyt) module with a pendant amine linker at N-1 has been prepared for applications in supramolecular quadruple hydrogen bonded array synthesis. Subsequent conjugation to four amine terminated telechelic polymers led to the formation of oligomeric DDAA arrays, as determined by NMR diffusion measurements, establishing that N-1 as well as N-9 ureidocytosine polymeric arrays can readily be accessed.

The Ndc80 kinetochore complex forms oligomeric arrays along microtubules.

The Ndc80 complex is a key site of regulated kinetochore-microtubule attachment, but the molecular mechanism underlying its function remains unknown. Here we present a subnanometer resolution cryo-electron microscopy reconstruction of the human Ndc80 complex bound to microtubules, sufficient for precise docking of crystal structures of the component proteins. We find that Ndc80 binds the microtubule with a tubulin monomer repeat, recognizing α- and β-tubulin at both intra- and inter-dimer interfaces in a manner that is sensitive to tubulin conformation. Furthermore, Ndc80 complexes self-associate along protofilaments via interactions mediated by the amino-terminal tail of the Ndc80 protein, the site of phospho-regulation by the Aurora B kinase. Ndc80's mode of interaction with the microtubule and its oligomerization suggest a mechanism by which Aurora B could regulate the stability of load-bearing Ndc80-microtubule attachments.

Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays

Few antivirals are effective against positive-strand RNA viruses, primarily because the high error rate during replication of these viruses leads to the rapid development of drug resistance. One of the favored current targets for the development of antiviral compounds is the active site of viral RNA-dependent RNA polymerases. However, like many subcellular processes, replication of the genomes of all positive-strand RNA viruses occurs in highly oligomeric complexes on the cytosolic surfaces of the intracellular membranes of infected host cells. In this study, catalytically inactive polymerases were shown to participate productively in functional oligomer formation and catalysis, as assayed by RNA template elongation. Direct protein transduction to introduce either active or inactive polymerases into cells infected with mutant virus confirmed the structural role for polymerase molecules during infection. Therefore, we suggest that targeting the active sites of polymerase molecules is not likely to be the best antiviral strategy, as inactivated polymerases do not inhibit replication of other viruses in the same cell and can, in fact, be useful in RNA replication complexes. On the other hand, polymerases that could not participate in functional RNA replication complexes were those that contained mutations in the amino terminus, leading to altered contacts in the folded polymerase and mutations in a known polymerase-polymerase interaction in the two-dimensional protein lattice. Thus, the functional nature of multimeric arrays of RNA-dependent RNA polymerase supplies a novel target for antiviral compounds and provides a new appreciation for enzymatic catalysis on membranous surfaces within cells.

Oligomerization of BenM, a LysR-type transcriptional regulator: structural basis for the aggregation of proteins in this family.

LysR-type transcriptional regulators comprise the largest family of homologous regulatory DNA-binding proteins in bacteria. A problematic challenge in the crystallization of LysR-type regulators stems from the insolubility and precipitation difficulties encountered with high concentrations of the full-length versions of these proteins. A general oligomerization scheme is proposed for this protein family based on the structures of the effector-binding domain of BenM in two different space groups, P4(3)22 and C222(1). These structures used the same oligomerization scheme of dimer-dimer interactions as another LysR-type regulator, CbnR, the full-length structure of which is available [Muraoka et al. (2003), J. Mol. Biol. 328, 555-566]. Evaluation of packing relationships and surface features suggests that BenM can form infinite oligomeric arrays in crystals through these dimer-dimer interactions. By extrapolation to the liquid phase, such dimer-dimer interactions may contribute to the significant difficulty in crystallizing full-length members of this family. The oligomerization of dimeric units to form biologically important tetramers appears to leave unsatisfied oligomerization sites. Under conditions that favor association, such as neutral pH and concentrations appropriate for crystallization, higher order oligomerization could cause solubility problems with purified proteins. A detailed model by which BenM and other LysR-type transcriptional regulators may form these arrays is proposed.

Organization of ETRAMPs and EXP‐1 at the parasite–host cell interface of malaria parasites

The parasite-host cell interface is a key compartment of vacuolated intracellular pathogens but little is known about its molecular composition and architecture. We used in vivo cross-linking to analyse the parasite-host cell interface of asexual stages of the most virulent human malaria parasite Plasmodium falciparum. We show that the integral membrane protein members of the early transcribed membrane protein (ETRAMP) family and exported protein 1 (EXP-1), which are components of the parasite-host cell interface, form complexes of oligomeric arrays in this compartment. The most notable feature is that each ETRAMP member and EXP-1 define separate arrays, demonstrating that the protein distribution in this membrane is non-random. Each of three recombinant ETRAMPs readily oligomerized in bacterial membranes, confirming that these arrays can form independently of other Plasmodium proteins. We propose that the malaria parasite-host cell interface contains patches of integral membrane proteins forming a mosaic of different microdomains in this membrane.

Solid state coordination chemistry: temperature dependence of the crystal chemistry of the oxovanadium-phenylphosphonate-copper(II)-2,2′-bipyridine system. Crystal structures of the one-dimensional [{Cu(bpy)}VO 2(O 3PC 6H 5)(HO 3PC 6H 5)], [{Cu 3(bpy) 3(H 2O)}V 4O 9(O 3PC 6H 5) 4], [{Cu(bpy)} 2V 3O 6(O 3PC 6H 5) 3(HO 3PC 6H 5)] and [{Cu(bpy)}VO(O 3PC 6H 5) 2

The temperature dependence of the hydrothermal chemistry of the oxovanadium-phenylphosphonate/copper(II)-bipyridine system has been investigated. Reactions of identical compositions of CuSO 4⋅5H 2O, bipyridine, C 6H 5PO 3H 2 and Na 3VO 4 in H 2O yielded [{Cu(bpy)}VO 2(O 3PC 6H 5)(HOC 6H 5)] ( 1), [{Cu 3(bpy) 3(H 2O)}V 4O 9(O 3PC 6H 5) 4]⋅3.9H 2O ( 2⋅3.9H 2O) and [{Cu(bpy)}VO(O 3PC 6H 5) 2] ( 4) at 120 °C, 150 °C and 180–230 °C, respectively. Furthermore, variations in stoichiometry at 150 °C provided yet a fourth one-dimensional phase [{Cu(bpy)} 2V 3O 6(O 3PC 6H 5) 3(HO 3PC 6H 5)] ( 3). Substitution of a methyl substituent for the phenyl group yielded another one-dimensional material [{Cu(bpy)}V 2O 4(O 3PCH 3) 2⋅H 2O ( 5⋅H 2O). The structural chemistry is discussed in terms of the polyhedral variability of vanadium oxides, the tendency of oxovanadium polyhedra to form oligomeric arrays, the variable modes of polyhedral connectivity associated with vanadium square pyramids and phosphorus tetrahedra and the steric influences of the copper(II)-bpy subunit.

Design of a real time quantitative PCR assay to assess global mRNA amplification of small size specimens for microarray hybridisation

Background: Low RNA yields from clinical samples are a limiting step for microarray technology. Aims: To design an accurate real time quantitative polymerase chain reaction (PCR) assay to assess the...