Structure and molecular organization of the centromere-kinetochore complex

For over a century, the terms centromere and kinetochore have been used interchangeably to describe a complex locus on eukaryotic chromosomes that attaches chromosomes to spindle fibres and facilitates chromosome movement in mitosis and meiosis. This region has become the focus of research aimed at defining the mechanism of chromosome segregation. A variety of new molecular probes and vastly improved optical-imaging technology have provided much new information on the structure of this locus and raised new hopes that an understanding of its function may soon be at hand.     

The molecular organization of the protein HC-IgA complex (HC-IgA)

Complexes of protein HC and monoclonal IgA1 or IgA2 or polyclonal IgA were isolated from human blood plasma. Dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that all complexes contain three types of chains: two light immunoglobulin chains, one regular IgA alpha-chain, and one chain with Mr = 90,000 carrying both alpha-chain and protein HC epitopes. The complexes were split into Fab alpha and Fc alpha fragments by bacterial IgA proteases. The protein HC epitopes were linked to the Fc fragments. Complexes of protein HC and an alpha-chain devoid of the variable region and the first heavy chain constant domain could also be demonstrated to be present in the blood plasma of a patient with alpha-heavy chain disease. Pepsin digestion of HC-IgA released a fragment containing all the protein HC epitopes and the C-terminal nonapeptide of the IgA alpha-chain. The light immunoglobulin chains, the regular alpha-chain, and the 90,000-Da chain from monoclonal HC-IgA1 were isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis and by repeated gel filtration in dodecyl sulfate-containing buffer. The N-terminal amino acid sequence of the alpha-chain was identical with that of a regular human heavy immunoglobulin chain of subgroup III. Subtractive degradations of the 90,000-Da chain displayed 2 amino acid residues in each position in a pattern suggesting simultaneous degradations of a chain identical with the regular alpha-chain of HC-IgA and of uncomplexed, low molecular weight, protein HC. All the results are compatible with a model for HC-IgA in which a single low molecular weight protein HC polypeptide chain is covalently linked, side by side, to the C-terminal nonapeptide of one of the two alpha-chains of a regular monomeric IgA unit.     

Crystal structure of Mdm12 reveals the architecture and dynamic organization of the ERMES complex

The endoplasmic reticulum–mitochondria encounter structure (ERMES) is a protein complex that plays a tethering role in physically connecting ER and mitochondria membranes. The ERMES complex is composed of Mdm12, Mmm1, and Mdm34, which have a SMP domain in common, and Mdm10. Here, we report the crystal structure of S. cerevisiae Mdm12. The Mdm12 forms a dimeric SMP structure through domain swapping of the β1‐strand comprising residues 1–7. Biochemical experiments reveal a phospholipid‐binding site located along a hydrophobic channel of the Mdm12 structure and that Mdm12 might have a binding preference for glycerophospholipids harboring a positively charged head group. Strikingly, both full‐length Mdm12 and Mdm12 truncated to exclude the disordered region (residues 74–114) display the same organization in the asymmetric unit, although they crystallize as a tetramer and hexamer, respectively. Taken together, these studies provide a novel understanding of the overall organization of SMP domains in the ERMES complex, indicating that Mdm12 interacts with Mdm34 through head‐to‐head contact, and with Mmm1 through tail‐to‐tail contact of SMP domains.     

Domain organization of the adenovirus preterminal protein.

In adenovirus-infected cells, the virus-encoded preterminal protein and DNA polymerase form a heterodimer that is directly involved in initiation of DNA replication. Monoclonal antibodies were raised against preterminal protein, and epitopes recognized by the antibodies were identified by using synthetic peptides. Partial proteolysis of preterminal protein reveals that it has a tripartite structure, with the three domains being separated by two protease-sensitive areas, located at sites processed by adenovirus protease. These areas of protease sensitivity are probably surface-exposed loops, as they are the sites, along with the C-terminal region of preterminal protein, recognized by the monoclonal antibodies. Preterminal protein is protected from proteolytic cleavage when bound to adenovirus DNA polymerase, suggesting either multiple contact points between the proteins or a DNA polymerase-induced conformational change in preterminal protein. Two of the preterminal protein-specific antibodies induced dissociation of the preterminal protein-adenovirus DNA polymerase heterodimer and inhibited initiation of adenovirus DNA replication in vitro. Antibodies binding close to the primary processing sites of adenovirus protease inhibited DNA binding, consistent with UV cross-linking results which reveal that an N-terminal, protease-resistant domain of preterminal protein contacts DNA. Monoclonal antibodies recognizing epitopes within the C-terminal 60 amino acids of preterminal protein stimulate DNA binding, an effect mediated through a decrease in the dissociation rate constant. These results suggest that preterminal protein contains a large, noncontiguous surface required for interaction with DNA polymerase, an N-terminal DNA binding domain, and a C-terminal regulatory domain.     

The molecular organization of the beta-globin complex of the deer mouse, Peromyscus maniculatus

Recombinant DNA clones have been isolated that contain 80 kb of thebeta-globin complex from the deer mouse, Peromyscus maniculatus.Comparisons of this complex with that from the laboratory mouse, Musdomesticus (with an order 5'-Hbby, Hbb-bhO, Hbb-bhl, Hbb-bh2, Hbb-bh3,Hbb-bl, Hbb-b2 3') highlight organizational trends in the beta-globincomplex since the two species diverged. Unlike other mammals studied thusfar, the deer mouse possesses three adult genes. Partial sequence analysisindicates that each of the three adult genes is intact and hence may befunctional. Hybridization of one of the two Mus pseudogenes, Hbb-bh3, togenomic blots from Peromyscus reveals that it has a homologous counterpartin Peromyscus. Homologous genes to the two gamma-like Mus genes, Hbb-bhOand Hbb-bhl, are also found in Peromyscus. The strong hybridization betweenthe Hbb-bhl genes and significant nucleotide similarity between the Hbb-bhOgenes suggest that both pairs are important for the ontogeny of these micealthough no known product has been identified for the Hbb-bhO genes. Thepresence of Hbb-bhO and Hbb-bhl in Peromyscus suggests that the duplicationthat created this related gene set occurred before the two lineagesdiverged. A single gene for Hbb-y has been isolated from Peromyscus. Theadult region in Peromyscus has undergone significant divergence from thesame region in Mus, having three rather than two adult genes, theacquisition of at least 15 kb of extra DNA relative to Mus, and possiblythe loss of the Hbb-bh2 pseudogene. The nonadult region of the complex, incontrast, contains the same set of genes apparently distributed over thesame amount of DNA as in the Mus beta- globin complex. This observationsuggests that the embryonic region of the complex is more evolutionarilystable than the adult region.     

Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.     

Cryo-EM asymmetric reconstruction of bacteriophage P22 reveals organization of its DNA packaging and infecting machinery

The mechanisms by which most double-stranded DNA viruses package and release their genomic DNA are not fully understood. Single particle cryo-electron microscopy and asymmetric 3D reconstruction reveal the organization of the complete bacteriophage P22 virion, including the protein channel through which DNA is first packaged and later ejected. This channel is formed by a dodecamer of portal proteins and sealed by a tail hub consisting of two stacked barrels capped by a protein needle. Six trimeric tailspikes attached around this tail hub are kinked, suggesting a functional hinge that may be used to trigger DNA release. Inside the capsid, the portal's central channel is plugged by densities interpreted as pilot/injection proteins. A short rod-like density near these proteins may be the terminal segment of the dsDNA genome. The coaxially packed DNA genome is encapsidated by the icosahedral shell. This complete structure unifies various biochemical, genetic, and crystallographic data of its components from the past several decades.     

Image reconstruction of fluorescent molecular tomography based on the tree structured Schur complement decomposition

Background  

The inverse problem of fluorescent molecular tomography (FMT) often involves complex large-scale matrix operations, which may lead to unacceptable computational errors and complexity. In this research, a tree structured Schur complement decomposition strategy is proposed to accelerate the reconstruction process and reduce the computational complexity. Additionally, an adaptive regularization scheme is developed to improve the ill-posedness of the inverse problem.     

Molecular organization of a high molecular weight multi-protease complex from rat liver

A latent multifunctional protease with a molecular weight of 722,000 to 760,000 purified from rat liver cytosol has been reported. This paper reports on the structure and subunit composition of the enzyme. Electron microscopy showed that the enzyme was a ring-shaped particle of 160(+/- 7) A diameter and 110(+/- 10) A height with a small hole of 10 to 30 A diameter (1 A = 0.1 nm). Small-angle X-ray scattering analysis indicated that the enzyme had a prolate ellipsoidal structure with an ellipsoid cavity in the center. The maximum dimension of the enzyme was estimated to be 210 A from a pair-distance distribution function. The radius of gyration obtained from a Guinier plot and the Stokes radius based on the ellipsoidal model were 66 A and 76 A, respectively. On two-dimensional gel electrophoresis, the purified enzyme separated into 13 to 15 characteristic components with molecular weights of 22,000 to 33,000 and isoelectric points of 4 to 9. These multiple components were not artifacts produced by limited proteolysis during purification of the enzyme, because the cell-free translation products in a reticulocyte lysate with poly(A)-mRNA of rat liver consisted of multiple components of similar sizes, and because peptide mapping analyses with lysylendopeptidase and V8 protease demonstrated clear differences in the primary structures of these components. The 13 main components were isolated from the purified enzyme by reverse-phase high performance liquid chromatography and shown to be non-identical. A model of the enzyme is proposed on the basis of these observations and previous physicochemical studies. Interestingly, the morphology of this protease is similar to that of the 16 to 22 S ring-shaped particles found in a variety of eukaryotic organisms. The structural similarity between this multi-protease complex and various reported subcellular particles is discussed.     

Solution structure of the dimerization domain of the eukaryotic stalk P1/P2 complex reveals the structural organization of eukaryotic stalk complex

The lateral ribosomal stalk is responsible for the kingdom-specific binding of translation factors and activation of GTP hydrolysis during protein synthesis. The eukaryotic stalk is composed of three acidic ribosomal proteins P0, P1 and P2. P0 binds two copies of P1/P2 hetero-dimers to form a pentameric P-complex. The structure of the eukaryotic stalk is currently not known. To provide a better understanding on the structural organization of eukaryotic stalk, we have determined the solution structure of the N-terminal dimerization domain (NTD) of P1/P2 hetero-dimer. Helix-1, -2 and -4 from each of the NTD-P1 and NTD-P2 form the dimeric interface that buries 2200 A(2) of solvent accessible surface area. In contrast to the symmetric P2 homo-dimer, P1/P2 hetero-dimer is asymmetric. Three conserved hydrophobic residues on the surface of NTD-P1 are replaced by charged residues in NTD-P2. Moreover, NTD-P1 has an extra turn in helix-1, which forms extensive intermolecular interactions with helix-1 and -4 of NTD-P2. Truncation of this extra turn of P1 abolished the formation of P1/P2 hetero-dimer. Systematic truncation studies suggest that P0 contains two spine-helices that each binds one copy of P1/P2 hetero-dimer. Modeling studies suggest that a large hydrophobic cavity, which can accommodate the loop between the spine-helices of P0, can be found on NTD-P1 but not on NTD-P2 when the helix-4 adopts an 'open' conformation. Based on the asymmetric properties of NTD-P1/NTD-P2, a structural model of the eukaryotic P-complex with P2/P1:P1/P2 topology is proposed.     

The dynamin A ring complex: molecular organization and nucleotide-dependent conformational changes

Here we show that Dictyostelium discoideum dynamin A is a fast GTPase, binds to negatively charged lipids, and self-assembles into rings and helices in a nucleotide-dependent manner, similar to human dynamin-1. Chemical modification of two cysteine residues, positioned in the middle domain and GTPase effector domain (GED), leads to altered assembly properties and the stabilization of a highly regular ring complex. Single particle analysis of this dynamin A* ring complex led to a three-dimensional map, which shows that the nucleotide-free complex consists of two layers with 11-fold symmetry. Our results reveal the molecular organization of the complex and indicate the importance of the middle domain and GED for the assembly of dynamin family proteins. Nucleotide-dependent changes observed with the unmodified and modified protein support a mechanochemical action of dynamin, in which tightening and stretching of a helix contribute to membrane fission.     

Image reconstruction from cryo-electron micrographs reveals the morphopoietic mechanism in the P2-P4 bacteriophage system.

The satellite bacteriophage P4 does not have genes coding for any major structural proteins, but assembles a capsid from the gene products of bacteriophage P2. The capsid assembled under control of P4 is smaller (45 nm) than the normal P2 capsid (60 nm). The low resolution (4.5 nm) structures of P2 and P4 capsids were determined by cryo-electron microscopy and image processing. The capsid of P2 shows T = 7 symmetry with most of the mass clustered as 12 pentamers and 60 hexamers. The P4 capsid has T = 4 symmetry with a similar distribution of mass to P2, but the hexamer geometry has changed. The major capsid protein has a two-domain structure. The major domains form the capsomers proper, while connecting domains form trivalent contacts between the capsomers. The size determination by P4 appears to function by altering hexamer geometry rather than by affecting the interdomain angle alone.     

Molecular organization of the uvomorulin-catenin complex

The Ca(2+)-dependent cell adhesion molecule uvomorulin is a member of the cadherin gene family. Its cytoplasmic region complexes with structurally defined proteins termed alpha-, beta-, and gamma-catenins. Here we show that A-CAM (N-cadherin), another member of this gene family, also associates with catenins suggesting that this complex formation may be a general property of the cadherins. For uvomorulin it has been found that this association with catenins is of crucial importance for the adhesive function, but little is known about the molecular organization of the uvomorulin-catenin complex. Using a combination of biochemical analyses we show that a single complex is composed of one molecule of uvomorulin, one or two molecules of beta-catenin, and one molecule of alpha-catenin. Furthermore, beta-catenin seems to interact more directly with uvomorulin. In pulse-chase experiments beta-catenin is already associated with the 135-kD uvomorulin precursor molecule but the assembly of the newly synthesized alpha-catenin into the complex is only detected around the time of endoproteolytic processing.     

Cytochrome b 6 f complex: Dynamic molecular organization,function and acclimation

The cytochrome b ₆ f complex occupies a central position in photosynthetic electron transport and proton translocation by linking PS II to PS I in linear electron flow from water to NADP⁺, and around PS I for cyclic electron flow. Cytochrome b ₆ f complexes are uniquely located in three membrane domains: the appressed granal membranes, the non-appressed stroma thylakoids and end grana membranes, and also the non-appressed grana margins, in contrast to the marked lateral heterogeneity of the localization of all other thylakoid multiprotein complexes. In addition to its vital role in vectorial electron transfer and proton translocation across the membrane, cytochrome b ₆ f complex is also involved in the regulation of balanced light excitation energy distribution between the photosystems, since its redox state governs the activation of LHC II kinase (the kinase that phosphorylates the mobile peripheral fraction of the chlorophyll a/b-proteins of LHC II of PS II). Hence, cytochrome b ₆ f complex is the molecular link in the interactive co-regulation of light-harvesting and electron transfer.The importance of a highly dynamic, yet flexible organization of the thylakoid membranes of plants and green algae has been highlighted by the exciting discovery that a lateral reorganization of some cytochrome b ₆ f complexes occurs in the state transition mechanism both in vivo and in vitro (Vallon et al. 1991). The lateral redistribution of phosphorylated LHC II from stacked granal membrane regions is accompanied by a concomitant movement of some cytochrome b ₆ f complexes from the granal membranes out to the PS I-containing stroma thylakoids. Thus, the dynamic movement of cytochrome b ₆ f complex as a multiprotein complex is a molecular mechanism for short-term adaptation to changing light conditions. With the concept of different membrane domains for linear and cyclic electron flow gaining credence, it is thought that linear electron flow occurs in the granal compartments and cyclic electron flow is localised in the stroma thylakoids at non-limiting irradiances. It is postulated that dynamic lateral reversible redistribution of some cytochrome b ₆ f complexes are part of the molecular mechanism involved in the regulation of linear electron transfer (ATP and NADPH) and cyclic electron flow (ATP only). Finally, the molecular significance of the marked regulation of cytochrome b ₆ f complexes for long-term regulation and optimization of photosynthetic function under varying environmental conditions, particularly light acclimation, is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - PS Photosystem     

Gene organization of the transforming region of adenovirus type 7 DNA

The sequence of the leftmost 11% of the weakly oncogenic human adenovirus type 7 (Ad7) DNA has been determined. This part of the Ad7 viral genome encompasses early region E1 which has been shown to be involved in the process of cell transformation in vitro (Dijkema et al., 1979). From the nucleotide sequence and determined coordinates of the E1 mRNAs, we are able to predict the primary structure of the polypeptides encoded by the transforming region of Ad7. The organization of the E1 region of Ad7 and of other adenovirus serotypes (Bos et al. 1981) leads to the proposal of a novel mechanism for gene regulation at the translational level in which protein synthesis can initiate at either the first or the second AUG triplet available in mRNA. The differences between the large E1b-specific tumor antigens of adenovirus types 12, 7 and 5 may explain the differences in oncogenicity of these viruses.     

Cytoskeletal organization at the postsynaptic complex

Postsynaptic densities and the adjacent cytoskeleton were examined in deep-etched, unfixed slices of guinea pig anteroventral cochlear nucleus. The postsynaptic density seen in conventional thin sections corresponds to a meshwork of 4-nm filaments associated with intramembrane particles at the postsynaptic active zone of inhibitory as well as excitatory synapses. These filaments intermesh with a lattice of 8- to 9-nm microfilaments, tentatively identified as F- actin, that is concentrated under the postsynaptic density. We postulate that the meshwork of 4-nm filaments anchors receptors to the adjacent microfilament lattice; this extended postsynaptic complex may limit the mobility of receptors and help maintain the curvature of the postsynaptic membrane.     

A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria

To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane-associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria.     

Structural organization and polypeptide composition of the avian adenovirus core.

CELO virus (fowl adenovirus 1) contained three core polypeptides of molecular weights 20,000, 12,000, and 9,500. The core was similar to that of human adenoviruses, with some evidence of compact subcore domains. Micrococcal nuclease digestion of CELO virus cores produced a smear of DNA fragments of gradually decreasing size, with no nucleosome subunit or repeat pattern. Moreover, when digested cores were analyzed without protease treatment, there was again no evidence of a nucleosome substructure; neither DNA fragments nor core proteins entered a 4% polyacrylamide gel. The organization of the core is thus quite unlike that of chromatin. Restriction endonuclease analysis of the DNA from digested cores showed that the right end was on the outside of the core. We suggest that adenovirus DNA is condensed into the core by cross-linking and neutralization by the core proteins, beginning with the packaging sequence at the center of the core and ending with the right end of the DNA on the outside.     

Pressure-induced changes in the molecular organization of a lipid-peptide complex. Polymyxin binding to phosphatidic acid membranes

The effect of 100 atm pressure on the organization of the lipid-peptide complex formed between polymyxin and dipalmitoyl phosphatidic acid has been investigated. Phase transition curves were obtained by electron paramagnetic resonance by measuring the partition coefficient of the spin label, 2, 2, 5, $\text{5-}\text{tetramethylpiperidine}\text{-N-}\text{oxyl}$. The three-step phase transition curve previously obtained with fluorescence polarization measurements was confirmed, demonstrating three distinct phosphatidic acid domains in the bilayer. Pressure increases binding of polymyxin to phosphatidic acid bilayers and alters the proportions of the two domains that differ in the mode of binding between phosphatidic acid and polymyxin. The binding curves of polymyxin to phosphatidic acid bilayers were determined and it was shown that application of pressure reduces the cooperativity of the binding curve.