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PubMed2005

Roles of the N-terminal domain on the function and quaternary structure of the ionotropic glutamate receptor.

The alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptors (iGluRs) mediates fast excitatory neurotransmission in the mammalian brain. Although the most N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain is suggested to play a role in the initial assembly of iGluR subunits, it is unclear how this domain is arranged and functions in intact iGluRs. Similarly, although recent crystallographic analyses indicate that the isolated ligand-binding lysine/arginine/ornithine-binding protein domain forms a 2-fold symmetric dimer, the subunit stoichiometry of intact iGluRs remains elusive. Here, we developed a new approach to address these issues. The LIVBP domain of the GluR1 subunit of AMPA receptors was replaced by leucine-zipper peptides designed to form stable symmetric dimers, trimers, tetramers, or pentamers. All these mutant GluR1s were expressed in human embryonic kidney 293 cells and were transported to the cell surface as well as wild type GluR1. Functional and biochemical analyses indicated that these oligomerizing peptides specifically controlled the formation of the expected number of subunits in a channel complex. However, the channel function was only restored by the tetramer-forming peptide. Although the purified LIVBP domain of GluR1 formed a dimmer in solution, a dimer-forming peptide could not restore the function of GluR1. Moreover, a cross-linking assay indicated that four LIVBP domains are located in proximity to each other. These results suggest that the function of the LIVBP domain is not simply to form initial dimers but to adopt a conformation compatible with the overall tetrameric arrangement of subunits in intact AMPA receptors.

Amino Acid Sequence↗

PubMed2000

Core structure of the outer membrane lipoprotein from Escherichia coli at 1.9 A resolution.

The outer membrane lipoprotein of the Escherichia coli cell envelope has characteristic lipid modifications at an amino-terminal cysteine and can exist in a form bound covalently to the peptidoglycan through a carboxyl-terminal lysine. The 56-residue polypeptide moiety of the lipoprotein, designated Lpp-56, folds into a stable, trimeric helical structure in aqueous solution. The 1.9 A resolution crystal structure of Lpp-56 comprises a parallel three-stranded coiled coil including a novel alanine-zipper unit and two helix-capping motifs. The amino-terminal motif forms a hydrogen-bonding network anchoring an umbrella-shaped fold. The carboxyl-terminal motif uses puckering of the tyrosine side-chains as a unique docking arrangement in helix termination. The structure provides an explanation for assembly and insertion of the lipoprotein molecules into the outer membrane of gram-negative bacteria and suggests a molecular target for antibacterial drug discovery.

Alanine↗

PubMed1996

Evidence for the direct interaction of the nifW gene product with the MoFe protein.

The Azotobacter vinelandii nifW gene, under control of the nifH promoter, was subcloned into the broad host range multicopy plasmid pKT230 for overexpression in both wild-type and delta nifW strains of A. vinelandii. Unlike the parent delta nifW strain, which grows slowly relative to wild-type under N2-fixing conditions, both overproduction strains grow at the same rate, showing that the overexpressed nifW product is functional in vivo. The approximately 40-fold overexpressed protein was purified, and sequence analysis confirmed its identity. During purification it was observed that NifW in crude extracts ran above the predicted molecular weight on denaturing gels and that as the purification proceeded lower molecular weight forms appeared. Mass spectrometry and studies with protease inhibitors revealed that this abnormal behavior was due to proteolysis. Native molecular weight determinations demonstrate that NifW is a homomultimer, most likely a trimer. Native gel electrophoresis analysis shows that the behavior of wild-type and overexpressed NifW are identical and that when extracts are prepared anaerobically only the homomultimeric forms of NifW are observed. When extracts are exposed to oxygen, however, NifW becomes part of a very high molecular weight complex. Immunoprecipitation with NifW antibodies demonstrate that under those conditions NifW specifically associates with the MoFe protein. These data are consistent with a model whereby NifW is not involved in the initial assembly of an active MoFe protein but rather is part of a system design to protect the MoFe protein from O2 damage.

Amino Acid Sequence↗

PubMed2002

Conformation-dependent side reactions in interstrand-disulfide bridging of trimeric collagenous peptides by regioselective cysteine chemistry.

Conversion of single-chain or disulfide-bridged dimeric collagenous peptides into Cys(Npys) derivatives as activated species for subsequent regioselective thiol/disulfide exchange reactions leads to side products whose origin and nature was determined by HPLC and ESI-MS. In both cases the high tendency of the educts to self-associate into triple-helical homotrimers, as assessed by their dichroic properties in the reaction media, is responsible for the failure of this well established cysteine chemistry. Only by optimizing the synthetic strategy or by exploiting a kinetic control of the reaction, could these conformation-dependent limitations be more or less efficiently bypassed for the regioselective assembly of heterotrimeric collagen model peptides crosslinked with artificial cystine knots.

Amino Acid Sequence↗

PubMed1995

A clathrin-binding site in the hinge of the beta 2 chain of mammalian AP-2 complexes.

The assembly of cytosolic clathrin into the cytoplasmic face of coated pits and coated vesicles appears to be driven by the clathrin-associated protein (AP) complexes. We have previously shown that one of the large chains of the AP complexes, the beta chain, is sufficient to drive coat assembly in vitro. This chain consists of two domains, the amino-terminal trunk and the carboxyl-terminal ear, linked by a "hinge." We report here that presence of the hinge in recombinant beta trunk or in recombinant beta ear fragments is essential for driving in vitro assembly of clathrin into coats. We have also used a binding assay to map the clathrin-binding site by nested deletion of hinge sequences to a 50-residue region in the center of the hinge. This sequence is conserved in all known beta sequences from multicellular organisms. The interaction of a single beta hinge with a clathrin triskelion is weak, and we propose that recruitment of cytosolic clathrin to a forming coated pit involves simultaneous contacts between the legs of single clathrin trimers and the beta hinges of two or three membrane-bound AP complexes. Uncoating is likely to require interruption of these contacts.

Adaptor Proteins, Vesicular Transport↗

PubMed1983

Collagen genes and brittle bones.

The heritable diseases of connective tissue are caused by known or putative defects in the synthesis of collagens, proteoglycans, glycoproteins, or attachment proteins of the extracellular matrix. Abnormal synthesis of type I collagen has been reported in several clinical variants of osteogenesis imperfecta. Because clinical classification of these variants is limited by genetic heterogenity and variable expression, biochemical criteria should be used for precise definition of the variants. Newly recognized molecular defects in osteogenesis imperfecta include the diminished formation of type I collagen and alpha-1[I] messenger RNA; abnormal synthesis or faulty assembly of alpha-2[I]; deletion or insertion of base pairs in the gene for alpha-1[I] or alpha-2[I] and failure to secrete type I procollagen; and substitution of cysteine for glycine in the triple helix. These molecular defects are characteristic of several variants. However, the molecular lesion in most cases of severe osteogenesis imperfecta has not been identified; synthesis of type I collagen and alpha-1: alpha-2 chain ratios appears to be normal. Production of an alpha-1 trimer may represent one such lesion in severe disease.

Bone Diseases, Developmental↗

PubMed1999

The leucine-based sorting motifs in the cytoplasmic domain of the invariant chain are recognized by the clathrin adaptors AP1 and AP2 and their medium chains.

Recognition of sorting signals within the cytoplasmic tail of membrane proteins by adaptor protein complexes is a crucial step in membrane protein sorting. The three known adaptor complexes, AP1, AP2, and AP3, have all been shown to recognize tyrosine- and leucine-based sorting signals, which are the most common sorting signals within membrane protein cytoplasmic tails. Although tyrosine-based signals are recognized by the micro-chains of adaptor complexes, the subunit recognizing leucine-based sorting signals is less clear. In this report we show by surface plasmon resonance that the two leucine-based sorting signals within the cytoplasmic tail of the invariant chain bind independently from each other to AP1 and AP2 but not to AP3. We also show that both motifs can be recognized by the micro-chains of AP1 and AP2. Moreover, by using monomeric as well as trimeric invariant chain constructs, we show that adaptor binding does not require trimerization of the invariant chain.

Adaptor Protein Complex 1↗

PubMed2004

Role of oligomerization domains in thrombospondins and other extracellular matrix proteins.

Coiled coils, collagen triple helices and globular oligomerization domains mediate the subunit assembly of many proteins in vertebrates and invertebrates. Oligomerization offers functional advantages including multivalency, increased binding strength and the combined function of different domains. These features are seen in natural proteins and may be introduced by protein engineering. The special focus of this review is on oligomerization domain of extracellular matrix proteins. For thrombospondins, initial interesting results on the functional role of oligomerization have been published. Other features remain to be explored. For example, it is not clear why thrombospondin-1 and thrombospondin-2 are trimers whereas thrombospondins-3 to -5 are pentamers. To stimulate this type of research, this review makes a survey of oligomerization domains and their functional role in extracellular matrix proteins.

Amino Acid Sequence↗

PubMed2003

Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix.

A ubiquitous feature of collagens is protein interaction, the trimerization of monomers to form a triple helix followed by higher order interactions during the formation of the mature extracellular matrix. The Caenorhabditis elegans cuticle is a complex extracellular matrix consisting predominantly of cuticle collagens, which are encoded by a family of approximately 154 genes. We identify two discrete interacting sets of collagens and show that they form functionally distinct matrix substructures. We show that mutation in or RNA-mediated interference of a gene encoding a collagen belonging to one interacting set affects the assembly of other members of that set, but not those belonging to the other set. During cuticle synthesis, the collagen genes are expressed in a distinct temporal series, which we hypothesize exists to facilitate partner finding and the formation of appropriate interactions between encoded collagens. Consistent with this hypothesis, we find for the two identified interacting sets that the individual members of each set are temporally coexpressed, whereas the two sets are expressed approximately 2 h apart during matrix synthesis.

Animals↗

PubMed2006

Stepwise disassembly and apparent nonstepwise reassembly for the oligomeric RbsD protein.

Many cellular proteins exist as homo-oligomers. The mechanism of the assembly process of such proteins is still poorly understood. We have previously observed that Hsp16.3, a protein exhibiting chaperone-like activity, undergoes stepwise disassembly and nonstepwise reassembly. Here, the disassembly and reassembly of a nonchaperone protein RbsD, from Escherichia coli, was studied in vitro. The protein was found to mainly exist as decamers with a small portion of apparently larger oligomeric forms, both of which are able to refold/reassemble effectively in a spontaneous way after being completely unfolded. Disassembly RbsD intermediates including pentamers, tetramers, trimers, dimers, and monomers were detected by using urea-containing pore gradient polyacrylamide gel electrophoresis, while only pentamers were detected for its reassembly. The observation of stepwise disassembly and apparent nonstepwise reassembly for both a chaperone protein (Hsp16.3) and a nonchaperone protein (RbsD) strongly suggests that such a feature is most likely general for homo-oligomeric proteins.

Bacterial Proteins↗

PubMed2005

Autotrophic cells of the Synechocystis psbH deletion mutant are deficient in synthesis of CP47 and accumulate inactive PS II core complexes.

Cells of the psbH deletion mutant IC7 of the cyanobacterium Synechocystis PCC 6803 grown in the absence of glucose contain strongly reduced levels of chlorophyll when compared with cells grown in the presence of glucose, or compared with wild-type (WT) cells. Low-temperature fluorescence emission spectra revealed decreased content of both active PS II (Photosystem II) and PS I (Photosystem I) complexes. Analysis of thylakoid membrane complexes of IC7 by native electrophoresis showed a similar set of chlorophyll-proteins, namely a PS II core complex and trimeric and monomeric PS II complexes, as in WT. However, in contrast to WT, the (35)S-methionine protein labeling pattern of the mutant exhibited no preferential labeling of the D1 protein in the PS II core complexes, and the labeled D1 and D2 proteins accumulated predominantly in the PS II reaction center lacking CP47. The results show that in autotrophically grown cells of the psbH deletion mutant, selective D1 turnover is inhibited and synthesis of CP47 becomes a limiting step in the PS II assembly.

Gene Deletion↗

PubMed1998

Structure of bacteriophage T4 fibritin M: a troublesome packing arrangement.

Fibritin, a 52 kDa product of bacteriophage T4 gene wac, forms 530 A long fibers, named whiskers, that attach to the phage neck and perform a helper function during phage assembly. Fibritin is a homotrimer, with its predominant central domain consisting of 12 consecutive alpha-helical coiled-coil segments linked together by loops. The central domain is flanked by small globular domains at both ends. Fibritin M is a genetically engineered fragment of the wild type and contains 74 amino-acid residues corresponding to the last coiled-coil segment and the complete carboxy-terminal domain. The crystals of fibritin M belong to the rare space group P3 with three crystallographically independent trimers in the unit cell. The structure has been established at 1.85 A resolution by combining molecular and isomorphous replacement techniques. One of the two heavy-atom derivatives used was gaseous xenon. A substantial fraction of residues in each independent trimer is disordered to various extents in proportion to the lack of restraints on the molecules provided by the lattice contacts. Accurate modeling of the solvent present in the crystals was crucial for achieving good agreement with experimental data.

Amino Acid Sequence↗

PubMed2004

Hydrogen bonding interactions in E- or Z-2-phenyl-3-( X'-pyridyl)propenoic acid ( X=2, 3 or 4) assemblies--a molecular modeling study.

The agglomeration properties of stereoisomeric 2-phenyl-3-(X'-pyridyl)propenoic acids (X=2, 3 or 4) were studied by the PM3 semiempirical quantum chemical method. Calculations revealed that dimers kept together by the intermolecular hydrogen bonding interactions of the carboxylic groups could be built from both stereoisomers irrespective of the position of the nitrogen heteroatom. The dimers of the Z-isomers could also be built through (aromatic)C-H...N hydrogen bonds between the dimer units. The longest agglomerate was the pentamer of the dimers when the nitrogen was in the 2' position. Longer hydrogen-bonded agglomerates than dimers could only be constructed from the E-isomer with the nitrogen in position 4'. Here, the trimer of the dimers proved to be the longest hydrogen-bonded entity and similarly to the Z-isomers, the dimer units are kept together by (aromatic)C-H...N intermolecular hydrogen bonds.

Acrylates↗

PubMed2005

Biochemical and functional evidence for heteromeric assembly of P2X1 and P2X4 subunits.

P2X receptors are ligand-gated ion channels activated by extracellular ATP. In expression systems, P2X subunits form homo- and heterotrimeric receptors. Heteromerization is also likely to occur in vivo as (i) most P2X subunits show overlapping distribution in different tissues and (ii) the functional properties of many native P2X receptors differ from those of heterologously expressed homomeric receptors. Here, we used the Xenopus laevis oocyte expression system to test for heteromerization of P2X1 and P2X4 subunits. Upon co-injection, P2X4 subunits were co-purified with hexahistidyl-tagged P2X1 subunits indicating heteromerization. Blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis of these P2X complexes excluded artificial aggregation and confirmed that both subunits were present in trimeric complexes of the same size. Two-electrode voltage-clamp experiments revealed functional P2X receptors with kinetic properties resembling homomeric P2X4 receptors and a pharmacological profile similar to homomeric P2X1 receptors. Thus, application of alpha,beta-methylene ATP evoked a slowly desensitizing current sensitive to the antagonists suramin and 2',3'-O-(2,4,6-trinitrophenyl)-ATP. This study provides for the first time biochemical and functional evidence for the formation of heteromeric P2X(1+4) receptors. These receptors may account for native P2X mediated responses that until now could not be correlated with previously described recombinant P2X receptors.

Adenosine Triphosphate↗

PubMed1995

Characterization of leucine zipper complexes by electrospray ionization mass spectrometry.

The development of "soft" ionization methods has enabled the mass spectrometric analysis of higher-order structural features of proteins. We have applied electrospray ionization mass spectrometry (ESI-MS) to the analysis of the number and composition of polypeptide chains in homomeric and heteromeric leucine zippers. In comparison with other methods that have been used to analyze leucine zippers, such as analytical ultracentrifugation, gel chromatography, or electrophoretic band shift assays, ESI-MS is very fast and highly sensitive and provides a straightforward way to distinguish between homomeric and heteromeric coiled-coil structures. ESI-MS analyses were carried out on the parallel dimeric leucine zipper domain GCN4-p1 of the yeast transcription factor GCN4 and on three synthetic peptides with the sequences Ac-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide: peptide LZ (X1, X2 = Leu), peptide LZ(12A) (X1 = Ala, X2 = Leu), and peptide LZ(16N) (X1 = Leu, X2 = Asn). Equilibrium ultracentrifugation analysis showed that LZ forms a trimeric coiled coil and this could be confirmed unequivocally by ESI-MS as could the dimeric nature of GCN4-p1. The formation of heteromeric two- and three-stranded leucine zippers composed of chains from LZ and LZ(12A), or from GCN4-p1 and LZ, was demonstrated by ESI-MS and confirmed by fluorescence quenching experiments on fluorescein-labeled peptides. The results illustrate the adaptability and flexibility of the leucine zipper motif, properties that could be useful to the design of specific protein assemblies by way of coiled-coil domains.

Amino Acid Sequence↗

PubMed1997

Monoclonal antibodies that distinguish between free and complexed heterotrimeric G protein beta subunits.

Heterotrimeric G proteins were purified from bovine brain by immunoaffinity chromatography on immobilized anti G protein monoclonal antibody 3C2. Release of betagamma subunits was effectuated by exposure of immobilized trimeric G proteins to MgAlF4. The resultant betagamma subunits were pure and biologically active. Following immunization of mice with purified betagamma subunits we obtained monoclonal anti beta antibodies showing broad species cross-reactivity. Characterization of the epitope recognized by one such monoclonal antibody, ARC9, indicated involvement of the extreme COOH-terminus, as assessed by its reactivity on beta subunits lacking the COOH-terminal 15 residues, obtained by in vitro translation. Although we used native betagamma subunits as immunogen, all monoclonal antibodies obtained failed to recognize assembled betagamma subunits, and were specific for free beta subunits. This property is useful in characterizing the assembly of G proteins from their subunits in living cells.

Amino Acid Sequence↗

PubMed2025

CCDC22 mutations that impair COMMD binding cause attenuated 3C/Ritscher-Schinzel syndrome.

The CCC complex, composed of CCDC22, CCDC93, and ten proteins of the COMMD family, coordinates several critical steps required to recycle internalized plasma membrane proteins from endosomes to the cell surface. CCC interacts with Retriever, a trimeric cargo recognition complex comprising VPS35L, VPS26C, and VPS29, and works closely with the WASH complex, a crucial regulator of branched actin polymerization at endosomal membranes. Mutations in genes encoding subunits of these three complexes, CCDC22, VPS35L, and WASHC5, have been linked with a developmental syndrome known as 3 C (cranio-cerebello-cardiac) or Ritscher-Schinzel syndrome. Here, we report a new CCDC22 missense mutation, p.E208K, that results in attenuated 3 C syndrome, without cardiac or neuroanatomical abnormalities. We show that this mutation impairs CCC complex assembly by disrupting a conserved interaction surface required for CCDC22-COMMD4 binding. We also review previously described cases and identify that CCDC22 p.P172R has a similar attenuated phenotype and impairs complex assembly in a similar fashion as p.E208K. The characterization of these mutations adds to our understanding of the clinical and molecular spectrum of these disorders.

Humans↗

PubMed2006

The early events of alpha-synuclein oligomerization revealed by photo-induced cross-linking.

Assembly of alpha-synuclein (alpha-Syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of Parkinson's disease. Studying the early events of alpha-Syn aggregation, such as oligomerization and nucleation, is indispensable to understanding the complicated process. Here, photo-induced cross-linking of unmodified proteins (PICUP) technique is applied to elucidate the early-stage oligomerization of alpha-Syn. Results show that alpha-Syn in solution exhibits a mixture of various species, including at least monomers, dimers and trimers. Aggregation of alpha-Syn probably originates from the dimeric and trimeric seeds. Furthermore, the N-terminal amphipathic region is proposed to be required for the oligomerization (dimerization and trimerization) process. This observation may extend our knowledge on the early events of alpha-Syn aggregation and the neurotoxic aggregation species.

Cross-Linking Reagents↗