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PubMed1998

Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off-pathway aggregation.

Off-pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self-association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off-pathway aggregation of the parallel beta-coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 degrees C prior to warming to 20 degrees C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature-shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5:517-523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off-pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450-455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide-bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly.

Bacteriophage P22↗
PubMed2004

Total chemical synthesis of N-myristoylated HIV-1 matrix protein p17: structural and mechanistic implications of p17 myristoylation.

The HIV-1 matrix protein p17, excised proteolytically from the N terminus of the Gag polyprotein, forms a protective shell attached to the inner surface of the plasma membrane of the virus. During the late stages of the HIV-1 replication cycle, the N-terminally myristoylated p17 domain targets the Gag polyprotein to the host-cell membrane for particle assembly. In the early stages of HIV-1 replication, however, some p17 molecules dissociate from the viral membrane to direct the preintegration complex to the host-cell nucleus. These two opposing targeting functions of p17 require that the protein be capable of reversible membrane interaction. It is postulated that a significant structural change in p17 triggered by proteolytic cleavage of the Gag polyprotein sequesters the N-terminal myristoyl group, resulting in a weaker membrane binding by the matrix protein than the Gag precursor. To test this "myristoyl switch" hypothesis, we obtained highly purified synthetic HIV-1 p17 of 131 amino acid residues and its N-myristoylated form in large quantity. Both forms of p17 were characterized by circular dichroism spectroscopy, protein chemical denaturation, and analytical centrifugal sedimentation. Our results indicate that although N-myristoylation causes no spectroscopically discernible conformational change in p17, it stabilizes the protein by 1 kcal/mol and promotes protein trimerization in solution. These findings support the premise that the myristoyl switch in p17 is triggered not by a structural change associated with proteolysis, but rather by the destabilization of oligomeric structures of membrane-bound p17 in the absence of downstream Gag subdomains.

Cell Membrane↗
PubMed1981

Arrangement of bacteriophage lambda receptor protein (LamB) in the cell surface of Escherichia coli: a reconstitution study.

The LamB protein purified in a solution of sodium dodecyl sulfate was assembled into an ordered hexagonal lattice structure with a lattice constant of about 7.8 nm in the presence of lipopolysaccharide. The LamB alone formed aggregates with some lattice structure. However, the regularity of the lattice was only maintained within a very small area. An ordered hexagonal lattice was also formed when the wild-type lipopolysaccharide was replaced by heptoseless lipopolysaccharide, lipid A, and even fatty acid. However, the lattice constants were appreciably smaller than that with the wild-type lipopolysaccharide. The results suggest that the heptose-containing polysaccharide region, as well as the fatty acid region, are involved in the interaction with the LamB protein. The LamB-lipopolysaccharide lattice was preferably formed on the peptidoglycan layer when the lipoprotein was covalently bound to this layer. These results indicate that the molecular arrangement of the LamB protein in the outer membrane is similar to that of matrix proteins, OmpC and OmpF, which exist as trimers. The ordered hexagonal lattice was active in the receptor function for lambda, resulting in phage adsorption and deoxyribonucleic acid ejection. Thus, this reconstitution system should provide a useful means of studying the mechanism of lambda infection.

Bacterial Outer Membrane Proteins↗
PubMed1998

Complementation of a fibre mutant adenovirus by packaging cell lines stably expressing the adenovirus type 5 fibre protein.

Adenovirus-based gene therapy vectors now in use cannot be targeted to specific cell types in vivo and are immunogenic, properties which limit their clinical utility. Improved vectors lacking the genes for viral structural proteins may overcome these limitations. We have developed cell lines which stably express the adenovirus type 5 (Ad5) fibre protein in its native trimeric form. These cells can complement an Ad5 mutant with a defect in the fibre gene, and are capable of incorporating the Ad5 fibre into particles of a different Ad serotype. As the fibre protein is responsible for the initial binding of virus to cells, packaging cell lines expressing different or modified fibre proteins will be useful in studying the mechanism by which adenovirus infects different cell types.

Adenoviridae↗
PubMed1999

Three-dimensional reconstruction of Lumbricus terrestris hemoglobin at 22 A resolution: intramolecular localization of the globin and linker chains.

A 3D reconstruction of the hemoglobin (Hb) of the earthworm Lumbricus terrestris was carried out by the 3D projection alignment method from electron microscopy images of a frozen-hydrated specimen at 22 A resolution. The results were analyzed by a new approach taking into account the evolution of the 210 densities forming the 3D volume as a function of the threshold of surface representation. The whole oligomer with D6point-group symmetry is comprised of 12 hollow globular substructures (HGS) with local 3-fold symmetry tethered to a complex network of linking subunits (linker complex). The 12 globin subunits of each HGS are distributed around local 3-fold axis in four layers of three subunits. The first layer, the most external, contains monomeric globin chains 2A, 3A, and 5A. The three trimers corresponding to the nine remaining subunits have one subunit in each of the second (2B, 3B, 5B), third (1A, 4A, 6A), and fourth (1B, 4B, 6B) layer. The distances between the centers of the globin chains forming the trimers are in the ranges 20-32 A and 45-52 A. The linker complex is made up of two types of linking units. The first type forms three loops connecting globin chains of the second, third and fourth layers. The average molecular mass (Mm) of these subunits was 25 kDa. The second type forms the central structure, termed hexagonal toroid, and its 12 connections to the HGS. This structure corresponds to a hexamer of a single linking unit with a Mm (31.2 kDa), size and a shape different from those of the HGS loops. A careful study of 3D volume architecture shows that each toroid linking unit is bound to the three loops of a HGS pair located in the upper and lower hexagonal layers, respectively. As shown in a model of architecture, hexagonal bilayered (HBL) Hbs can be built very simply from 144 globin chains and 42 linker chains belonging to two different types. We also propose a simple assembly sequence for the construction of HBL Hbs based on the architecture model.

Animals↗
PubMed2006

Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus.

The critical viral components for packaging DNA, recognizing and binding to host cells, and injecting the condensed DNA into the host are organized at a single vertex of many icosahedral viruses. These component structures do not share icosahedral symmetry and cannot be resolved using a conventional icosahedral averaging method. Here we report the structure of the entire infectious Salmonella bacteriophage epsilon15 (ref. 1) determined from single-particle cryo-electron microscopy, without icosahedral averaging. This structure displays not only the icosahedral shell of 60 hexamers and 11 pentamers, but also the non-icosahedral components at one pentameric vertex. The densities at this vertex can be identified as the 12-subunit portal complex sandwiched between an internal cylindrical core and an external tail hub connecting to six projecting trimeric tailspikes. The viral genome is packed as coaxial coils in at least three outer layers with approximately 90 terminal nucleotides extending through the protein core and the portal complex and poised for injection. The shell protein from icosahedral reconstruction at higher resolution exhibits a similar fold to that of other double-stranded DNA viruses including herpesvirus, suggesting a common ancestor among these diverse viruses. The image reconstruction approach should be applicable to studying other biological nanomachines with components of mixed symmetries.

Bacteriophages↗
PubMed2005

Protein phosphatase 5 is a negative modulator of heat shock factor 1.

The major stress protein transcription factor, heat shock factor (HSF1), is tightly regulated through a multilayered activation-deactivation process involving oligomerization, post-translational modification, and interaction with the heat shock protein (Hsp90)-containing multichaperone complex. Conditions of proteotoxic stress, such as heat shock, trigger reversible assembly of latent HSF1 monomers into DNA-binding homotrimers that bind with high affinity to cognate heat shock elements. Transactivation is a second and independently regulated function of HSF1 that is accompanied by hyperphosphorylation and appears to involve a number of signaling events. Association of HSF1 with Hsp90 chaperone complexes provides additional regulatory complexity, however, not all the co-chaperones have been identified, and the specific molecular interactions throughout the activation/deactivation pathway remain to be determined. Here we demonstrate that protein phosphatase 5 (PP5), a tetratricopeptide domain-containing component of Hsp90-steroid receptor complexes, functions as a negative modulator of HSF1 activity. Physical interactions between PP5 and HSF1-Hsp90 complexes were observed in co-immunoprecipitation and gel mobility supershift experiments. Overexpression of PP5 or activation of endogenous phosphatase activity resulted in diminished HSF1 DNA binding and transcriptional activities, and accelerated recovery. Conversely, microinjection of PP5 antibodies, or inhibition of its phosphatase activity in vivo, significantly delayed trimer disassembly after heat shock. Inhibition of PP5 activity did not activate HSF1 in unstressed cells. These results indicate that PP5 is a negative modulator of HSF1 activity.

Animals↗
PubMed2000

Multiple roles for the twin arginine leader sequence of dimethyl sulfoxide reductase of Escherichia coli.

Dimethyl sulfoxide (Me(2)SO) reductase of Escherichia coli is a terminal electron transport chain enzyme that is expressed under anaerobic growth conditions and is required for anaerobic growth with Me(2)SO as the terminal electron acceptor. The trimeric enzyme is composed of a membrane extrinsic catalytic dimer (DmsAB) and a membrane intrinsic anchor (DmsC). The amino terminus of DmsA has a leader sequence with a twin arginine motif that targets DmsAB to the membrane via a novel Sec-independent mechanism termed MTT for membrane targeting and translocation. We demonstrate that the Met-1 present upstream of the twin arginine motif serves as the correct translational start site. The leader is essential for the expression of DmsA, stability of the DmsAB dimer, and membrane targeting of the reductase holoenzyme. Mutation of arginine 17 to aspartate abolished membrane targeting. The reductase was labile in the leader sequence mutants. These mutants failed to support growth on glycerol-Me(2)SO minimal medium. Replacing the DmsA leader with the TorA leader of trimethylamine N-oxide reductase produced a membrane-bound DmsABC with greatly reduced enzyme activity and inefficient anaerobic respiration indicating that the twin arginine leaders may play specific roles in the assembly of redox enzymes.

Amino Acid Sequence↗
PubMed1997

The number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin.

Calnexin and calreticulin are homologous molecular chaperones that promote proper folding, oligomeric assembly, and quality control of newly synthesized glycoproteins in the endoplasmic reticulum (ER). Both are lectins that bind to substrate glycoproteins that have monoglucosylated N-linked oligosaccharides. Their binding to newly translated influenza virus hemagglutinin (HA), and various mutants thereof, was analyzed in microsomes after in vitro translation and expression in live CHO cells. A large fraction of the HA molecules was found to occur in ternary HA- calnexin-calreticulin complexes. In contrast to calnexin, calreticulin was found to bind primarily to early folding intermediates. Analysis of HA mutants with different numbers and locations of N-linked glycans showed that although the two chaperones share the same carbohydrate specificity, they display distinct binding properties; calreticulin binding depends on the oligosaccharides in the more rapidly folding top/hinge domain of HA whereas calnexin is less discriminating. Calnexin's binding was reduced if the HA was expressed as a soluble anchor-free protein rather than membrane bound. When the co- and posttranslational folding and trimerization of glycosylation mutants was analyzed, it was observed that removal of stem domain glycans caused accelerated folding whereas removal of the top domain glycans (especially the oligosaccharide attached to Asn81) inhibited folding. In summary, the data established that individual N-linked glycans in HA have distinct roles in calnexin/calreticulin binding and in co- and posttranslational folding.

Amino Acid Substitution↗
PubMed1977

Spatial relationships of the proteins of vesicular stomatitis virus: induction of reversible oligomers by cleavable protein cross-linkers and oxidation.

To delineate the proximity and spatial arrangement of the major structural proteins of intact vesicular stomatitis (VS) virions, protein complexes formed by oxidation or by bivalent cross-linkers were analyzed by two-dimensional electrophoresis on polyacrylamide slab gels. H2O2 oxidation of VS virions produced an N-polypeptide dimer (molecular weight, approximately equal to 110,000) on a first dimension gel that could be reduced to N monomers (molecular weight, approximately equal to 50,000). Proteins extracted from unreduced and unoxidized VS virions contained dimeric and trimeric forms of M-protein complexes as well as a heterodimer of M and N protein. Qualitatively similar VS viral protein complexes were generated by exposing VS virions to the reversible protein cross-linkers methyl-4-mercaptobutyrimidate (MMB), tartryl diazide (TDA), and dithiobis(succinimidyl proprionate) (DTBSP); cross-linked complexes on first-dimension gels were cleaved by reduction with 2-mercaptoethanol (MMB or DTBSP cross-linked) or by periodate oxidation (TDA cross-linked). In addition to covalently linked homodiamers of M and N proteins and a protein M-N heterodimer, the protein cross-linkers also generated homo-oligomers of G protein and a G-M heterodimer. These data suggest that the glycoprotein spike of VS virus is composed of more than one G protein. The existence of N-M and G-M heterodimers is consistent with the hypothesis that the matrix (M) protein may serve as a bridge between the G and N proteins in assembly of the VS virion.

Cell Line↗
PubMed2025

Modeling Alternative Conformational States in CASP16.

The CASP16 Ensemble Prediction experiment assessed advances in methods for modeling proteins, nucleic acids, and their complexes in multiple conformational states. Targets included systems with experimental structures determined in two or three states, evaluated by direct comparison to experimental coordinates, as well as domain-linker-domain (D-L-D) targets assessed against statistical models from NMR and SAXS data. This paper focuses on the former class of multi-state targets. Ten ensembles were released as community challenges, including ligand-induced conformational changes, protein-DNA complexes, a trimeric protein, a stem-loop RNA, and multiple oligomeric states of a single RNA. For five targets, some groups produced reasonably accurate models of both reference states (best TM-score >0.75). However, with the exception of one protein-ligand complex (T1214), where an apo structure was available as a template, predictors generally failed to capture key structural details distinguishing the states. Overall, accuracy was significantly lower than for single-state targets in other CASP experiments. The most successful approaches generated multiple AlphaFold2 models using enhanced multiple sequence alignments and sampling protocols, followed by model quality based selection. While the AlphaFold3 server performed well on several targets, individual groups outperformed it in specific cases. By contrast, predictions for one protein-DNA complex, three RNA targets, and multiple oligomeric RNA states consistently fell short (TM-score <0.75). These results highlight both progress and persistent challenges in multi-state prediction. Despite recent advances, accurate modeling of conformational ensembles, particularly RNA and large multimeric assemblies, remains a critical frontier for structural biology.

AlphaFold2↗
PubMed2001

Interaction of Proteus mirabilis urease apoenzyme and accessory proteins identified with yeast two-hybrid technology.

Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)(3). To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.

Antibodies, Bacterial↗
PubMed1994

The role of cysteine residues in the folding and association of the COOH-terminal propeptide of types I and III procollagen.

Procollagen chains assemble in a type-specific manner forming either homo- or heterotrimers. The molecular mechanisms underlying procollagen chain selectivity are unknown, although it is thought that the C-propeptide (COOH-terminal propeptide) is responsible for directing chain recognition and assembly. To define the processes involved in chain selection we reconstituted the initial stages of procollagen folding and assembly in a cell-free system. Using human pro-alpha 1(III) and pro-alpha 2(I) chains as prototypes of chains that are either capable or incapable of forming homotrimeric molecules, respectively, we constructed two minigenes (p alpha 1(III) delta 1 and p alpha 2(I) delta 1) that lacked most of the triple helical domains. The minigenes were transcribed in vitro and translated in a rabbit reticulocyte lysate supplemented with microsomal membranes under conditions that favored disulfide bond formation. Both pro-alpha 1(III) delta 1 and pro-alpha 2(I) delta 1 chains formed intrachain disulfide bonds within the C-propeptide. However, only pro-alpha 1(III) delta 1 chains were able to self-associate forming homotrimers stabilized by interchain disulfide bonds. The C-propeptide of the pro-alpha 1(III) chain contains 8 cysteine residues (Cys-1-8). We used a site-directed mutagenesis to investigate the role of specific cysteine residues in trimer formation and found that substitution of serine for Cys-1, Cys-2, Cys-3, and Cys-4 prevented interchain disulfide bonding and trimerization. Furthermore, mutations in Cys-1 and Cys-4 also prevented intrachain disulfide bond formation within the C-propeptide. The C-propeptide of the pro-alpha 2(I) chain contains only 7 cysteine residues, lacking cysteine at position 2. Substitution of the existing Ser residue with Cys did not produce a homotrimeric phenotype, indicating that additional recognition signals are required to determine chain selection.

Animals↗
PubMed1996

Collagen-based structures containing the peptoid residue N-isobutylglycine (Nleu): synthesis and biophysical studies of Gly-Pro-Nleu sequences by circular dichroism, ultraviolet absorbance, and optical rotation.

A peptoid residue N-isobutylglycine (Nleu) was introduced as a proline surrogate in collagen-like triple helical structures. A series of single chain and template-assembled collagen-based peptide-peptoid structures composed of Gly-Pro-Nleu sequences were prepared by solid-phase segment condensation methods. Both a synthetic route in solution and a solid phase method were employed to couple the KTA (cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid, also known as the Kemp triacid) based template, KTA-(Gly-OH)3, to peptide-peptoid chains. Biophysical studies using CD, uv absorbance, and optical rotation measurements demonstrated that these compounds form triple-helical structures when the chains are longer than critical lengths. Results from melting curve measurements indicated that the Gly-Pro-Nleu sequence is comparable to the Gly-Pro-Pro sequence in stabilizing a triple-helical conformation. The KTA-based template stabilized triple-helical structures as can be seen by the increased melting temperatures as compared to equivalent single chain molecules. In addition, the template reduced the minimum chain length necessary to form a triple helix from six to only three trimer repeats.

Biophysical Phenomena↗
PubMed2002

Assembly of photosystem I. I. Inactivation of the rubA gene encoding a membrane-associated rubredoxin in the cyanobacterium Synechococcus sp. PCC 7002 causes a loss of photosystem I activity.

A 4.4-kb HindIII fragment, encoding an unusual rubredoxin (denoted RubA), a homolog of the Synechocystis sp. PCC 6803 gene slr2034 and Arabidopsis thaliana HCF136, and the psbEFLJ operon, was cloned from the cyanobacterium Synechococcus sp. PCC 7002. Inactivation of the slr2034 homolog produced a mutant with no detectable phenotype and wild-type photosystem (PS) II levels. Inactivation of the rubA gene of Synechococcus sp. PCC 7002 produced a mutant unable to grow photoautotrophically. RubA and PS I electron transport activity were completely absent in the mutant, although PS II activity was approximately 80% of the wild-type level. RubA contains a domain of approximately 50 amino acids with very high similarity to the rubredoxins of anaerobic bacteria and archaea, but it also contains a region of about 50 amino acids that is predicted to form a flexible hinge and a transmembrane alpha-helix at its C terminus. Overproduction of the water-soluble rubredoxin domain in Escherichia coli led to a product with the absorption and EPR spectra of typical rubredoxins. RubA was present in thylakoid but not plasma membranes of cyanobacteria and in chloroplast thylakoids isolated from spinach and Chlamydomonas reinhardtii. Fractionation studies suggest that RubA might transiently associate with PS I monomers, but no evidence for an association with PS I trimers or PS II was observed. PS I levels were significantly lower than in the wild type ( approximately 40%), but trimeric PS I complexes could be isolated from the rubA mutant. These PS I complexes completely lacked the stromal subunits PsaC, PsaD, and PsaE but contained all membrane-intrinsic subunits. The three missing proteins could be detected immunologically in whole cells, but their levels were greatly reduced, and degradation products were also detected. Our results indicate that RubA plays a specific role in the biogenesis of PS I.

Amino Acid Sequence↗
PubMed1999

Probing activation of the prokaryotic arginine transcriptional regulator using chimeric proteins.

The major transcription factors controlling arginine metabolism in Escherichia coli and Bacillus subtilis, ArgR and AhrC, respectively, are homologous multimeric proteins that form l -arginine-dependent DNA-binding complexes capable of repressing transcription of the biosynthetic genes (both), activating transcription of catabolic genes (AhrC only) or facilitating plasmid dimer resolution (both). Multimerisation and l -arginine binding are associated with the C-terminal 70-80 residues; the N-terminal regions contain a winged helix-turn-helix DNA-binding domain. We have constructed chimeric genes in which the sequences for the N and C-terminal domains have been swapped. The resultant chimeric proteins and their corresponding native proteins have been analysed for their ability to multimerise and bind DNA operator sites in an L-arginine-dependent fashion. Gel filtration and equilibrium sedimentation analysis are consistent with the formation of hexamers by all four proteins in the presence of L-arginine and at high protein concentrations (>100 nM monomer). The hexamer sedimentation coefficients suggest that there is a reduction in molecular volume upon binding L-arginine, consistent with a conformational change accompanying an allosteric activation of DNA-binding. In the absence of L-arginine or at lower protein concentrations, the hexamers are clearly in rapid equilibrium with smaller subunits, whose dominant species appear to be based on trimers, as expected from the crystal structure of the ArgR C-terminal fragment, with the exception of the ArgR-C chimera, which apparently dissociates into dimers, suggesting that in the intact protein the DNA-binding domains may have a significant dimeric interaction. The hexamer-trimer Kdis in the micromolar range, suggesting that trimers are the principal species at in vivo concentrations.DNA binding by all four proteins has been probed by gel retardation and DNase I footprinting analysis using all three types of naturally occurring operators: biosynthetic sites encompassing two 18 bp ARG boxes separated by 2 bp; biosynthetic sites containing two such boxes and a third 18 bp ARG box at a distance of 100 bp downstream, i.e. within the structural gene; and finally a catabolic operator which contains a single ARG box site. The data show that all four proteins bind to the operators at the expected regions in an L-arginine-dependent fashion. From the apparent affinities of the chimeras for each target site, there is no obvious sequence-specificity associated with the N-terminal domains; rather the data can be interpreted in terms of differential allosteric activation, including DNA binding in the absence of L-arginine.Remarkably, the proteins show apparent "anti-competition" in the presence of excess, specific DNA fragments in gel retardation. This appears to be due to assembly of an activated form of the protein, probably hexamers, on the operator DNA. The data are discussed in terms of the current models for the mode of action of both native proteins.

Amino Acid Sequence↗
PubMed1997

Primary structure and expression of matrilin-2, the closest relative of cartilage matrix protein within the von Willebrand factor type A-like module superfamily.

A mouse cDNA encoding a novel member of the von Willebrand factor type A-like module superfamily was cloned. The protein precursor of 956 amino acids consists of a putative signal peptide, two von Willebrand factor type A-like domains connected by 10 epidermal growth factor-like modules, a potential oligomerization domain, and a unique segment, and it contains potential N-glycosylation sites. A sequence similarity search indicated the closest relation to the trimeric cartilage matrix protein (CMP). Since they constitute a novel protein family, we introduce the term matrilin-2 for the new protein, reserving matrilin-1 as an alternative name for CMP. A 3. 9-kilobase matrilin-2 mRNA was detected in a variety of mouse organs, including calvaria, uterus, heart, and brain, as well as fibroblast and osteoblast cell lines. Expressed human and rat cDNA sequence tags indicate a high degree of interspecies conservation. A group of 120-150-kDa bands was, after reduction, recognized specifically with an antiserum against the matrilin-2-glutathione S-transferase fusion protein in media of the matrilin-2-expressing cell lines. Assuming glycosylation, this agrees well with the predicted minimum Mr of the mature protein (104,300). Immunolocalization of matrilin-2 in developing skeletal elements showed reactivity in the perichondrium and the osteoblast layer of trabecular bone. CMP binds both collagen fibrils and aggrecan, and because of the similar structure and complementary expression pattern, matrilin-2 is likely to perform similar functions in the extracellular matrix assembly of other tissues.

Amino Acid Sequence↗
PubMed2001

Differential roles of two conserved glycine residues in the fusion peptide of Semliki Forest virus.

Semliki Forest Virus (SFV) is an enveloped alphavirus that infects cells by a low-pH-dependent membrane fusion reaction. SFV fusion is catalyzed by the spike protein E1 subunit, which contains a putative fusion peptide between residues 79 and 97. Prior mutagenesis studies demonstrated that an E1 G91D mutation blocks both virus-membrane fusion and the formation of a highly stable E1 trimer believed to be a critical fusion intermediate. We have here demonstrated that the G91D mutant was also inactive in hemifusion, suggesting that the E1 homotrimer is important in the initial stages of lipid mixing. Revertant analysis of a G91 deletion mutant indicated that G91 was crucial for the viability of SFV. In contrast, a G83D mutation produced infectious virus with both efficient fusion and homotrimer formation. Thus, the G83 position, although highly conserved among alphaviruses, was functional if replaced with a charged amino acid.

Amino Acid Sequence↗