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PubMed2006

Molecular recognition in the assembly of collagens: terminal noncollagenous domains are key recognition modules in the formation of triple helical protomers.

The alpha-chains of the collagen superfamily are encoded with information that specifies self-assembly into fibrils, microfibrils, and networks that have diverse functions in the extracellular matrix. A key self-organizing step, common to all collagen types, is trimerization that selects, binds, and registers cognate alpha-chains for assembly of triple helical protomers that subsequently oligomerize into specific suprastructures. In this article, we review recent findings on the mechanism of chain selection and infer that terminal noncollagenous domains function as recognition modules in trimerization and are therefore key determinants of specificity in the assembly of suprastructures. This mechanism is also illustrated with computer-generated animations.

Biopolymers↗
PubMed1993

Folding and assembly of phage P22 tailspike endorhamnosidase lacking the N-terminal, head-binding domain.

Tryptic digestion of a thermal unfolding intermediate of the phage P22 tailspike endorhamnosidase produces an N-terminally shortened protein fragment comprising amino-acid residues 108-666 [Chen, B.-L. & King, J. (1991) Biochemistry 30, 6260-6269]. In the present work, the 60-kDa C-terminal fragment was purified to homogeneity from the tryptic digest by gel-fitration chromatography. As in the case for the whole tailspike protein (72 kDa), the purified fragment was found to remain stably folded as a highly soluble, SDS-resistant, enzymatically active trimer. However, its unfolding in the presence of guanidinium chloride was accelerated at least 10-fold compared to the complete, native tailspike protein. Shortened tailspike trimers reconstituted spontaneously and with high yield after diluting a solution containing acid-urea-unfolded fragment polypeptides with neutral buffer. Upon recombinant expression of the 60-kDa polypeptide in Escherichia coli, it also assembled efficiently and formed SDS-resistant trimers. The refolding and assembly pathway of the N-terminally shortened tailspike paralleled that of the complete protein with slightly, but significantly, accelerated folding reactions, at both the subunit and the trimer levels. As found for the complete tailspike protein, yields of refolding and assembly of the 60-kDa fragments into SDS-resistant trimers decreased with increasing temperature. The refolding yield of fragments derived from a temperature-sensitive mutant (Gly244-->Arg) tailspike protein was affected in similar fashion as shown for the whole protein. We conclude that the N-terminal domain (residues 1-107) is dispensable for folding and assembly of the P22 tailspike endorhamnosidase both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1996

Mechanism of laminin chain assembly into a triple-stranded coiled-coil structure.

Laminin, a basement membrane glycoprotein, is a heterotrimer with alpha, beta, and gamma chains held together by a triple-stranded alpha-helical coiled-coil structure. Recently, a short peptide sequence at the C-terminus of the alpha-helical domain of each chain was identified as a critical site for the initiation of laminin chain assembly. Synthetic peptides, B1 and B2 (51-mers from the mouse laminin beta 1 and gamma 1 chains respectively) and M (55-mer from the laminin alpha 2 chain), containing these sites were able to assemble into a triple-stranded coiled-coil structure with chain-specific interactions [Nomizu, M., Otaka, A., Utani, A., Roller, P. P., & Yamada, Y. (1994) J. Biol. Chem. 269, 30386-30392]. Here we focus on the mechanism of laminin assembly and examine the conformation and stability of the peptides under various conditions using circular dichroism (CD) spectroscopy. Dependence on chain length for the conformation and stability of trimers suggests that 51-mers for laminin beta 1 and gamma 1 chains and a 55-mer for the laminin alpha 2 chain are critical to attain high thermal stability (T m = 62 degrees C), similar to the larger fragments (approximately 200-mers) and to intact laminins. Since the conformation and stability are dependent on pH and the B1 and B2 monomers and the B1-B2 dimer conformations are partially destroyed at neutral pH, it is likely that they contain intra- and/or interchain repulsions by acidic residues. Moreover, the B1-B2 dimer was significantly more stable under acidic conditions, while the B1-B2/M trimer appears to dissociate into separate B1-B2 and M peptides at pH 2. Urea-induced denaturation showed that the B1-B2/M was more stable than the B1-B2, while both complexes showed virtually identical guanidine hydrochloride denaturation curves. Our data indicate that ionic interactions between B1-B2 and M are critical for the specific trimer formation. We propose a mechanism for laminin assembly: (1) A heterodimer B1-B2 is preferentially formed and creates an acidic pocket which provides a less stable structure due to intra- and intermolecular repulsions between acidic amino acids. (2) A basic site in the M peptide interacts specifically with the acidic pocket of the B1-B2 dimer and results in assembly into a more stable triple-stranded coiled-coil structure.

Amino Acid Sequence↗
PubMed2000

Self-assembly and supramolecular organization of EMILIN.

The primary structure of human Elastin microfibril interface-located protein (EMILIN), an elastic fiber-associated glycoprotein, consists of a globular C1q domain (gC1q) at the C terminus, a short collagenous stalk, a long region with a high potential for forming coiled-coil alpha helices, and a cysteine-rich N-terminal sequence. It is not known whether the EMILIN gC1q domain is involved in the assembly process and in the supramolecular organization as shown for the similar domain of collagen X. By employing the yeast two-hybrid system the EMILIN gC1q domains interacted with themselves, proving for the first time that this interaction occurs in vivo. The gC1q domain formed oligomers running as trimers in native gels that were less stable than the comparable trimers of the collagen X gC1q domain since they did not withstand heating. The collagenous domain was trypsin-resistant and migrated at a size corresponding to a triple helix under native conditions. In reducing agarose gels, EMILIN also migrated as a trimer, whereas under non-reducing conditions it formed polymers of many millions of daltons. A truncated fragment lacking gC1q and collagenous domains assembled to a much lesser extent, thus deducing that the C-terminal domain(s) are essential for the formation of trimers that finally assemble into large EMILIN multimers.

Cell Line↗
PubMed2006

Structure, stability, and biological activity of bacteriophage T4 gene product 9 probed with mutagenesis and monoclonal antibodies.

Gene product (gp) 9 connects the long tail fibers and triggers the structural transition of T4 phage baseplate at the beginning of infection process. Gp9 is a parallel homotrimer with 288 amino acid residues per chain that forms three domains. To investigate the role of the gp9 amino terminus, we have engineered a set of mutants with deletions and random substitutions in this part. The structure of the mutants was probed using monoclonal antibodies that bind to either N-terminal, middle, or C-terminal domains. Deletions of up to 12 N-terminal residues as well as random substitutions of the second, third and fourth residues yielded trimers that failed to incorporate in vitro into the T4 9(-)-particles and were not able to convert them into infectious virions. As detected using monoclonal antibodies, these mutants undergo structural changes in both N-terminal and middle domains. Furthermore, deletion of the first twenty residues caused profound structural changes in all three gp9 domains. In addition, N-terminally truncated proteins and randomized mutants formed SDS-resistant "conformers" due to unwinding of the N-terminal region. Co-expression of the full-length gp9 and the mutant lacking first 20 residues clearly shows the assembly of heterotrimers, suggesting that the gp9 trimerization in vivo occurs post-translationally. Collectively, our data indicate that the aminoterminal sequence of gp9 is important to maintain a competent structure capable of incorporating into the baseplate, and may be also required at intermediate stages of gp9 folding and assembly.

Amino Acid Sequence↗
PubMed1996

Assembly of the Sindbis virus spike protein complex.

The Sindbis virus glycoproteins E1 and E2 are organized into 80 trimers of heterodimers within the virus envelope. Using pulse-chase protocols and chemical crosslinkers, we have found that E1 and E2 precursor, PE2, rapidly assemble into heterodimers and then into trimers of heterodimers after translocation into the endoplasmic reticulum. E1 folds into its mature conformation within the endoplasmic reticulum via at least three intermediates differing in the configurations of their disulfide bonds. PE2 can pair with the second of these E1 folding intermediates. The remaining E1 folding steps, therefore, occur after E1-PE2 multimers begin to form. Quaternary interactions between E1 and PE2 may help guide the folding of E1. While no PE2 folding intermediates have yet been detected, we have found that PE2 transiently enters into large, noncovalent complexes or aggregates with other PE2 molecules and/or with unknown host factors prior to pairing with E1.

Animals↗
PubMed2002

Identification of rotavirus VP6 residues located at the interface with VP2 that are essential for capsid assembly and transcriptase activity.

Rotavirus has a complex triple-layered icosahedral capsid. The external layer consists of VP7 and VP4, the intermediate layer consists of VP6 trimers, and the internal layer consists of VP2. Double-layered particles (DLP) derived from the virus by solubilization of VP4 and VP7 are transcriptionally competent and extrude capped mRNA from their vertices. Analysis of the pseudoatomic model of the VP6 layer, obtained by placing the atomic structure of VP6 into electron microscopy reconstructions of the DLP, has identified the regions of the protein involved in interactions with the internal layer. To study the role of VP6 both in the assembly of DLP and in transcription, 13 site-specific substitution mutations of VP6, targeting the contacts between the two inner layers, were constructed and expressed in the baculovirus system. The effects of these mutations on VP6 expression, trimerization, and formation of macromolecular assemblies were investigated. Using either in vitro reconstituted DLP derived from purified viral cores and recombinant VP6 or in vivo self-assembled virus-like particles resulting from the coexpression of VP2 and VP6 in the baculovirus-Sf9 system (VLP2/6), we have identified the amino acids essential for recovery of transcription or assembly. All VP6 mutants formed stable trimers which, like wild-type VP6, assembled into tubular structures. The ability of VP6 to interact with VP2 was examined by several assays, including electron microscopy, coimmunoprecipitation, purification of VLP2/6, and monitoring of the transcriptase activity of reconstituted DLP. Of the 13 VP6 mutants examined, 3 were unable to assemble with VP2 and 3 others partially assembled. These mutants either did not rescue the transcriptase activity of core particles or did so only marginally. Four mutants as well as the wild-type VP6 assembled and transcribed very well. Three mutants assembled well on cores but, surprisingly, did not rescue the transcriptase activity of reconstituted DLP. Our results indicate that hydrophobic interactions between VP6 and VP2 residues are responsible for the stability of the DLP. They also show that subtle electrostatic interactions between VP6 and the underlying transcriptase machinery can be essential for mRNA synthesis.

Amino Acid Sequence↗
PubMed1989

Identification of the clathrin-binding domain of assembly protein AP-2.

The clathrin binding domain of the assembly protein AP-2 has been identified by proteolytically cleaving AP-2 into 2 discrete moieties, termed light and heavy mero-AP (LM-AP and HM-AP), and testing their ability to bind to clathrin assembled into cage structures or to clathrin trimers immobilized on Sepharose. The smaller product (LM-AP), which contains 20-40-kD fragments of the parent 100-kD polypeptides and which comprises two small appendages in the native AP-2 molecule, did not significantly interact with clathrin under either condition. In contrast, the HM-AP complex, which forms the larger central mass of the native AP-2 structure and contains uncleaved 50-kD and 16-kD polypeptides as well as 60-66-kD fragments of the parent 100-kD polypeptides, retained binding activity for both dissociated and assembled clathrin.

Adaptor Proteins, Vesicular Transport↗
PubMed1988

Monoclonal antibodies localize events in the folding, assembly, and intracellular transport of the influenza virus hemagglutinin glycoprotein.

We used monoclonal antibodies that recognize monomeric and/or trimeric forms of the influenza virus hemagglutinin (HA) to study biosynthesis of this integral membrane protein in influenza virus-infected cells. We find the following: First, the globular head of the HA folds into its mature conformation in the endoplasmic reticulum prior to the assembly of HA monomers into trimers. Second, trimerization begins within 1 to 2 min following synthesis, with a half-time of approximately 5 min. Third, trimerization occurs only after the HA has been transported from the endoplasmic reticulum. Fourth, newly formed trimers are sensitive to acid-induced conformational alterations associated with viral fusion activity.

Animals↗
PubMed2001

Evidence for a mechanism of repression of heat shock factor 1 transcriptional activity by a multichaperone complex.

In the absence of stress, human heat shock factor 1 (hHSF1) is in its unactivated form. hHSF1 polypeptide is in a dynamic heterocomplex with Hsp90 and is incapable of specifically binding DNA. When cells are stressed, heterocomplex assembly is disrupted. Unbound hHSF1 homotrimerizes, acquires DNA binding activity, and concentrates in the nucleus, but remains transcriptionally inactive. A subsequent reaction converts this inactive, trimeric form into the active, hyperphosphorylated transcription factor. Subsequent to the stressful event, hHSF1 is deactivated and eventually returned to its unactivated form. Evidence is presented herein that trimeric hHSF1 has the propensity to dynamically associate with an Hsp90-immunophilin-p23 complex through its regulatory domain. Formation of this heterocomplex results in repression of the transcriptional activity of trimeric hHSF1. Stress-denatured proteins effectively compete with trimeric hHSF1 for Hsp90-immunophilin-p23 complex, counteracting assembly of the heterocomplex and repression of hHSF1 transcriptional activity. This repression mechanism may be required for a proportional transcriptional response to stress. Formation of the heterocomplex may also represent the first step toward returning the hHSF1 to its unactivated form.

Base Sequence↗
PubMed1982

Assembly of adenovirus major capsid protein is mediated by a nonvirion protein.

The assembly of hexon, the major capsid protein of adenovirus, was investigated with the use of conformation-specific monoclonal antibodies. The hexon capsomere is a trimer of three identical monomers folded into a highly conserved and stable structure. The unique nature of this structure is indicated by the lack of common antigenic determinants between the capsomere and either monomeric or denatured hexon. The assembly of the trimer requires the action of a nonvirion protein, the 100K protein. The virus-encoded 100K protein forms a tight complex with hexon polypeptides. This 100K-hexon complex can form on the polyribosomes while hexon is a nascent chain. Exclusion chromatography revealed that the complex has a molecular weight of 800,000. The complex contains only pretrimer hexon; no mature hexon capsomeres can be found bound to 100K. Kinetic analysis of hexon trimerization and hexon-100K binding indicated that trimerization and the release of hexon from the complex occur concomitantly.

Adenoviruses, Human↗
PubMed2005

Proliferating cell nuclear antigen (PCNA) may function as a double homotrimer complex in the mammalian cell.

The diverse function of proliferating cell nuclear antigen (PCNA) may be regulated by interactions with different protein partners. Interestingly, the binding sites for all known PCNA-associating proteins are on the outer surface or the C termini ("front") sides of the PCNA trimer. Using cell extracts and purified human PCNA protein, we show here that two PCNA homotrimers form a back-to-back doublet. Mutation analysis suggests that the Arg-5 and Lys-110 residues on the PCNA back side are the contact points of the two homotrimers in the doublet. Furthermore, short synthetic peptides encompassing either Arg-5 or Lys-110 inhibit double trimer formation. We also found that a PCNA double trimer, but not a homotrimer alone, can simultaneously accommodate chromatin assembly factor-1 and polymerase delta. Together, our data supports a model that chromatin remodeling by chromatin assembly factor-1 (and, possibly, many other cellular activities) are tightly coupled with DNA replication (and repair) through a PCNA double trimer complex.

Animals↗
PubMed2002

The interdigitated beta-helix domain of the P22 tailspike protein acts as a molecular clamp in trimer stabilization.

The P22 tailspike adhesin is an elongated thermostable trimer resistant to protease digestion and to denaturation in sodium dodecyl sulfate. Monomeric, dimeric, and protrimeric folding and assembly intermediates lack this stability and are thermolabile. In the native trimer, three right-handed parallel beta-helices (residues 143-540), pack side-by-side around the three-fold axis. After residue 540, these single chain beta-helices terminate and residues 541-567 of the three polypeptide chains wrap around each other to form a three-stranded interdigitated beta-helix. Three mutants located in this region -- G546D, R563Q, and A575T -- blocked formation of native tailspike trimers, and accumulated soluble forms of the mutant polypeptide chains within cells. The substitutions R563Q and A575T appeared to prevent stable association of partially folded monomers. G546D, in the interdigitated region of the chain, blocked tailspike folding at the transition from the partially-folded protrimer to the native trimer. The protrimer-like species accumulating in the G546D mutant melted out at 42 degrees C and was trypsin and SDS sensitive. The G546D defect was not corrected by introduction of global suppressor mutations, which correct kinetic defects in beta-helix folding. The simplest interpretation of these results is that the very high thermostability (T(m) = 88 degrees C), protease and detergent resistance of the native tailspike acquired in the protrimer-to-trimer transition, depends on the formation of the three-stranded interdigitated region. This interdigitated beta-helix appears to function as a molecular clamp insuring thermostable subunit association in the native trimer.

Bacteriophage P22↗
PubMed2004

Nucleic acid binding properties and intermediates of HCV core protein multimerization in Pichia pastoris.

Little is known about the in vivo assembly pathway or structure of the hepatitis C virus nucleocapsid. In this work the intermediates of HCcAg multimerization in Pichia pastoris cells and the nucleic acid binding properties of structured nucleocapsid-like particles (NLPs) were studied. Extensive cross-linking was observed for HCcAg after glutaraldehyde treatment. Data suggest that HCcAg exists in dimeric forms probably representing P21-P21, P21-P23, and P23-P23 dimers. In addition, the presence of HCcAg species that might represent trimers and multimers was observed. After sucrose equilibrium density gradient purification and nuclease digestion, NLPs were shown to contain both RNA and DNA molecules. Finally, the analysis by electron microscopy indicated that native NLPs were resistant to nuclease treatment. These results indicated that HCcAg assembles through dimers, trimers, and multimers' intermediates into capsids in P. pastoris cells. Assembly of NLPs in its natural environment might confer stability to these particles by adopting a compact structure.

Binding Sites↗
PubMed1994

Three-dimensional structure of membrane-bound annexin V. A correlative electron microscopy-X-ray crystallography study.

We have used electron microscopy to analyse the structure of wild-type human annexin V (recombinant and placental) and of several mutants (single and double point mutants) bound to monolayers composed of DOPS, DOPE, or brain extract (Folch fraction III). On these phospholipids and on DOPS/DOPC (3:1, w/w) protein trimers, as also found in 3-D crystals, assemble to form a hexagonal lattice with a unit vector length of about 18 nm. The resolution obtained in projection is 1.7 to 2.2 nm for wild-type and mutants. There are no significant differences between the annexin V mutants and the wild-type protein at this resolution. All proteins bind as trimers with their convex side harbouring the Ca(2+)-binding sites facing the membrane. A comparison of the 3-D reconstruction of annexin V wild-type with the high resolution crystal structure shows that the domain structure is preserved but the relative orientation of the modules (II/III) and (I/IV) is slightly changed so that the Ca(2+)-binding sites in all four domains (including the recently observed binding site in domain III) become coplanar to the membrane. The thickness of the molecule obtained in the 3-D reconstruction corresponds well with the thickness of the high resolution crystal structure indicative of peripheral binding of annexin V without substantial penetration of the membrane.

Annexin A5↗
PubMed1991

The extracellular hemoglobin of the earthworm, Lumbricus terrestris. Oxygenation properties of isolated chains, trimer, and a reassociated product.

The extracellular hemoglobin of the earthworm Lumbricus terrestris has a two-tiered hexagonal structure that can be dissociated into 1/12 subunits. The Hb contains four major kinds of oxygen-binding chains, a, b, c, and d, of which a-c form a disulfide-linked trimer. Additional non-heme chains are necessary for the assembly of the intact 3800-kDa molecule of approximately 200 subunits. Oxygen equilibria have been measured for chains c and d, the abc trimer, the partially reassembled product of addition of chain d to the trimer, and the intact molecule. The results show that oxygenation of the trimer but not the isolated c or d subunits is modulated by both pH and Ca2+ ions. Cooperativity of oxygen binding by the trimer is low (Hill coefficient approximately 1.3). However, addition of chain d results in a substantial decrease in oxygen affinity and a large increase in cooperativity so that the oxygen equilibrium becomes indistinguishable from that of the intact native molecule at pH 6.8. Light-scattering data show that the smallest observed trimeric abc unit is the dimer (abc)2 at pH 6.8. Analysis of the major sedimentation velocity boundary of the product of the abc unit and chain d in the CO form in the absence of calcium surprisingly can be accounted for entirely in terms of a nondissociating dimer, (abc)2, and chain d. The data for the CO form in the presence of calcium are best fitted in terms of (abc)2.d. Although both subunits c and d also form dimers, oxygen binding by subunit c, but not d, is highly cooperative. These observations, taken together, suggest that the two dimers (abc)2 and d2 are likely to be the major participants in forming the primary functional unit, (abcd)2, which at pH 7.4 is partially dissociated when in the CO form. Subunit d is clearly necessary for the formation of a cooperative unit. The hypothesis that (abcd)2 is a primary functional unit is consistent with a stoichiometry of 2 (abcd)2 units per 1/12 subunit or 24 such units in each molecule of Hb which would contain, in all, 192 heme-containing chains.

Animals↗
PubMed2004

Constitutive expression of pea Lhcb 1-2 in tobacco affects plant development, morphology and photosynthetic capacity.

Lhcb1-2 from pea was constitutively expressed in transgenic tobacco plants and assessed for functional impact. The successful assembly of the encoded proteins into LHCII trimers was confirmed by electrospray tandem mass spectrometry. Constitutive production of LHCb1-2 led to increased number of thylakoid membranes per chloroplast, increased grana stacking, higher chloroplast numbers per palisade cell and increased photosynthetic capacity at low irradiance, both on a chlorophyll and leaf area basis. The transgenic plants also displayed increased cell volume, larger leaves, higher leaf number per plant at flowering, increased biomass and increased seed weight, when grown under low irradiance levels. Under high irradiance, both transgenic and wild type plants displayed similar photosynthetic rates when tested at 25 degrees C; however, the non-photochemical quenching (NPQ) and qE values increased in the transgenic plants. The exposure of transgenic plants to a photoinhibitory treatment (4 degrees C for 4 h, under continuous illumination) resulted in more detrimental impairment of photosynthesis, since recovery was slower than the non-transgenic plants. These data indicate that constitutive expression of additional Lhcb1-2 transgenes led to a series of changes at all levels of the plant (cellular, leaf and whole organism), and a delay in flowering and senescence. The additional production of the pea protein appears to be accommodated by increasing cellular structures such as the number of thylakoids per chloroplast, organelle volume, organelles per cell, and leaf expansion. The presence of the trimeric pea protein in the tobacco LHCII, however, caused a possible change in the organization of the associated super-complex, that in turn limited photosynthesis at low temperature.

Amino Acid Sequence↗
PubMed2006

Genetic targeting of adenovirus vectors using a reovirus sigma1-based attachment protein.

Targeting adenovirus vectors (AdV's) for selective transduction of specific cell types requires ablation of native adenovirus tropism and introduction of a unique target-binding moiety. To bring these requirements within reach, we developed a novel strategy to target AdV's genetically that relies on replacement of the entire adenovirus fiber protein with a fusion molecule comprising the virion-anchoring domain of fiber and the oligomerization domain of reovirus attachment protein sigma1. The chimeric molecule forms trimers, is transported to the nucleus, and assembles onto the adenovirus capsid. In contrast to previously reported genetically targeted vectors, the AdV presented herein propagates efficiently without a requirement for complementing fiber. Due to ablation of the native adenovirus tropism, the infectivity of this AdV was at least 35-fold reduced on 293 cells. Importantly, a His tag incorporated into the chimeric attachment protein conferred His-tag-dependent tropism to the AdV, which resulted in a 12- to 40-fold greater transduction efficiency on two different cell lines expressing a His-tag-binding receptor. In addition, the infection efficiency was strongly reduced by preincubation with a His-tag-specific Ab. Thus, this sigma1-based chimeric attachment molecule provides a promising new platform for the generation of truly targeted AdV's.

Adenoviridae↗