Smooth muscle tropomyosin coiled-coil dimers. Subunit composition, assembly, and end-to-end interaction

Subunits of gizzard smooth muscle tropomyosin, dissociated by guanidinium chloride and reassociated by high salt dialysis, form a 1:1 mixture of the beta beta and gamma gamma homodimers (Graceffa, P. (1989) Biochemistry 28, 1282-1287). The homodimers have now been separated by anion-exchange chromatography and native gel electrophoresis, enabling us to show that the native protein is composed of more than 90% heterodimer. The in vitro equilibrium distribution of heterodimer and homodimers, at close to physiological temperature and ionic conditions, was calculated from thermal unfolding profiles of separated homodimers and heterodimer, as monitored by circular dichroism. The results, for an equal proportion of beta and gamma chains, indicate a predominant formation of heterodimer via chain dissociation and chain exchange, although the proportion of heterodimer was much less than the 90-100% found in the native protein. However, the proportion of heterodimer for actin-bound tropomyosin, determined by analyzing tropomyosin sedimented with actin, was greater than 90%, which may provide a model for assembly in vivo. The end-to-end interactions of the homodimers are about the same but are much less than that of the native heterodimer, as determined by viscometry. The greater end-to-end interaction of heterodimers may lead to stronger binding to actin compared to homodimers and thus would further shift the equilibrium between heterodimer and homodimers toward heterodimer and possibly account for the almost exclusive population of heterodimer in the presence of actin. The greater end-to-end interaction of the heterodimer may also provide a functional advantage for its preferred assembly. This study also shows that the two-step thermal unfolding of the homodimer mixture is due to the formation of heterodimer via an intermediate which is a new type of tropomyosin species which forms a gel in low salt. This tropomyosin is also present in small amounts in native tropomyosin preparations.     

Expression and chain assembly of human laminin-332 in an insect cell-free translation system

Laminins are a family of large heterotrimeric glycoproteins comprising alpha, beta, and gamma chains. To determine the molecular mechanisms underlying chain assembly in vitro, we expressed human laminin-332 subunits in an insect cell-free translation system. We successfully produced the beta3-gamma2 heterodimer and the alpha3-beta3-gamma2 heterotrimer of the laminin coiled-coil (LCC) domain following co-translation of each chain. The alpha3-beta3 and the alpha3-gamma2 heterodimer were not detected, suggesting that the alpha3 chain can assemble with only beta3-gamma2 heterodimer to form a heterotrimer via disulfide bonds. These results are consistent with those of a previous report indicating that laminin chain assembly proceeds through the beta-gamma heterodimer to the alpha-beta-gamma heterotrimer in vivo. We suggest that the cell-free translation system is a valid system with which to study the mechanisms underlying laminin chain assembly.     

Processing of hepatocyte growth factor to the heterodimeric form is required for biological activity.

Hepatocyte growth factor is a plasminogen-like molecule with diverse biological effects. Although it is synthesized as a single chain polypeptide, it was originally purified as a disulfide-linked heterodimer which was generated by an internal proteolytic event. Subsequent work indicated that preparations consisting largely of the monomeric form also exhibited potent activity. By using a combination of protease inhibition and site-directed mutagenesis, we established that conversion of the single chain polypeptide to the heterodimer occurred during the bioassay and was required for mitogenic and motogenic activity.     

A major part of platelet-derived growth factor purified from human platelets is a heterodimer of one A and one B chain

Platelet-derived growth factor, PDGF, purified from human platelets is a disulfide-bonded dimer consisting of two homologous polypeptide chains denoted A and B; it has not been known whether it is a heterodimer or a mixture of homodimers. We present here evidence that a major part of PDGF has a heterodimer structure. A highly homogeneous, 31-kDa PDGF was purified in the presence of protease inhibitors and shown to contain both chains by means of immunoprecipitations with peptide antisera specific for the A and B chains, respectively. The susceptibility of PDGF to mild acid treatment and its chromatographic behavior in reversed-phase high performance liquid chromatography and immobilized metal ion affinity chromatography, as compared to A and B chain homodimers, is consistent with a heterodimer structure. Analysis of PDGF purified according to our routine, large scale procedure revealed the major part to have a heterodimer structure. In addition, B chain homodimers were also found. With the demonstration that a major part of PDGF purified from human platelets occurs as a heterodimer, all three dimeric forms of PDGF have been identified. The following nomenclature to distinguish the various forms is suggested: PDGF-AA, a homodimer of A chains; PDGF-AB, a heterodimer; PDGF-BB, a homodimer of B chains; PDGF, any dimeric form of A or B chains.     

Assembly and dissociation of human leukocyte antigen (HLA)-A2 studied by real-time fluorescence resonance energy transfer

Class I major histocompatibility complex (MHC) heterodimer, composed of human leukocyte antigen (HLA)-A2 heavy chain and human beta(2)-microglobulin (beta(2)m), was produced by denaturation and gel filtration of the recombinant water-soluble HLA-A2/beta(2)m/peptide ternary complex in 8 M urea Tris-HCl buffer, followed by refolding of the separated chains without peptide. Peptide affinity and kinetics of the ternary complex formation and dissociation were investigated in real time by monitoring the fluorescence resonance energy transfer (FRET) from intrinsic HLA-A2 heavy-chain tryptophans to a dansyl fluorophore conjugated to the bound peptide. Peptide binding to the heterodimer was a second order process with rate constants linearly dependent upon temperature in Arrhenius coordinates over 0-20 degrees C. The binding rate constant of pRT6C-dansyl [ILKEPC(dansyl)HGV] at 37 degrees C evaluated by extrapolation of the Arrhenius plot was (2.0 +/- 0.5) x 10(6) M(-1) s(-1). Association of the heavy chain with beta(2)m was a first order process, apparently controlled by a conformational transition in the heavy chain. One of these conformations bound to beta(2)m to form the heavy chain/beta(2)m heterodimer whereas the second conformer oligomerized. Peptide dissociation from the ternary complex was a first-order reaction over the temperature range 20-37 degrees C, suggesting that the ternary complex also exists in two conformations. Taken together, the present data suggest that association of beta(2)m changes the HLA-A2 heavy-chain conformation thereby promoting peptide binding. Peptide dissociation from the ternary complex induces dissociation of the heavy-chain/beta(2)m heterodimer thereby causing oligomerization of the heavy chain. The lability of the HLA-A2/beta(2)m heterodimer and the strong tendency of the "free" heavy chain to oligomerize may provide an efficient mechanism for control of antigen presentation under physiological conditions by reducing the direct loading of HLA with exogenous peptide at the cell surface.     

A novel integrin (alpha E beta 4) from human epithelial cells suggests a fourth family of integrin adhesion receptors.

A new member of the integrin superfamily of adhesion receptors was isolated from human epithelial cells. Analogously to other integrins, this molecule is a heterodimer comprised of structurally unrelated subunits, both glycosylated. Unequivocal amino-acid sequence homologies were observed between these subunits and integrin alpha and beta chain sequences, indicating that this epithelial heterodimer is a novel integrin. No obvious serologic cross-reactivities were detected with other integrins. The beta chain of the epithelial integrin displayed a mol. wt significantly higher than other integrin beta chains, possibly due to a large sialic acid content. Integrin heterodimers are grouped into three families, based on which of three beta chains (beta 1, beta 2 and beta 3) they contain. Therefore, the epithelial integrin may represent the prototype of a fourth integrin family, because it contains a structurally distinct beta chain. The designation alpha E beta 4 is proposed for this novel human integrin.     

Radical formation in the dimeric small subunit of ribonucleotide reductase requires only one tyrosine 122

The small subunit of ribonucleoside-diphosphate reductase (EC 1.17.4.1) is a homodimer. Its catalytic site contains one tyrosyl radical, which is localized to Tyr-122 in one of its polypeptide chains. The engineered Tyr-122----Phe protein was used to demonstrate that it is possible to form a correct ferric iron center in vitro in the absence of Tyr-122. Heterodimers, consisting of one Tyr-122-containing polypeptide chain and one Phe-122-containing polypeptide chain, were constructed. The heterodimer population contained one-half the amount of tyrosyl radical as compared to a homodimer with Tyr-122, i.e. every second heterodimer contains a tyrosyl radical. Thus, one Tyr-122 is sufficient for radical formation. Radical-containing heterodimers are catalytically competent.     

Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity

The expression of immunoglobulin heavy and light chain variable regions in the cytoplasm of Escherichia coli and formation of a functional heterodimer has been demonstrated. Variable domain sequences were taken from the heavy and light chain cDNAs of the monoclonal antibody Gloop 2 and engineered for expression in a dual origin expression vector. The engineered genes vhg2 and vlg2 were separately subcloned into the vector, creating two expression plasmids. Expression of the heavy and light chain variable region genes (encoding 116 and 109 amino acids respectively) was investigated in eight E. coli strains; the polypeptides were rapidly degraded in a host strain optimized for expression and in E. coli strains deficient in the major protease La (lon-). Accumulation was permitted in severely protease-deficient E. coli having a defective heat-shock response. A lon- mutation in this genetic background permitted even higher accumulation. Expression levels were 7 and 1% of total bacterial protein for light and heavy chain variable regions respectively. Expression of the heavy chain variable region gene was increased by including a longer Shine-Dalgarno sequence. Similar constructions in the light chain vector had no effect on expression levels. The insoluble variable region polypeptides were reconstituted into a heterodimer possessing the full antigen binding characteristics of both the parent monoclonal antibody and its Fab fragment.     

Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM

Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-alpha) and 39 kd (Ig-beta), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain.     

An allosteric mechanism controls antigen presentation by the H-2K(b) complex.

The mechanism of assembly/dissociation of a recombinant water-soluble class I major histocompatibility complex (MHC) H-2Kb molecule was studied by a real-time fluorescence resonance energy transfer method. Like the H-2Kd ternary complex [Gakamsky et al. (1996) Biochemistry 35, 14841-14848], the interactions among the heavy chain, beta2-microglobulin (beta2m), and antigenic peptides were found to be controlled by an allosteric mechanism. Association of the heavy chain with beta2m increased peptide binding rate constants by more than 2 orders of magnitude and enhanced affinity of the heavy-chain molecule for peptides. Interaction of peptides with the heavy-chain binding site, in turn, increased markedly the affinity of the heavy chain for beta2m. Binding of peptide variants of the ovalbumin sequence (257-264) to the heavy chain/beta2m heterodimer was found to be a biphasic reaction. The fast phase was a second-order process with nearly the same rate constants as those of binding of peptides derived from the influenza virus nucleoprotein 147-155 to the H-2Kd heavy chain/beta2m heterodimer [(3.0 +/- 1.0) x 10(-6) M-1 s-1 at 37 degrees C]. The slow phase was a result of both the ternary complex assembly from the "free" heavy chain, beta2m, and peptide as well as an intramolecular conformational transition within the heavy chain/beta2m heterodimer to a peptide binding conformation. Biexponential kinetics of peptide or beta2m dissociation from the ternary complex were observed. They suggest that it can exist in two conformations. The rate constants of beta2m dissociation from the H-2Kb ternary complex were, in the limits of experimental accuracy, independent of the structure of the bound peptide, though their affinities differed by an order of magnitude. Dissociation of peptides from the Kb heavy chain was always faster than from the ternary complexes, yet the heavy chain/peptide complexes were considerably more stable compared with their Kd/nucleoprotein peptide counterparts.     

Sequence analyses of Type I and Type II chains in human hair and epithelial keratin intermediate filaments: promiscuous obligate heterodimers, Type II template for molecule formation and a rationale for heterodimer formation

Sequence comparisons have been undertaken for all hair and epithelial keratin IF chains from a single species--human. The results lead to several new proposals. First, it is clear that not only is the chain structure of the molecule an obligate heterodimer but promiscuous association of Type I and Type II chains must occur in vivo. Second, the higher predicted content of alpha-helix in Type II chains in solution relative to that expected for Type I chains suggests that it is the Type II chains that precede their Type I counterparts and that they may serve as templates for molecule formation. Third, heterodimer formation leads naturally to greater structural and functional specificity, and this may be required not only because keratin IF have more interacting partners in its cell type than other types of IF have in theirs but also because hair and skin IF have two distinct structures that relate to the "reducing" or "oxidizing" environment in which they can find themselves. The transition between the two forms may require specific head/tail interactions and this, it is proposed, would be more easily accomplished by a heterodimer structure with its greater in-built specificity.     

Structure of the GMPPNP-stabilized NG domain complex of the SRP GTPases Ffh and FtsY

Ffh and FtsY are GTPase components of the signal recognition particle co-translational targeting complex that assemble during the SRP cycle to form a GTP-dependent and pseudo twofold symmetric heterodimer. Previously the SRP GTPase heterodimer has been stabilized and purified for crystallographic studies using both the non-hydrolysable GTP analog GMPPCP and the pseudo-transition state analog GDP:AlF4, revealing in both cases a buried nucleotide pair that bridges and forms a key element of the heterodimer interface. A complex of Ffh and FtsY from Thermus aquaticus formed in the presence of the analog GMPPNP could not be obtained, however. The origin of this failure was previously unclear, and it was thought to have arisen from either instability of the analog, or, alternatively, from differences in its interactions within the tightly conscribed composite active site chamber of the complex. Using insights gained from the previous structure determinations, we have now determined the structure of the SRP GTPase targeting heterodimer stabilized by the non-hydrolysable GTP analog GMPPNP. The structure demonstrates how the different GTP analogs are accommodated within the active site chamber despite slight differences in the geometry of the phosphate chain. It also reveals a K+ coordination site at the highly conserved DARGG loop at the N/G interdomain interface.     

The internal propeptide of the ricin precursor carries a sequence-specific determinant for vacuolar sorting

Ricin is a heterodimeric toxin that accumulates in the storage vacuoles of castor bean (Ricinus communis) endosperm. Proricin is synthesized as a single polypeptide precursor comprising the catalytic A chain and the Gal-binding B chain joined by a 12-amino acid linker propeptide. Upon arrival in the vacuole, the linker is removed. Here, we replicate these events in transfected tobacco (Nicotiana tabacum) leaf protoplasts. We show that the internal linker propeptide is responsible for vacuolar sorting and is sufficient to redirect the ricin heterodimer to the vacuole when fused to the A or the B chain. This internal peptide can also target two different secretory protein reporters to the vacuole. Moreover, mutation of the isoleucine residue within an NPIR-like motif of the propeptide affects vacuolar sorting in proricin and in the reconstituted A-B heterodimer. This is the first reported example of a sequence-specific vacuolar sorting signal located within an internal propeptide.     

The unfolding story of T cell receptor gamma

Antigen-specific, major histocompatibility complex-restricted recognition by classical T cells is mediated by a T cell receptor (TCR) consisting of a disulfide-linked alpha beta heterodimer. During the search for the genes encoding the alpha and beta proteins, a third immunoglobulin-like gene, termed gamma, was uncovered. Like the TCR alpha and beta genes, the TCR gamma gene consists of variable and constant segments that rearrange during T cell development in the thymus. Although the physiological role of TCR gamma remains an enigma, much has been learned with the recent identification of the protein products of this gene family in both mice and humans. The gamma chain is associated with a partner chain, termed delta. The gamma delta heterodimer is associated with an invariant T3 complex, very similar to that associated with the alpha beta heterodimer, and appears predominantly, if not exclusively, on cells with a CD4-, CD8- phenotype both in the thymus and in the periphery. TCR gamma delta is the first T3-associated receptor to appear during thymocyte development and defines a separate T cell lineage distinct from alpha beta-bearing cells. Although TCR alpha beta-bearing cells and TCR gamma delta-bearing cells follow parallel developmental pathways, the diversity of expressed gamma delta receptors is extremely limited relative to that of alpha beta receptors.     

pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+

Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5-6.75 (Kd approximately 4 nM), whereas a Kd approximately 20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 microM total Cu2+) on maximal activity regenerated was negligible at pH 6.25-8.0. However, this level of Cu2+ increased the inter-chain affinity by approximately 5-fold at pH 7.25. This effect resulted from an approximately 1.5-fold increased association rate constant (k(on)) and an approximately 3-fold reduced dissociation rate constant (k(off)). High affinity (Kd=5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.     

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.     

Dimer formation of octaprenyl-diphosphate synthase (IspB) is essential for chain length determination of ubiquinone

Ubiquinone (Q), composed of a quinone core and an isoprenoid side chain, is a key component of the respiratory chain and is an important antioxidant. In Escherichia coli, the side chain of Q-8 is synthesized by octaprenyl-diphosphate synthase, which is encoded by an essential gene, ispB. To determine how IspB regulates the length of the isoprenoid, we constructed 15 ispB mutants and expressed them in E. coli and Saccharomyces cerevisiae. The Y38A and R321V mutants produced Q-6 and Q-7, and the Y38A/R321V double mutant produced Q-5 and Q-6, indicating that these residues are involved in the determination of chain length. E. coli cells (ispB::cat) harboring an Arg-321 mutant were temperature-sensitive for growth, which indicates that Arg-321 is important for thermostability of IspB. Intriguingly, E. coli cells harboring wild-type ispB and the A79Y mutant produced mainly Q-6, although the activity of the enzyme with the A79Y mutation was completely abolished. When a heterodimer of His-tagged wild-type IspB and glutathione S-transferase-tagged IspB(A79Y) was formed, the enzyme produced a shorter length isoprenoid. These results indicate that although the A79Y mutant is functionally inactive, it can regulate activity upon forming a heterodimer with wild-type IspB, and this dimer formation is important for the determination of the isoprenoid chain length.