N-terminal PDZ domain is required for NHERF dimerization

NHERF, a 55 kDa PDZ-containing protein, binds receptors and ion transporters to mediate signal transduction at the plasma membrane. Recombinant NHERF demonstrated an apparent size of 150 kDa on gel filtration, which could be reduced to approximately 55 kDa by protein denaturing agents, consistent with the formation of NHERF dimers. Biosensor studies established the time- and concentration-dependent dimerization of NHERF. Overlays of recombinant NHERF fragments suggested that NHERF dimerization was principally mediated by the N-terminal PDZ-I domain. In PS120 cells, reversible protein phosphorylation modulated NHERF dimerization and suggested a role for NHERF dimers in hormonal signaling.     

Epidermal growth factor receptor dimerization monitored in live cells

We present a method for monitoring receptor dimerization at the membrane of live cells. Chimeric proteins containing the epidermal growth factor (EGF) receptor extracellular and transmembrane domains fused to weakly complementing beta-galactosidase (beta-gal) deletion mutants were expressed in cells in culture. Treatment of the cells with EGF-like compounds for as little as 15 s resulted in chimeric receptor dimerization detectable as beta-gal enzymatic activity. The dose response of chimeric receptors was ligand specific. beta-galactosidase complementation was reversible upon removal of ligand and could be reinduced. Antibodies that block ligand binding inhibited receptor dimerization and beta-gal complementation. These results demonstrate that beta-gal complementation provides a rapid, simple, and sensitive assay for protein interactions and for detecting and monitoring the kinetics of receptor dimerization.     

Dimerization of human growth hormone in the presence of metal ions

Zn(2+) and Co(2+) ions are known to promote human growth hormone reversible dimerization. In these studies, dimerization was also shown to be initiated by nine other metal ions: Cd(2+), Hg(2+), Cu(2+), Ag+, Au(3+), Au+, Pd(2+), Ni(2+), and Pt(4+). In some cases (Hg(2+), Ag(+), Au(3+), and Ni(2+)) formation of higher oligomers also took place. In addition further detailed investigation of dimerization in the presence of Zn(2+) ions was carried out.     

大肠杆菌表达的重组葡激酶-水蛭素融合蛋白的分离纯化及其二聚体分析

采用阴离子交换和凝胶过滤色谱法纯化大肠杆菌高密度培养所表达的重组葡激酶－水蛭素融合蛋白（rSFH）,经SDS-PAGE和RP-HPLC分析纯度达到98％以上,每升发酵液得率约0.7g。同时利用疏水色谱、MALDI-TOF对纯化过程中出现的rSFH同源二聚体的分析和表面疏水面积的计算及利用高效排阻色谱（HPSEC）分析NaCl、温度对rSFH的可逆二聚化行为的影响,认为疏水作用在rSFH的可逆二聚化行为中发挥着重要作用。     

Self organization of membrane proteins via dimerization

Protein-protein dimerization is ubiquitous in biology, but its role in self-organization remains unexplored. Here we use Monte Carlo simulations to demonstrate that under diffusion-limited conditions, reversible dimerization alone can cause membrane proteins to cluster into oligomer-like structures. When multiple distinct protein species are able to form dimers, then heterodimerization and homodimerization can organize proteins into structured clusters that can affect cellular physiology. As an example, we demonstrate how receptor dimerization could provide a physical mechanism for regulating information flow by controlling receptor-receptor cross talk. These results are physically realistic for some membrane proteins, including members of the G-protein coupled receptor family, and may provide a physiological reason as to why many proteins dimerize.     

Dimerization of the 3'UTR of bicoid mRNA involves a two-step mechanism.

The proper localization of bicoid (bcd) mRNA requires cis-acting signals within its 3' untranslated region (UTR) and trans-acting factors such as Staufen. Dimerization of bcd mRNA through intermolecular base-pairing between two complementary loops of domain III of the 3'UTR was proposed to be important for particle formation in the embryo. The participation in the dimerization process of each domain building the 3'UTR was evaluated by thermodynamic and kinetic analysis of various mutated and truncated RNAs. Although sequence complementarity between the two loops of domain III is required for initiating mRNA dimerization, the initial reversible loop-loop complex is converted rapidly into an almost irreversible complex. This conversion involves parts of RNA outside of domain III that promote initial recognition, and dimerization can be inhibited by sense or antisense oligonucleotides only before conversion has proceeded. Injection of the different bcd RNA variants into living Drosophila embryos shows that all elements that inhibit RNA dimerization in vitro prevent formation of localized particles containing Staufen. Particle formation appeared to be dependent on both mRNA dimerization and other element(s) in domains IV and V. Domain III of bcd mRNA could be substituted by heterologous dimerization motifs of different geometry. The resulting dimers were converted into stable forms, independently of the dimerization module used. Moreover, these chimeric RNAs were competent in forming localized particles and recruiting Staufen. The finding that the dimerization domain of bcd mRNA is interchangeable suggests that dimerization by itself, and not the precise geometry of the intermolecular interactions, is essential for the localization process. This suggests that the stabilizing interactions that are formed during the second step of the dimerization process might represent crucial elements for Staufen recognition and localization.     

The dimerization interface of the metastasis-associated protein S100A4 (Mts1): in vivo and in vitro studies

The S100 calcium-binding proteins are implicated in signal transduction, motility, and cytoskeletal dynamics. The three-dimensional structure of several S100 proteins revealed that the proteins form non-covalent dimers. However, the mechanism of the S100 dimerization is still obscure. In this study we characterized the dimerization of S100A4 (also named Mts1) in vitro and in vivo. Analytical ultracentrifugation revealed that apoS100A4 was present in solution as a mixture of monomers and dimers in a rapidly reversible equilibrium (K(d) = 4 +/- 2 microm). The binding of calcium promoted dimerization. Replacement of Tyr-75 by Phe resulted in the stabilization of the dimer. Helix IV is known to form the major part of the dimerization interface in homologous S100 proteins. By using the yeast two-hybrid system we showed that only a few residues of helix IV, namely Phe-72, Tyr-75, Phe-78, and Leu-79, are essential for dimerization in vivo. A homology model demonstrated that these residues form a hydrophobic cluster on helix IV. Their role is to stabilize the structure of individual subunits rather than provide specific interactions across the dimerization surface. Our mutation data showed that the specificity at the dimerization surface is not particularly stringent, which is consistent with recent data indicating that S100 proteins can form heterodimers.     

Mechanism of PKR activation: dimerization and kinase activation in the absence of double-stranded RNA

The kinase PKR is a central component of the interferon antiviral pathway. PKR is activated upon binding double-stranded (ds) RNA to undergo autophosphorylation. Although PKR is known to dimerize, the relationship between dimerization and activation remains unclear. Here, we directly characterize dimerization of PKR in free solution using analytical ultracentrifugation and correlate self-association with autophosphorylation activity. Latent, unphosphorylated PKR exists predominantly as a monomer at protein concentrations below 2 mg/ml. A monomer sedimentation coefficient of s(20,w)(0)=3.58 S and a frictional ratio of f/f(0)=1.62 indicate an asymmetric shape. Sedimentation equilibrium measurements indicate that PKR undergoes a weak, reversible monomer-dimer equilibrium with K(d)=450 microM. This dimerization reaction serves to initiate a previously unrecognized dsRNA-independent autophosphorylation reaction. The resulting activated enzyme is phosphorylated on the two critical threonine residues present in the activation loop and is competent to phosphorylate the physiological substrate, eIF2alpha. Dimer stability is enhanced by approximately 500-fold upon autophosphorylation. We propose a chain reaction model for PKR dsRNA-independent activation where dimerization of latent enzyme followed by intermolecular phosphorylation serves as the initiation step. Subsequent propagation steps likely involve phosphorylation of latent PKR monomers by activated enzyme within high-affinity heterodimers. Our results support a model whereby dsRNA functions by bringing PKR monomers into close proximity in a manner that is analogous to the dimerization of free PKR.     

Kinetic and thermodynamic study of the tetramerization equilibrium of phosphorylase b

The association equilibrium of phosphorylase b, induced by AMP and in the presence of Mg2+, has been shown to be a reversible process that follows second order and first order reversible rate laws in the direction of tetramerization and dimerization respectively, this fact being independent of temperature and of enzyme and AMP concentrations. Moreover, rate and equilibrium constants have been evaluated and their dependence on temperature and AMP concentration studied in this work. An important role that the existence of two classes of AMP binding sites per enzymatic subunit plays in the aggregation properties of the enzyme has also been emphasized. In the presence of 0.1 and 1 mM AMP (binding to the high affinity site), the values of the change in enthalpy, activation energy of dimerization and activation energy of tetramerization are: -36 kcal/mol, +36 kcal/mol, and 0 kcal/mol respectively. Binding of AMP to the low affinity site (10 mM AMP) yields significant changes in the self-association equilibrium, since the preceding parameters reach the following values: -18, +32, and +14 kcal/mol.     

Constitutive and agonist-induced dimerizations of the P2Y1 receptor: relationship to internalization and scaffolding

In living cells, P2Y(1) receptor dimerization was quantitated by an improved version of fluorescence resonance energy transfer donor photobleaching analysis. 44% of the P2Y(1) receptors expressed in HEK293 cell membranes exist as dimers in the resting state, inducible by agonist exposure to give 85-100% dimerization. Monomer and constitutive dimers are fully active. Agonist-induced dimerization follows desensitization and is fully reversible upon withdrawal of agonist. Receptor dimers are required for internalization at 37 degrees C but are not sufficient; at 20 degrees C dimerization also occurs, but endocytosis is abolished. Removal of the C-terminal 19 amino acids abolished both dimerization and internalization, whereas full activation by agonists was retained up to a loss of 39 amino acids, confirming active monomers. This receptor is known to bind through its last four amino acids (DTSL) to a scaffolding protein, Na/H exchanger regulatory factor-2, which was endogenous here, and DTSL removal blocked constitutive dimerization specifically. Distinction should therefore be made between the following: 1) constitutive dimers tethered to a scaffolding protein, together with effector proteins, within a signaling micro-domain, and 2) free dimers in the cell membrane, which here are inducible by agonist exposure. For the class A G-protein-coupled receptors, we suggest that the percentages of free monomers, and in many cases of induced free dimers, commonly become artifactually increased; this would arise from an excess there of the receptor over its specific scaffold and from a lack of the native targeting of the receptor to that site.     

Lipid Raft-Mediated Regulation of G-Protein Coupled Receptor Signaling by Ligands which Influence Receptor Dimerization: A Computational Study

G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors; they activate heterotrimeric G-proteins in response to ligand stimulation. Although many GPCRs have been shown to form homo- and/or heterodimers on the cell membrane, the purpose of this dimerization is not known. Recent research has shown that receptor dimerization may have a role in organization of receptors on the cell surface. In addition, microdomains on the cell membrane termed lipid rafts have been shown to play a role in GPCR localization. Using a combination of stochastic (Monte Carlo) and deterministic modeling, we propose a novel mechanism for lipid raft partitioning of GPCRs based on reversible dimerization of receptors and then demonstrate that such localization can affect GPCR signaling. Modeling results are consistent with a variety of experimental data indicating that lipid rafts have a role in amplification or attenuation of G-protein signaling. Thus our work suggests a new mechanism by which dimerization-inducing or inhibiting characteristics of ligands can influence GPCR signaling by controlling receptor organization on the cell membrane.     

Cytoplasmic domain of P-selectin glycoprotein ligand-1 facilitates dimerization and export from the endoplasmic reticulum

P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric transmembrane mucin on leukocytes. During inflammation, reversible interactions of PSGL-1 with selectins mediate leukocyte rolling on vascular surfaces. The transmembrane domain of PSGL-1 is required for dimerization, and the cytoplasmic domain propagates signals that activate β(2) integrins to slow rolling on integrin ligands. Leukocytes from knock-in "ΔCD" mice express a truncated PSGL-1 that lacks the cytoplasmic domain. Unexpectedly, they have 10-fold less PSGL-1 on their surfaces than WT leukocytes. Using glycosidases, proteases, Western blotting, confocal microscopy, cell-surface cross-linking, FRET, and pulse-chase metabolic labeling, we demonstrate that deleting the cytoplasmic domain impaired dimerization and delayed export of PSGL-1 from the endoplasmic reticulum (ER), markedly increasing a monomeric precursor in the ER and decreasing mature PSGL-1 on the cell surface. A monomeric full-length PSGL-1 made by substituting the transmembrane domain with that of CD43 exited the ER normally, revealing that dimerization was not required for ER export. Thus, the transmembrane and cytoplasmic domains cooperate to promote dimerization of PSGL-1. Furthermore, the cytoplasmic domain provides a key signal to export precursors of PSGL-1 from the ER to the Golgi apparatus en route to the cell surface.     

Lipase adsorption as a factor of lipolysis regulation

Sorption isotherms of pancreatic lipase on solid supports were studied. It was shown that the enzyme adsorption can be described by Langmuir equation for hydrophobic surface and by the equation which takes into account reversible dimerization of the protein in the absorption layer for hydrophilic surface. The catalytic properties of adsorbed lipase depend on the nature of solid support. The significant role of the structure of adsorption layer in heterogeneous activation of the enzyme on hydrophobic surface was suggested.     

Mutation of Glu-166 Blocks the Substrate-Induced Dimerization of SARS Coronavirus Main Protease

The maturation of SARS coronavirus involves the autocleavage of polyproteins 1a and 1ab by the main protease (Mpro) and a papain-like protease; these represent attractive targets for the development of anti-SARS drugs. The functional unit of Mpro is a homodimer, and each subunit has a His-41?Cys-145 catalytic dyad. Current thinking in this area is that Mpro dimerization is essential for catalysis, although the influence of the substrate binding on the dimer formation has never been explored. Here, we delineate the contributions of the peptide substrate to Mpro dimerization. Enzyme kinetic assays indicate that the monomeric mutant R298A/L exhibits lower activity but in a cooperative manner. Analytical ultracentrifugation analyses indicate that in the presence of substrates, the major species of R298A/L shows a significant size shift toward the dimeric form and the monomer-dimer dissociation constant of R298A/L decreases by 12- to 17-fold, approaching that for wild-type. Furthermore, this substrate-induced dimerization was found to be reversible after substrates were removed. Based on the crystal structures, a key residue, Glu-166, which is responsible for recognizing the Gln-P1 of the substrate and binding to Ser-1 of another protomer, will interact with Asn-142 and block the S1 subsite entrance in the monomer. Our studies indicate that mutation of Glu-166 in the R298A mutant indeed blocks the substrate-induced dimerization. This demonstrates that Glu-166 plays a pivotal role in connecting the substrate binding site with the dimer interface. We conclude that protein-ligand and protein-protein interactions are closely correlated in Mpro.     

Sequence-specific dimerization of the transmembrane domain of the "BH3-only" protein BNIP3 in membranes and detergent

Mitochondria-mediated apoptosis is regulated by proteins of the Bcl-2 superfamily, most of which contain a C-terminal hydrophobic domain that plays a role in membrane targeting. Experiments with BNIP3 have implicated the transmembrane (TM) domain in its proapoptotic function, homodimerization, and interactions with Bcl-2 and Bcl-xL. We show that the BNIP3 TM domain self-associates strongly in Escherichia coli cell membranes and causes reversible dimerization of a soluble protein in the detergent SDS when expressed as an in-frame fusion. Limited mutational analysis identifies specific residues that are critical for BNIP3 TM self-association in membranes, and these residues are also important for dimerization in SDS micelles, suggesting that the self-association observed in membranes is preserved in detergent. The effects of sequence changes at positions Ala176 and Gly180 suggest that the BNIP3 TM domain associates using a variant of the GXXXG motif previously shown to be important in the dimerization of glycophorin A. The importance of residue His173 in BNIP3 TM domain dimerization indicates that polar residues, which have been implicated in self-association of model TM peptides, can act in concert with the AXXXG motif to stabilize TM domain interactions. Our results demonstrate that the hydrophobic C-terminal TM domain of the pro-apoptotic BNIP3 protein dimerizes tightly in lipidic environments, and that this association has a strong sequence dependence but is independent of the identity of flanking regions. Thus, the transmembrane domain represents another region of the Bcl-2 superfamily of proteins that is capable of mediating strong and specific protein-protein interactions.     

Homodimerization of human mu-opioid receptor overexpressed in Sf9 insect cells

In this study, we demonstrate that human mu-opioid receptors do form SDS-resistant homodimers and examine the ability of human mu-opioid receptors to dimerize and the role of agonists in the dimerization. Increasing concentrations and longer exposure of agonists reduce the levels of dimmer with a corresponding increase in the levels of monomer. This effect is achieved with both peptide and alkaloid opioid agonists and it is antagonist reversible. These results suggest that human mu-opioid receptors are present as receptor oligomers and interconversion between dimeric and monomeric forms may be important for biological activity.     

Mechanism of dimerization of bicoid mRNA: initiation and stabilization

Dimerization of bcd mRNA was shown to be important for the formation of ribonucleoprotein particles and their localization in Drosophila embryo. The cis-element responsible for dimerization is localized in a stem-loop domain (domain III) containing two essential complementary 6-nucleotide sequences in a hairpin loop (LIIIb) and an interior loop (LIIIa). Such an RNA element can potentially generate single or double "hand-by-arm" interactions leading to open and closed complexes, respectively. The former retains the possibility of forming multimers, whereas the latter does not. We showed previously that dimerization proceeds through a two-step mechanism, which includes a transition from the reversible initiation complex into a very stable one. Here we have addressed the nature of the initial interactions and the mechanism of transition. We engineered a series of different RNA fragments with the capacity to form defined open dimers, multimers, or closed dimers. We compared their thermodynamic and kinetic behavior and mapped nucleotides involved in intermolecular interactions by enzymatic and chemical footprinting experiments and chemical modification interference. Our results indicate that the initiation step leads to a reversible open dimer, involving a more limited number of intermolecular base pairs than expected. The two loops play distinct roles in this process, and the structure of loop IIIb is more constrained than that of loop IIIa. Thus, loop IIIa appears to be the driving element of the recognition process. The initial open dimer is then converted into a stable closed dimer, possibly through a kinetically controlled mechanism.     

Kinetics and thermodynamics of dimer formation and dissociation for a recombinant humanized monoclonal antibody to vascular endothelial growth factor.

The recombinant humanized antibody (rhuMAb) VEGF has a high affinity for vascular endothelial growth factor and is currently being evaluated in clinical trials as a cancer therapeutic. Under acidic pH and low ionic strength conditions, the antibody was predominantly present as monomer. Under physiological conditions, the appearance of significant amounts of a noncovalent, reversible dimer were observed by size-exclusion chromatography. The kinetics and thermodynamics of the reversible self-association for rhuMAb VEGF monomer were investigated as a function of pH, temperature, and ionic strength by size-exclusion chromatography using the concentration jump method. The rate constant for dimer formation ranged 23-112 M(-)(1) min(-)(1) under the conditions studied, values that are significantly lower than those reported in the literature for other proteins that self-associate. The rate constant for dissociation ranged 0.0039-0.021 min(-)(1). Gibbs' free energies, enthalpies, entropies, and activation energies were determined and revealed that dimer formation is optimal at pH 7.5-8.0, which may be reflective of charge shielding occurring near the pI of the protein. There was a negative change in entropy for dissociation (values from -18.1 to -12.8 cal/mol K). In the presence of D(2)O or 1 M NaCl, dimerization was enhanced. The results of the kinetic and thermodynamic analysis of this study indicate that rhuMAb VEGF dimerization occurs primarily through hydrophobic interactions.