Role Of Dimerization Research Articles

Heterologous quaternary structure of CXCL12 and its relationship to the CC chemokine family

X-ray crystallographic studies reveal that CXCL12 is able to form multiple dimer types, a traditional CXC dimer and a 'CC-like' form. Phylogenetic analysis of all known human chemokines demonstrates CXCL12 is more closely related to the CC chemokine class than other CXC chemokines. These observations indicate that CXCL12 contains genomic and structural elements characteristic of both CXC and CC chemokines.Chemokines are members of a superfamily of proteins involved in the migration of cells to the proper anatomical position during embryonic development or in response to infection or stress during an immune response. There are two major (CC and CXC) and two minor (CX3C and XC) families based on the sequence around the first conserved cysteine. The topology of all structures is essentially identical with a flexible N-terminal region of 3-8 amino acids, a 10-20 residue N-terminal loop, a short 3{sub 10}-helix, three {beta}-strands, and a {alpha}-helix. The major consequence of the subtle difference between the families occurs at the oligomeric level. Monomers of the CC, CXC, and CX3C families form dimers in a family-specific manner. The XCL1 chemokine is a monomer that can interconvert between two folded states. All chemokines activate GPCRs according to family-specificity, however there are a fewmore » examples of chemokines crossing the family boundary to function as antagonists. A two-stage mechanism for chemokine activation of GPCRs has been proposed. The N-terminal region of the receptor interacts with the chemokine, followed by receptor activation by the chemokine N-terminal region. Monomeric chemokines have been demonstrated to be the active form for receptor function. There are numerous examples of both chemokines and their receptors forming dimers. While family-specific dimerization may be an attractive explanation for why specific chemokines only activate GPCRs within their own family, the role of dimers in the function of chemokines has not been resolved. Given that CXCL12 is in the CXC family, the CXC dimer is considered the physiologic dimer in all previous studies based on crystallographic evidence. NMR and mutational studies agree with the CXC dimer form in solution. The CXC form of the dimer is seen in recent structures of CXCL12 bound to a heparin disaccharide and several CXCR4 peptides. In one case, crystals of the CXC-type dimer were soaked in a heparin disaccharide solution to determine the interactions between this dimer and bound disaccharide. In another case, in order to overcome NMR chemical shift line broadening when CXCR4 peptides are added, a 'locked' dimer was constructed by introducing a cysteine mutant that linked subunits as a CXC dimer through an inter-subunit disulfide bond. The solution structures of the locked CXC dimer with CXCR4 peptides were determined. The locked CXC dimer retained Ca{sup 2+} mobilization yet lost chemotaxis activity, presumably because the monomer is the active form. In addition to existing as a monomer and CXC dimer, CXCL12 is now demonstrated to have the capacity to form CC type dimers in the presence of a CXCR4 peptide.« less

Charge stabilisation by dimer formation of an endcapped thiophene tetramer—an in situ NMR spectroelectrochemical study

The role of dimers in the charge stabilisation of 5,5′′′-dicyano-3,3′′-dibutyl 2,2′:5′,2′′:5′′,2′′′-quaterthiophene (DCNDBQT) via an electrochemical reaction is studied in detail by in situ NMR spectroelectrochemistry. Among the two oxidation steps the first was found to be reversible while the second is completely irreversible. By in situ NMR spectroelectrochemistry we are able to demonstrate the formation of a π-dimer via the cation generated in the first electron transfer as well as the formation of a σ-dimer by a follow-up reaction of the dication generated in the second oxidation step. As the thiophene oligomer is blocked in the 2- and 5-positions by the cyanide group the monomer undergoes a dimerisation in the 4′-position. In situ NMR spectroelectrochemistry delivers proof of the coupling reaction of the thiophene rings in the electrochemistry of α,ω-endcapped oligothiophenes although such endcapped structures are expected to be prevented from chemical follow-up reactions. Furthermore, substitution by cyano groups opens the route to a cathodic reduction of the structure. The dicyanodibutylquaterthiophene gives a single reduction step which turns out to be quasi-reversible but the anionic structure formed in the reduction was not detectable by in situ NMR spectroelectrochemistry. This and the follow-up reactions are discussed in detail.

Biochemical and Functional Characterization of Crystallographic Ca2+/CaM-Cav1.2 A-C-IQ Complex Dimer

The ubiquitous calcium sensor calmodulin (CaM) mediates two important voltage-gated calcium channel (CaV) calcium-dependent modulatory mechanisms through its interaction with the CaV C-terminal tail. Here, we report the structure of Ca2+/CaM bound to a portion of the CaV1.2 C-terminus with three consecutive CaM binding motifs (A-C-IQ domain). The structure reveals two channel chains dimerized via bridging CaMs and interactions between two long, antiparallel helices. Unlike the crystal structure, the CaM peptide complex is a monomer in solution that corresponds to a single channel chain and two CaMs. Disruption of the crystallographic inter-helix interactions had minimal effect on CDI and CDF of full-length CaV1.2. Moreover, subunit counting experiments using CaV1.2-EGFP fusion proteins clearly indicate that CaV1.2 is a monomer in cell membranes. Thus, contrary to previously proposed models, there appears to be no role for dimerization in channel function.

Role of Dimerization in Poly-Ubiquitin Chain Formation

The ubiquitin proteolysis pathway is responsible for protein degradation utilizing three enzymes, the ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2), and a ligating enzyme (E3), that respectively activate, transfer and ligate ubiquitin (Ub) onto a target protein. Repeated cycles of this process results in a poly-ubiquitinated target protein that is degraded by the 26S proteasome. How the poly-Ub chains are formed remains unknown. One suggested model involves the dimerization of an E2 enzyme allowing Ub passage and ligation between adjacent E2 enzymes. We are examining this mechanism for the E2 enzymes Ubc1 and HIP2, which contain C-terminal UBA domains that allows for non-covalent Ub binding in addition to a thioester-bound Ub. The dimerization of these E2 enzymes was tested using sedimentation equilibrium and small angle x-ray scattering and showed that both are monomeric. Disulphide-bound E2-Ub complexes were used to mimic the thiolester, and these complexes had a weak propensity to dimerize. This was supported in ubiquitination assays that showed a thioester-bound Ub on an E2 could be transferred to a non-hydrolyzable, disulphide-bound Ub molecule on a second E2 enzyme. This suggests weak dimerization is likely sufficient to allow the first step of poly-Ub chain formation. We tested whether the length of the poly-Ub chains on HIP2 stimulates dimerization by creating HIP2-Ub2 and HIP2-Ub4 complexes. The ability of these species to dimerize was assessed via sedimentation equilibrium and NMR spectroscopy. The isolated UBA domain from HIP2 was used in competition experiments to determine how it might influence poly-Ub chain formation. This work provides the first structural evidence for poly-Ub chain formation as assembled on the E2 enzyme.

Cyclic Nucleotide Binding GAF Domains from Phosphodiesterases: Structural and Mechanistic Insights

GAF domains regulate the catalytic activity of certain vertebrate cyclic nucleotide phosphodiesterases (PDEs) by allosteric, noncatalytic binding of cyclic nucleotides. GAF domains arranged in tandem are found in PDE2, -5, -6, -10, and -11, all of which regulate the cellular concentrations of the second messengers cAMP and/or cGMP. Nucleotide binding to GAF domains affects the overall conformation and the catalytic activity of full-length PDEs. The cyclic nucleotide-bound GAF domains from PDE2, -5, -6, and -10 all adopt a conserved fold but show subtle differences within the binding pocket architecture that account for a large range of nucleotide affinities and selectivity. NMR data and details from the structure of full-length nucleotide-free PDE2A reveal the dynamic nature and magnitude of the conformational change that accompanies nucleotide binding. The discussed GAF domain structures further reveal differences in dimerization properties and highlight the structural diversity within GAF domain-containing PDEs.

Identification of Specific Transmembrane Residues and Ligand-Induced Interface Changes Involved In Homo-Dimer Formation of a Yeast G Protein-Coupled Receptor

The Saccharomyces cerevisiae alpha-factor pheromone receptor, Ste2p, has been studied as a model for G protein-coupled receptor (GPCR) structure and function. Dimerization has been demonstrated for many GPCRs, although the role(s) of dimerization in receptor function is disputed. Transmembrane domains one (TM1) and four (TM4) of Ste2p were shown previously to play a role in dimerization. In this study, single cysteine substitutions were introduced into a Cys-less Ste2p, and disulfide-mediated dimerization was assessed. Six residues in TM1 (L64 to M69) that had not been previously investigated and 19 residues in TM7 (T278 to A296) of which 15 were not previously investigated were mutated to create 25 single Cys-containing Ste2p molecules. Ste2p mutants V68C in TM1 and nine mutants in TM7 (cysteine substituted into residues 278, 285, 289, and 291 to 296) showed increased dimerization upon addition of an oxidizing agent in comparison to the background dimers formed by the Cys-less receptor. The formation of dimers was decreased for TM7 mutant receptors in the presence of alpha-factor indicating that ligand binding resulted in a conformational change that influenced dimerization. The effect of ligand on dimer formation suggests that dimers are formed in the resting state and the activated state of the receptor by different TM interactions.

Mutated ERBB4: a novel drug target in metastatic melanoma?

Mutated <i>ERBB4</i>: a novel drug target in metastatic melanoma?

Role of Dimerization in the Catalytic Properties of the Escherichia coli Disulfide Isomerase DsbC

The bacterial protein-disulfide isomerase DsbC is a homodimeric V-shaped enzyme that consists of a dimerization domain, two alpha-helical linkers, and two opposing thioredoxin fold catalytic domains. The functional significance of the two catalytic domains of DsbC is not well understood yet. We have engineered heterodimer-like DsbC derivatives covalently linked via (Gly(3)-Ser) flexible linkers. We either inactivated one of the catalytic sites (CGYC), or entirely removed one of the catalytic domains while maintaining the putative binding area intact. Variants having a single active catalytic site display significant levels of isomerase activity. Furthermore, mDsbC[H45D]-dim[D53H], a DsbC variant lacking an entire catalytic domain but with an intact dimerization domain, also showed isomerase activity, albeit at lower levels. In addition, the absence of the catalytic domain allowed this protein to catalyze in vivo oxidation. Our results reveal that two catalytic domains in DsbC are not essential for disulfide bond isomerization and that a determining feature in isomerization is the availability of a substrate binding domain.

Role of dimerization and substrate exclusion in the regulation of bone morphogenetic protein-1 and mammalian tolloid.

The bone morphogenetic protein (BMP)-1/tolloid metalloproteinases are evolutionarily conserved enzymes that are fundamental to dorsal-ventral patterning and tissue morphogenesis. The lack of knowledge regarding how these proteinases recognize and cleave their substrates represents a major hurdle to understanding tissue assembly and embryonic patterning. Although BMP-1 and mammalian tolloid (mTLD) are splice variants, it is puzzling why BMP-1, which lacks 3 of the 7 noncatalytic domains present in all other family members, is the most effective proteinase. Using a combination of single-particle electron microscopy, small-angle X-ray scattering, and other biophysical measurements in solution, we show that mTLD, but not BMP-1, forms a calcium-ion-dependent dimer under physiological conditions. Using a domain deletion approach, we provide evidence that EGF2, which is absent in BMP-1, is critical to the formation of the dimer. Based on a combination of structural and functional data, we propose that mTLD activity is regulated by a substrate exclusion mechanism. These results provide a mechanistic insight into how alternative splicing of the Bmp1 gene produces 2 proteinases with differing biological activities and have broad implications for regulation of BMP-1/mTLD and related proteinases during BMP signaling and tissue assembly.

Synergistic nuclear import of NeuroD1 and its partner transcription factor, E47, via heterodimerization

The transition from undifferentiated pluripotent cells to terminally differentiated neurons is coordinated by a repertoire of transcription factors. NeuroD1 is a type II basic helix loop helix (bHLH) transcription factor that plays critical roles in neuronal differentiation and maintenance in the central nervous system. Its dimerization with E47, a type I bHLH transcription factor, leads to the transcriptional regulation of target genes. Mounting evidence suggests that regulating the localization of transcription factors contributes to the regulation of their activity during development as defects in their localization underlie a variety of developmental disorders. In this study, we attempted to understand the nuclear import mannerisms of NeuroD1 and E47. We found that the nuclear import of NeuroD1 and E47 is energy-dependent and involves the Ran-mediated pathway. Herein, we demonstrate that NeuroD1 and E47 can dimerize inside the cytoplasm before their nuclear import. Moreover, this dimerization promotes nuclear import as the nuclear accumulation of NeuroD1 was enhanced in the presence of E47 in an in vitro nuclear import assay, and NLS-deficient NeuroD1 was successfully imported into the nucleus upon E47 overexpression. NeuroD1 also had a similar effect on the nuclear accumulation of NLS-deficient E47. These findings suggest a novel role for dimerization that may promote, at least partially, the nuclear import of transcription factors allowing them to function efficiently in the nucleus.

Identification and Role of the Homodimerization Interface of the Glycosylphosphatidylinositol-anchored Membrane Type 6 Matrix Metalloproteinase (MMP25)

The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reduction-sensitive form of M(r) 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys(532) is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys(530) and Cys(534) form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of alpha-1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.

Negative Regulation of the Yeast ABC Transporter Ycf1p by Phosphorylation within Its N-terminal Extension

The yeast vacuolar membrane protein Ycf1p and its mammalian counterpart, MRP1, belong to the ABCC subfamily of ATP-binding cassette (ABC) transporters that rid cells of toxic endogenous and xenobiotic compounds. Like most members of the ABCC subfamily, Ycf1p contains an N-terminal extension in addition to its ABC "core" domain and transports substrates in the form of glutathione conjugates. Ycf1p is subject to complex regulation to ensure its optimal function. Previous studies showed that Ycf1p activity is stimulated by a guanine nucleotide exchange factor, Tus1p, and is positively regulated by phosphorylation in its ABC core domain at residues Ser-908 and Thr-911. Here we provide evidence that phosphorylation of Ser-251 in the Ycf1p N-terminal extension negatively regulates activity. Mutant Ycf1p-S251A exhibits increased resistance to cadmium in vivo and increased Ycf1p-dependent transport of [(3)H]estradiol-beta-17-glucuronide in vitro as compared with wild-type Ycf1p. Activity is restored to the wild-type level for Ycf1-S251E. To identify kinase(s) that negatively regulate Ycf1p function, we conducted an integrated membrane yeast two-hybrid (iMYTH) screen and identified two kinase genes, CKA1 and HAL5, deletion of which increases Ycf1p function. Genetic evidence suggests that Cka1p may regulate Ycf1p function through phosphorylation of Ser-251 either directly or indirectly. Overall, this study provides compelling evidence that negative, as well as positive, regulation of Ycf1p is mediated by phosphorylation.

Regions in the Cytosolic C-Terminus of the Secretory Na+-K+-2Cl- Cotransporter NKCC1 Are Required for Its Homodimerization

The "secretory" Na+-K+-2Cl- cotransporter, NKCC1, is a member of a small gene family of electroneutral cation-chloride cotransporters (CCCs) with 9 homologues in vertebrates. A number of these transporters, including NKCC1 itself, have been shown to exist as homodimers in the membrane, suggesting that this may be a common feature of the CCCs. Here we employ chemical cross-linking studies, a novel co-immunoprecipition assay, and NKCC1/CCC chimeras to further explore the basis and significance of NKCC1 dimerization. An N-terminally truncated NKCC1 (nttNKCC1), in which the first 20 kDa of the 28 kDa cytosolic N-terminus are deleted, forms homodimers as well as heterodimers with full-length NKCC1, indicating that this region of N-terminus is not required for dimerization. On the other hand, replacing the 50 kDa NKCC1 C-terminus with that of several other non-NKCC1 homologues results in chimeric proteins that form homodimers but show little or no heterodimerization with NKCC1, demonstrating that the C-terminus of NKCC1 plays an essential role in dimerization and that NKCC1 dimerization exhibits definite homologue-specificity. Using additional chimeras we find that the residues required for dimer formation lie between amino acids 751 and 998 of (rat) NKCC1. We also show that dramatically overexpressing the nonfunctional truncated protein nttNKCC1 relative to the endogenous NKCC1 in the HEK293 cells results in a modest inhibition of fluxes via the endogenous transporter and a change in its sensitivity to the specific inhibitor bumetanide. These latter results indicate that there is a functional interaction between dimer subunits but that nonfunctional subunits do not necessarily have a dominant negative effect as has been previously proposed.

Impaired RNA incorporation and dimerization in live attenuated leader-variants of SIVmac239

BackgroundThe 5' untranslated region (UTR) or leader sequence of simian immunodeficiency virus (SIVmac239) is multifunctional and harbors the regulatory elements for viral replication, persistence, gene translation, expression, and the packaging and dimerization of viral genomic RNA (vRNA). We have constructed a series of deletions in the SIVmac239 leader sequence in order to determine the involvement of this region in both the packaging and dimerization of viral genomic RNA. We also assessed the impact of these deletions upon viral infectiousness, replication kinetics and gene expression in cell lines and monkey peripheral blood mononuclear cells (PBMC).ResultsRegions on both sides of the major splice donor (SD) were found to be necessary for the efficiency and specificity of viral genome packaging. However, stem-loop1 is critical for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the initiation site of SIV-Gag have additive effects on RNA packaging and contribute to a lesser degree to RNA dimerization. The targeted disruption of structures on both sides of the SD also severely impacts viral infectiousness, gene expression and replication in both CEMx174 cells and rhesus PBMC.ConclusionIn the leader region of SIVmac239, stem-loop1 functions as the primary determinant for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the translational initiation site of SIV-Gag are classified as secondary determinants and play a role in dimerization. Collectively, these data signify a linkage between the primary encapsidation determinant of SIVmac239 and RNA dimerization.

In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N- and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

Activation function 1 domain plays a negative role in dimerization of estrogen receptor beta

Transcriptional potential of estrogen receptor beta (ERβ) depends on the ligand binding and subsequent dimerization of the receptor protein. In order to examine the role of N-terminally located activation function 1 (AF-1) protein domain in the dimerization process of ERβ, we used yeast SOS-Recruitment System (SRS). Two variants of ERβ protein were expressed in the yeast cells: full length receptor and a truncated form, lacking AF-1. We observed that upon 17β-estradiol treatment only the shorter form of the receptor dimerized, whereas the full-length one did not. This result suggests an inhibitory function of AF-1 in dimer formation and supports previous studies showing that N-terminal domain of ERβ suppresses transcriptional activity.

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<h3>Background</h3> Neuropilin-1 (NRP1) is a co-receptor that complexes with diverse ligands and their cognate receptors. As such, it plays a role in multiple different biological processes, including axon guidance and angiogenesis. NRP1 contains two CUB domains (a1 and a2) involved in binding the ligand Semaphorin3A (SEMA3A), two Factor V/VIII domains (b1 and b2) involved in VEGF ligand binding and one MAM domain (c domain). While functional antibodies with anti-tumor activity have been generated against the SEMA3A and VEGF binding domains, little attention has been paid to the c domain of NRP1, which has been implicated in the dimerization of NRP1, a prerequisite for functionality. We therefore hypothesized that c domain-binding antibodies would offer an opportunity to generate functional inhibitors of both SEMA3A and VEGF signaling and therefore improved anti-tumor activity. <h3>Methods</h3> Recombinant human NRP1 comprising all subdomains was used to identify fully human anti-NRP1 antibodies. Specific antibodies were tested for their ability to block NRP1 interactions with recombinant SEMA3A and VEGF protein in vitro. Blocking antibodies were subsequently assessed for their functional effects, such as inhibition of SEMA3A-mediated growth cone collapse. Antibodies with diverse binding characteristics were then tested for in vivo anti-tumor activity in multiple cancer models of interest. <h3>Results</h3> Recombinant NRP1 containing the a1, a2, b1, b2 and c subdomains was used to successfully identify a series of specific monoclonal antibodies that cross-reacted with Cynomolgus monkey and mouse NRP1, but not human NRP2. Except for the a2 domain, epitope mapping showed an even distribution of mAbs for binding to each of the NRP1 subdomains, including the c domain that has been proposed to play a role in dimerization. Using biolayer interferometry, we identified antibody classes with direct SEMA3A and/or VEGF blocking properties. Further optimization of these antibodies yielded mAbs with subnanomolar affinities that showed significant tumor growth inhibition in multiple mouse models, including anti-PD1 non-responsive models. <h3>Conclusions</h3> Here we demonstrate the identification of fully human monoclonal antibodies that specifically bind to the c domain of human NRP1. A subset of these c domain binders do not block either SEMA3A or VEGF binding to NRP1 but do show in vivo efficacy, suggesting a role for the c domain of NRP1 in the formation of functional (dimeric) complexes. Thus, c domain binding antibodies show remarkable inhibition of tumor growth in mouse cancer models and offer a novel means of therapeutic intervention in patients who are refractory to immune checkpoint inhibition.

L55P Transthyretin Accelerates Subunit Exchange and Leads to Rapid Formation of Hybrid Tetramers

Transthyretin is a tetrameric protein associated with the commonest form of systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry, we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, L55P. Wild-type subunit exchange occurs via both monomers and dimers, whereas exchange via dimers is the dominant mechanism for the L55P variant. Because patients with the L55P mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and L55P tetramers. We found that hybrid tetramers containing two or three L55P subunits dominate in the early stages of the reaction. Surprisingly, we also found that, in the presence of L55P transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidosis.

The ErbB-4 s80 intracellular domain is a constitutively active tyrosine kinase

The ErbB-4 receptor tyrosine kinase homo- and heterodimerizes following heregulin binding, which provokes increased levels of tyrosine autophosphorylation. Unique to the ErbB family, ErbB-4 is then proteolytically cleaved by alpha- and gamma-secretase to produce an 80 kDa intracellular domain (s80 ICD) fragment. This fragment is found in both the cytoplasm and nucleus of many normal and cancer cells and can interact with transcription factors in the cytoplasm and nucleus. Since the s80 ICD lacks ectodomain sequences known to play a major role in dimerization of ErbB family members, we asked whether the s80 ICD is an active tyrosine kinase. Here, we demonstrate that the s80 ICD is a constitutively active tyrosine kinase and can form homodimers. The s80 ICD is autophosphorylated in cells and can phosphorylate an exogenous substrate in vitro. Also, the s80 ICD can coassociate and dimers are detected by chemical crosslinking. This is the first example of constitutive kinase activation and dimerization totally within the cytoplasmic domain of an ErbB receptor and suggests that the s80 ICD may function to phosphorylate substrates in the cytoplasm or nucleus.

The Unstructured N-terminal Tail of ParG Modulates Assembly of a Quaternary Nucleoprotein Complex in Transcription Repression

ParG is the prototype of a group of small (<10 kDa) proteins involved in accurate plasmid segregation. The protein is a dimeric DNA binding factor, which consists of symmetric paired C-terminal domains that interleave into a ribbon-helix-helix fold that is crucial for the interaction with DNA, and unstructured N-terminal domains of previously unknown function. Here the ParG protein is shown to be a transcriptional repressor of the parFG genes. The protein assembles on its operator site initially as a tetramer (dimer of dimers) and, at elevated protein concentrations, as a pair of tetramers. Progressive deletion of the mobile N-terminal tails concomitantly decreased transcriptional repression by ParG and perturbed the DNA binding kinetics of the protein. The flexible tails are not necessary for ParG dimerization but instead modulate the organization of a higher order nucleoprotein complex that is crucial for proper transcriptional repression. This is achieved by transient associations between the flexible and folded domains in complex with the target DNA. Numerous ParG homologs encoded by plasmids of Gram-negative bacteria similarly are predicted to possess N-terminal disordered tails, suggesting that this is a common feature of partition operon autoregulation. The results provide new insights into the role of natively unfolded domains in protein function, the molecular mechanisms of transcription regulation, and the control of plasmid segregation.