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PubMed2000

A distinct seven-residue trigger sequence is indispensable for proper coiled-coil formation of the human macrophage scavenger receptor oligomerization domain.

We have recently identified a distinct 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils but not in trimers, tetramers, or pentamers. This coiled-coil trigger pattern was demonstrated to be indispensable for the assembly of the oligomerization domain of the actin-bundling protein cortexillin I from Dictyostelium discoideum and the leucine zipper domain of the yeast transcriptional activator GCN4. With the aim to extend our knowledge on trigger sequences we have investigated the human macrophage scavenger receptor type A oligomerization domain as a representative of three-stranded coiled coils. We prepared a variety of recombinant N- and C-terminal deletion mutants from the full-length oligomerization domain by heterologous gene expression in Escherichia coli and assessed their ability to form trimeric coiled-coil structures by circular dichroism spectroscopy and analytical ultracentrifugation. Deletion mapping identified a distinct seven-residue sequence that was absolutely required for proper coiled-coil formation, supporting our previous results that heptad repeats alone are not sufficient for oligomerization. The finding that all fragments containing this particular sequence exhibited similar thermal stabilities indicates primarily a stabilizing function of the coiled-coil trigger. Based on sequence similarity, we suggest that functionally related sites are present in other three-stranded coiled-coil proteins.

Amino Acid Sequence↗
PubMed2000

Activation of the Ikappa B kinases by RIP via IKKgamma /NEMO-mediated oligomerization.

To understand the mechanism of activation of the IkappaB kinase (IKK) complex in the tumor necrosis factor (TNF) receptor 1 pathway, we examined the possibility that oligomerization of the IKK complex triggered by ligand-induced trimerization of the TNF receptor 1 complex is responsible for activation of the IKKs. Gel filtration analysis of the IKK complex revealed that TNFalpha stimulation induces a large increase in the size of this complex, suggesting oligomerization. Substitution of the C-terminal region of IKKgamma, which interacts with RIP, with a truncated DR4 lacking its cytoplasmic death domain, produced a molecule that could induce IKK and NF-kappaB activation in cells in response to TRAIL. Enforced oligomerization of the N terminus of IKKgamma or truncated IKKalpha or IKKbeta lacking their serine-cluster domains can also induce IKK and NF-kappaB activation. These data suggest that IKKgamma functions as a signaling adaptor between the upstream regulators such as RIP and the IKKs and that oligomerization of the IKK complex by upstream regulators is a critical step in activation of this complex.

Animals↗
PubMed2000

Oligomerization of Rac1 gtpase mediated by the carboxyl-terminal polybasic domain.

The Rho family GTPase Rac1 mediates a variety of signal transduction processes leading to activation of NADPH oxidase, actin cytoskeleton reorganization, transcription activation, and stimulation of DNA synthesis. In this study, Rac1 was found to form a reversible monomer and oligomer in both the GDP- and GTP-bound states in vitro and in cells. Mutational analysis and peptide competition experiments showed that the unique C-terminal domain of Rac1 consisting of six consecutive basic residues (amino acids 183-188) is required for the homophilic interaction. Oligomerization of Rac1-GTP led to a self-stimulatory GTPase-activating protein (GAP) activity, resulting in a significantly enhanced intrinsic GTP hydrolysis rate of Rac1-GTP. Deletion or mutation of the polybasic residues drastically decreased its intrinsic GTPase activity and resulted in a loss of the self-stimulatory GAP activity. In the oligomeric state, Rac1 became insensitive to the RhoGAP stimulation, albeit maintaining the responsiveness to the guanine nucleotide exchange factor. The ability of the Rac1 C-terminal mutants to activate the effector p21(cdc42/rac)-activated kinase-1 correlated with their oligomerization states, suggesting that oligomer formation potentiates effector activation. Furthermore, the oligomer-to-monomer transition of Rac1-GDP could be driven effectively by interaction with the Rho guanine nucleotide dissociation inhibitor. Building on previous characterizations of Rac1 interaction with regulatory proteins and effectors, these results suggest that Rac1 may employ yet another means of regulation by cycling between the monomeric and oligomeric states to effectively generate a transient and augmented signal.

Amino Acid Sequence↗
PubMed2002

Ceramide biosynthesis is required for the formation of the oligomeric H+-ATPase Pma1p in the yeast endoplasmic reticulum.

The yeast plasma membrane H(+)-ATPase Pma1p is one of the most abundant proteins to traverse the secretory pathway. Newly synthesized Pma1p exits the endoplasmic reticulum (ER) via COPII-coated vesicles bound for the Golgi. Unlike most secreted proteins, efficient incorporation of Pma1p into COPII vesicles requires the Sec24p homolog Lst1p, suggesting a unique role for Lst1p in ER export. Vesicles formed with mixed Sec24p-Lst1p coats are larger than those with Sec24p alone. Here, we examined the relationship between Pma1p biosynthesis and the requirement for this novel coat subunit. We show that Pma1p forms a large oligomeric complex of >1 MDa in the ER, which is packaged into COPII vesicles. Furthermore, oligomerization of Pma1p is linked to membrane lipid composition; Pma1p is rendered monomeric in cells depleted of ceramide, suggesting that association with lipid rafts may influence oligomerization. Surprisingly, monomeric Pma1p present in ceramide-deficient membranes can be exported from the ER in COPII vesicles in a reaction that is stimulated by Lst1p. We suggest that Lst1p directly conveys Pma1p into a COPII vesicle and that the larger size of mixed Sec24pLst1p COPII vesicles is not essential to the packaging of large oligomeric complexes.

COP-Coated Vesicles↗
PubMed2002

Ca(2+)-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP-alpha-actinin-4 complex for oligomerization and endocytosis.

Two PDZ domain-containing proteins, NHERF and E3KARP are necessary for cAMP-dependent inhibition of Na(+)/H(+) exchanger 3 (NHE3). In this study, we demonstrate a specific role of E3KARP, which is not duplicated by NHERF, in Ca(2+)-dependent inhibition of NHE3 activity. NHE3 activity is inhibited by elevation of intracellular Ca(2+) ([Ca(2+)](i)) in PS120 fibroblasts stably expressing E3KARP but not those expressing NHERF. In addition, this Ca(2+)-dependent inhibition requires Ca(2+)-dependent association between alpha-actinin-4 and E3KARP. NHE3 is indirectly connected to alpha-actinin-4 in a protein complex through Ca(2+)-dependent interaction between alpha-actinin-4 and E3KARP, which occurs through the actin-binding domain plus spectrin repeat domain of alpha-actinin-4. Elevation of [Ca(2+)](i) results in oligomerization and endocytosis of NHE3 as well as in inhibition of NHE3 activity. Overexpression of alpha-actinin-4 potentiates the inhibitory effect of ionomycin on NHE3 activity by accelerating the oligomerization and endocytosis of NHE3. In contrast, overexpression of the actin-binding domain plus spectrin repeat domain acts as a dominant-negative mutant and prevents the inhibitory effect of ionomycin on NHE3 activity as well as the oligomerization and internalization of NHE3. From these results, we propose that elevated Ca(2+) inhibits NHE3 activity through oligomerization and endocytosis of NHE3, which occurs via formation of an NHE3-E3KARP-alpha-actinin-4 complex.

Actinin↗
PubMed2002

Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability.

Genetic evidence predicts a causative role for amyloid-beta (A beta) in Alzheimer's disease. Recent debate has focused on whether fibrils (amyloid) or soluble oligomers of A beta are the active species that contribute to neurodegeneration and dementia. We developed two aggregation protocols for the consistent production of stable oligomeric or fibrillar preparations of A beta-(1-42). Here we report that oligomers inhibit neuronal viability 10-fold more than fibrils and approximately 40-fold more than unaggregated peptide, with oligomeric A beta-(1-42)-induced inhibition significant at 10 nm. Under A beta-(1-42) oligomer- and fibril-forming conditions, A beta-(1-40) remains predominantly as unassembled monomer and had significantly less effect on neuronal viability than preparations of A beta-(1-42). We applied the aggregation protocols developed for wild type A beta-(1-42) to A beta-(1-42) with the Dutch (E22Q) or Arctic (E22G) mutations. Oligomeric preparations of the mutations exhibited extensive protofibril and fibril formation, respectively, but were not consistently different from wild type A beta-(1-42) in terms of inhibition of neuronal viability. However, fibrillar preparations of the mutants appeared larger and induced significantly more inhibition of neuronal viability than wild type A beta-(1-42) fibril preparations. These data demonstrate that protocols developed to produce oligomeric and fibrillar A beta-(1-42) are useful in distinguishing the structural and functional differences between A beta-(1-42) and A beta-(1-40) and genetic mutations of A beta-(1-42).

Alzheimer Disease↗
PubMed2002

Phospholipid-induced monomerization and signal-peptide-induced oligomerization of SecA.

The SecA ATPase drives the processive translocation of the N terminus of secreted proteins through the cytoplasmic membrane in eubacteria via cycles of binding and release from the SecYEG translocon coupled to ATP turnover. SecA forms a physiological dimer with a dissociation constant that has previously been shown to vary with temperature and ionic strength. We now present data showing that the oligomeric state of SecA in solution is altered by ligands that it interacts with during protein translocation. Analytical ultracentrifugation, chemical cross-linking, and fluorescence anisotropy measurements show that the physiological dimer of SecA is monomerized by long-chain phospholipid analogues. Addition of wild-type but not mutant signal sequence peptide to these SecA monomers redimerizes the protein. Physiological dimers of SecA do not change their oligomeric state when they bind signal sequence peptide in the compact, low temperature conformational state but polymerize when they bind the peptide in the domain-dissociated, high-temperature conformational state that interacts with SecYEG. This last result shows that, at least under some conditions, signal peptide interactions drive formation of new intermolecular contacts distinct from those stabilizing the physiological dimer. The observations that signal peptides promote conformationally specific oligomerization of SecA while phospholipids promote subunit dissociation suggest that the oligomeric state of SecA could change dynamically during the protein translocation reaction. Cycles of SecA subunit recruitment and dissociation could potentially be employed to achieve processivity in polypeptide transport.

Adenosine Triphosphatases↗
PubMed2002

Oligomerization-dependent association of the SAM domains from Schizosaccharomyces pombe Byr2 and Ste4.

SAM (sterile alpha motif) domains are protein-protein interaction modules found in a large number of regulatory proteins. Byr2 and Ste4 are two SAM domain-containing proteins in the mating pheromone response pathway of the fission yeast, Schizosaccharomyces pombe. Byr2 is a mitogen-activated protein kinase kinase kinase that is regulated by Ste4. Tu et al. (Tu, H., Barr, M., Dong, D. L., and Wigler, M. (1997) Mol. Cell. Biol. 17, 5876-5887) showed that the isolated SAM domain of Byr2 binds a fragment of Ste4 that contains both a leucine zipper (Ste4-LZ) domain as well as a SAM domain, suggesting that Byr2-SAM and Ste4-SAM may form a hetero-oligomer. Here, we show that the individual SAM domains of Ste4 and Byr2 are monomeric at low concentrations and bind to each other in a 1:1 stoichiometry with a relatively weak dissociation constant of 56 +/- 3 microm. Inclusion of the Ste4-LZ domain, which determines the oligomeric state of Ste4, has a dramatic effect on binding affinity, however. We find that the Ste4-LZ domain is trimeric and, when included with the Ste4-SAM domain, yields a 3:1 Ste4-LZ-SAM:Byr2-SAM complex with a tight dissociation constant of 19 +/- 4 nm. These results suggest that the Ste4-LZ-SAM protein may recognize multiple binding sites on Byr2-SAM, indicating a new mode of oligomeric organization for SAM domains. The fact that high affinity binding occurs only with the addition of an oligomerization domain suggests that it may be necessary to include ancillary oligomerization modules when searching for binding partners of SAM domains.

Binding Sites↗
PubMed2002

Changes in oligomerization are essential for the chaperone activity of a small heat shock protein in vivo and in vitro.

The ability of small heat shock proteins (sHSPs) to prevent thermal aggregation of other proteins may require disassembly and reassembly of sHSP oligomers. We investigated the role of changes in sHSP oligomerization by studying a mutant with reduced oligomeric stability. In HSP16.6, the single sHSP in the cyanobacterium Synechocystis sp. PCC 6803, the mutation L66A causes oligomer instability and reduced chaperone activity in vitro. Because thermotolerance of Synechocystis depends on HSP16.6, a phenotype that is enhanced in a deltaClpB1 strain, the effect of mutations can also be assayed in vivo. L66A causes severe defects in thermotolerance, suggesting that oligomeric stability of sHSPs is required for cellular function. This hypothesis was supported by a selection for intragenic suppressors of L66A, which identified mutations that stabilize oligomers of both L66A and wild-type HSP16.6. Analysis of both over- and under-oligomerizing mutants suggests that sHSPs must disassemble before they can release substrates. Furthermore, the suppressor mutations not only restore in vivo activity to L66A, they also ameliorate chaperone defects in vitro, and thus provide the first direct evidence for a chaperone function of an sHSP in cellular thermotolerance.

Biopolymers↗
PubMed2003

Oligomerization-induced modulation of TPR-MET tyrosine kinase activity.

Phosphorylation, although necessary, may not be sufficient to fully activate many receptor tyrosine kinases (RTKs). Oligomerization-induced conformational changes may be necessary to modulate the kinetic properties of RTKs and render them fully functional. To investigate this regulatory mechanism, recombinant TPR-MET, a functionally active oncoprotein derivative of the RTK c-MET, has been expressed and purified for quantitative enzymatic analysis. This naturally occurring oncoprotein contains the cytoplasmic domain of c-MET fused to a coiled coil motif from the nuclear pore complex (TPR). cytoMET, the monomeric analog of TPR-MET, has also been expressed and purified for comparative enzymatic analysis. ATP and peptide substrates have been kinetically characterized for both TPR-MET and cytoMET. Significantly, phosphorylated TPR-MET has smaller Km values for ATP (Km,ATP) and peptide substrates (Km,peptide) and a larger kcat relative to phosphorylated cytoMET. This provides the first direct evidence that receptor oligomerization and not simply activation loop phosphorylation modulates RTK enzymatic activity. The ATP dissociation constants (Kd,ATP) for the two enzymes also displayed significant differences. In contrast, the KI values for the ATP competitive inhibitor staurosporin are similar for the two phosphorylated enzymes. These results suggest that much of the oligomerization-induced kinetic changes occur with respect to peptide substrate binding or catalytic efficiency. The possibility that oligomerization-induced conformational changes occur within the cytoplasmic domain of receptor tyrosine kinases has significant implications for structure-based design of RTK inhibitors and the development of a detailed mechanistic model of RTK activation.

Adenosine Triphosphate↗
PubMed2003

BCL-2 selectively interacts with the BID-induced open conformer of BAK, inhibiting BAK auto-oligomerization.

Caspase-8 cleaves BID to tBID, which targets mitochondria and induces oligomerization of BAX and BAK within the outer membrane, resulting in release of cytochrome c from the organelle. Here, we have initiated these steps in isolated mitochondria derived from control and BCL-2-overexpressing cells using synthetic BH3 peptides and subsequently analyzed the BCL members by chemical cross-linking. The results show that the BH3 domain of BID interacts with and induces an "open" conformation of BAK, exposing the BAK N terminus. This open (activated) conformer of BAK potently induces oligomerization of non-activated ("closed") conformers, causing a cascade of BAK auto-oligomerization. Induction of the open conformation of BAK occurs even in the presence of excess BCL-2, but BCL-2 selectively interacts with this open conformer and blocks BAK oligomerization and cytochrome c release, dependent on the ratio of BID BH3 and BCL-2. This mechanism of inhibition by BCL-2 also occurs in intact cells stimulated with Fas or expressing tBID. Although BID BH3 interacts with both BCL-2 and BAK, the results indicate that when BCL-2 is in excess it can sequester the BID BH3-induced activated conformer of BAK, effectively blocking downstream events. This model suggests that the primary mechanism for BCL-2 blockade targets activated BAK rather than sequestering tBID.

Amino Acid Sequence↗
PubMed2004

Oligomerization of wild type and nonfunctional mutant angiotensin II type I receptors inhibits galphaq protein signaling but not ERK activation.

The 7-transmembrane or G protein-coupled receptors relay signals from hormones and sensory stimuli to multiple signaling systems at the intracellular face of the plasma membrane including heterotrimeric G proteins, ERK1/2, and arrestins. It is an emerging concept that 7-transmembrane receptors form oligomers; however, it is not well understood which roles oligomerization plays in receptor activation of different signaling systems. To begin to address this question, we used the angiotensin II type 1 (AT(1)) receptor, a key regulator of blood pressure and fluid homeostasis that in specific context has been described to activate ERKs without activating G proteins. By using bioluminescence resonance energy transfer, we demonstrate that AT(1) receptors exist as oligomers in transfected COS-7 cells. AT(1) oligomerization was both constitutive and receptor-specific as neither agonist, antagonist, nor co-expression with three other receptors affected the bioluminescence resonance energy transfer 2 signal. Furthermore, the oligomerization occurs early in biosynthesis before surface expression, because we could control AT(1) receptor export from the endoplasmic reticulum or Golgi by using regulated secretion/aggregation technology (RPD trade mark ). Co-expression studies of wild type AT(1) and AT(1) receptor mutants, defective in either ligand binding or G protein and ERK activation, yielded an interesting result. The mutant receptors specifically exerted a dominant negative effect on Galpha(q) activation, whereas ERK activation was preserved. These data suggest that distinctly active conformations of AT(1) oligomers can couple to each of these signaling systems and imply that oligomerization plays an active role in supporting these distinctly active conformations of AT(1) receptors.

Animals↗
PubMed2004

Mutants in a small heat shock protein that affect the oligomeric state. Analysis and allele-specific suppression.

Oligomerization is an essential property of small heat shock proteins (sHSPs) that appears to regulate their chaperone activity. We have examined the role of conserved hydrophobic residues that are postulated to stabilize sHSP oligomers. We identified a mutation of Synechocystis Hsp16.6 that impairs function in vivo and in vitro. The V143A mutation is in the C-terminal extension, a region predicted to form an oligomeric interaction with a hydrophobic region that includes the site of a previously characterized mutation, L66A. Both mutants were dimeric, but V143A had a stronger oligomerization defect than L66A. However, V143A protected a model substrate better than L66A. This suggests that although the two regions both play a role in oligomerization, they are not equivalent. Nevertheless, the addition of either dimeric sHSP enhanced the in vitro chaperone activity of wild type Hsp16.6, consistent with models that the sHSP dimers initiate interactions with substrates. Suppressor analysis of V143A identified mutations in the N terminus that restored activity by restabilizing the oligomer. These mutants were allele-specific and unable to suppress L66A, although they suppressed a dimeric C-terminal truncation of Hsp16.6. Conversely, suppressors of L66A were unable to suppress either V143A or the truncation, although they, like suppressors of V143A, stabilize the Hsp16.6 oligomer. We interpret these data as evidence that the mutations V143A and L66A stabilize two different dimeric structures and as further support that sHSP dimers are active species.

Alleles↗
PubMed2004

Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel.

The insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis undergo several conformational changes from crystal inclusion protoxins to membrane-inserted channels in the midgut epithelial cells of the target insect. Here we analyzed the stability of the different forms of Cry1Ab toxin, monomeric toxin, pre-pore complex, and membrane-inserted channel, after urea and thermal denaturation by monitoring intrinsic tryptophan fluorescence of the protein and 1-anilinonaphthalene-8-sulfonic acid binding to partially unfolded proteins. Our results showed that flexibility of the monomeric toxin was dramatically enhanced upon oligomerization and was even further increased by insertion of the pre-pore into the membrane as shown by the lower concentration of chaotropic agents needed to achieve unfolding of the oligomeric species. The flexibility of the toxin structures is further increased by alkaline pH. We found that the monomer-monomer interaction in the pre-pore is highly stable because urea promotes oligomer denaturation without disassembly. Partial unfolding and limited proteolysis studies demonstrated that domains II and III were less stable and unfold first, followed by unfolding of the most stable domain I, and also that domain I is involved in monomer-monomer interaction. The thermal-induced unfolding and analysis of energy transfer from Trp residues to bound 1-anilinonaphthalene-8-sulfonic acid dye showed that in the membrane-inserted pore domains II and III are particularly sensitive to heat denaturation, in contrast to domain I, suggesting that only domain I may be inserted into the membrane. Finally, the insertion into the membrane of the oligomeric pre-pore structure was not affected by pH. However, a looser conformation of the membrane-inserted domain I induced by neutral or alkaline pH correlates with active channel formation. Our studies suggest for the first time that a more flexible conformation of Cry toxin could be necessary for membrane insertion, and this flexible structure is induced by toxin oligomerization. Finally the alkaline pH found in the midgut lumen of lepidopteran insects could increase the flexibility of membrane-inserted domain I necessary for pore formation.

Anilino Naphthalenesulfonates↗
PubMed2004

Oligomerization of the {gamma}-aminobutyric acid transporter-1 is driven by an interplay of polar and hydrophobic interactions in transmembrane helix II.

The available evidence indicates that members of the neurotransmitter:sodium symporter family form constitutive oligomers. Their second transmembrane helix (TM2) contains a leucine heptad repeat proposed to be involved in oligomerization. In artificial transmembrane segments, interhelical interactions are stabilized by polar residues. We searched for these hydrogen bond donors in TM2 by mutating the five polar residues in TM2 of the gamma-aminobutyric acid transporter-1 (GAT1). We tested the ability of the resulting mutants to oligomerize by fluorescence microscopy, Foerster resonance energy transfer, and beta-lactamase fragment complementation. Of all generated mutants, only Y86A- (but not Y86F-), E101A-, E101Q-, and E101D-GAT1 were judged by these criteria to be deficient in oligomerization and were retained intracellularly. The observations are consistent with a model where the leucine heptad repeat in TM2 drives a homophilic association that is stabilized by Tyr(86) and Glu(101); Tyr(86) participates in hydrophobic stacking. Glu(101) is in the a-position of the leucine heptad repeat (where positions 1-7 are denoted a-g, and each leucine is in the central d-position). Thus, Glu(101) is in the position predicted for the hydrogen bond donor (i.e. sandwiched between Leu(97) and Leu(104), which are one helical turn above and below Glu(101)). These key residues, namely Tyr(86) and Glu(101), are conserved in related transporters from archaeae to humans; they are therefore likely to support oligomeric assembly in transporter orthologs and possibly other proteins with multiple transmembrane segments.

Amino Acid Sequence↗
PubMed2004

The box VII motif of Escherichia coli DnaA protein is required for DnaA oligomerization at the E. coli replication origin.

Escherichia coli DnaA protein initiates DNA replication from the chromosomal origin, oriC, and regulates the frequency of this process. Structure-function studies indicate that the replication initiator comprises four domains. Based on the structural similarity of Aquifex aeolicus DnaA to other AAA+ proteins that are oligomeric, it was proposed that Domain III functions in oligomerization at oriC (Erzberger, J. P., Pirruccello, M. M., and Berger, J. M. (2002) EMBO J. 21, 4763-4773). Because the Box VII motif within Domain III is conserved among DnaA homologues and may function in oligomerization, we substituted conserved Box VII amino acids of E. coli DnaA with alanine by site-directed mutagenesis to examine the role of this motif. All mutant proteins are inactive in initiation from oriC in vivo and in vitro, but they support RK2 plasmid DNA replication in vivo. Thus, RK2 requires only a subset of DnaA functions for plasmid DNA replication. Biochemical studies on a mutant DnaA carrying an alanine substitution at arginine 281 (R281A) in Box VII show that it is inactive in in vitro replication of an oriC plasmid, but this defect is not from the failure to bind to ATP, DnaB in the DnaB-DnaC complex, or oriC. Because the mutant DnaA is also active in the strand opening of oriC, whereas DnaB fails to bind to this unwound region, the open structure is insufficient by itself to load DnaB helicase. Our results show that the mutant fails to form a stable oligomeric DnaA-oriC complex, which is required for the loading of DnaB.

Adenosine Triphosphatases↗
PubMed2005

ATRIP oligomerization is required for ATR-dependent checkpoint signaling.

The ATM and ATR kinases signal cell cycle checkpoint responses to DNA damage. Inactive ATM is an oligomer that is disrupted to form active monomers in response to ionizing radiation. We examined whether ATR is activated by a similar mechanism. We found that the ATRIP subunit of the ATR kinase and ATR itself exist as homooligomers in cells. We did not detect regulation of ATR or ATRIP oligomerization after DNA damage. The predicted coiled-coil domain of ATRIP is essential for ATRIP oligomerization, stable ATR binding, and accumulation of ATRIP at DNA lesions. Additionally, the ATRIP coiled-coil is also required for ATRIP to support ATR-dependent checkpoint signaling to Chk1. Replacing the ATRIP coiled-coil domain with a heterologous dimerization domain restored stable binding to ATR and localization to damage-induced intranuclear foci. Thus, the ATR-ATRIP complex exists in higher order oligomeric states within cells and ATRIP oligomerization is essential for its function.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The core histone N-terminal tail domains function independently and additively during salt-dependent oligomerization of nucleosomal arrays.

Salt-dependent oligomerization of nucleosomal arrays is related to fiber-fiber interactions and global chromosome structure. Previous studies have shown that the H2A/H2B and H3/H4 N-terminal domain (NTD) pairs are able to mediate array oligomerization. However, because of technical barriers, the function(s) of the individual core histone NTDs have not been investigated. To address this question, all possible combinations of "tailless" nucleosomal arrays were assembled from native and NTD-deleted recombinant Xenopus core histones and tandemly repeated 5 S rDNA. The recombinant arrays were characterized by differential centrifugation over the range of 0-50 mm MgCl2 to determine how each NTD affects salt-dependent oligomerization. Results indicate that all core histone NTDs participate in the oligomerization process and that the NTDs function additively and independently. These observations provide direct biochemical evidence linking all four core histone NTDs to the assembly and maintenance of global chromatin structures.

Animals↗