Formation Of Oligomeric Complexes Research Articles

Functional in vitro diversity of an intrinsically disordered plant protein during freeze-thawing is encoded by its structural plasticity.

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix-helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.

Isotopic Substitution Reveals the Importance of Aluminate Diffusion Dynamics in Gibbsite (Al(OH)3) Crystallization from Alkaline Aqueous Solution

Understanding molecular-scale factors governing the precipitation of aluminum hydroxides, such as gibbsite, under alkaline conditions is important for the formation of laterite deposits, as well as aluminum processing. However, mechanisms enabling tetrahedral aluminate ions to assemble into octahedral sites of the gibbsite lattice remain unclear. Formation of oligomeric complexes has been hypothesized as a critical intermediate step. Here, we report a study of gibbsite solubility in highly alkaline solutions using deuterium substitution to probe equilibrium and kinetic factors that could affect oligomeric intermediate formation, including the reactivity and the diffusivity of aluminate ions. When substituting sodium hydroxide with sodium deuteroxide, solution analysis shows a nearly 40% and 50% increase in gibbsite solubility in 2.4 and 3.3 mol·kg–1 total sodium solutions, respectively. Raman spectroscopy indicated that monomeric aluminate ions are the predominant species in solution irrespective of deuteration. However, both 27Al and 23Na nuclear magnetic resonance (NMR) spectroscopy revealed significant differences in chemical shifts in deuterated solutions, and analysis of 1H, 23Na, and 27Al diffusion coefficients with pulsed-field gradient, stimulated echo NMR spectroscopy shows a decrease in ion diffusivity with increasing total deuterium in solution, consistent with the notion of increasing strength in the hydrogen/deuterium bonding network. Because the relative change in 1H diffusion coefficients are commensurate with the difference in apparent solubility constants, there is evidence for an oligomeric intermediate whose steady-state concentration is maintained by the collision frequency of aluminate ions.

A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion

The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD, and MakE. MakA, MakB, and MakE were readily detected in culture supernatants of wild-type V. cholerae, whereas secretion was very much reduced from a flagellum-deficient mutant. Crystal structures of MakA, MakB, and MakE revealed a structural relationship to a superfamily of bacterial pore-forming toxins. Expression of MakA/B/E in Escherichia coli resulted in toxicity toward Caenorhabditis elegans used as a predatory model organism. None of these Mak proteins alone or in pairwise combinations were cytolytic, but an equimolar mixture of MakA, MakB, and MakE acted as a tripartite cytolytic toxin invitro, causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining the assembly of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity toward C. elegans Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen Vibrio anguillarum We suggest that the hitherto-unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms.

MRNA Targeting, Transport and Local Translation in Eukaryotic Cells: From the Classical View to a Diversity of New Concepts.

Spatial organization of protein biosynthesis in the eukaryotic cell has been studied for more than fifty years, thus many facts have already been included in textbooks. According to the classical view, mRNA transcripts encoding secreted and transmembrane proteins are translated by ribosomes associated with endoplasmic reticulum membranes, while soluble cytoplasmic proteins are synthesized on free polysomes. However, in the last few years, new data has emerged, revealing selective translation of mRNA on mitochondria and plastids, in proximity to peroxisomes and endosomes, in various granules and at the cytoskeleton (actin network, vimentin intermediate filaments, microtubules and centrosomes). There are also long-standing debates about the possibility of protein synthesis in the nucleus. Localized translation can be determined by targeting signals in the synthesized protein, nucleotide sequences in the mRNA itself, or both. With RNA-binding proteins, many transcripts can be assembled into specific RNA condensates and form RNP particles, which may be transported by molecular motors to the sites of active translation, form granules and provoke liquid-liquid phase separation in the cytoplasm, both under normal conditions and during cell stress. The translation of some mRNAs occurs in specialized “translation factories,” assemblysomes, transperons and other structures necessary for the correct folding of proteins, interaction with functional partners and formation of oligomeric complexes. Intracellular localization of mRNA has a significant impact on the efficiency of its translation and presumably determines its response to cellular stress. Compartmentalization of mRNAs and the translation machinery also plays an important role in viral infections. Many viruses provoke the formation of specific intracellular structures, virus factories, for the production of their proteins. Here we review the current concepts of the molecular mechanisms of transport, selective localization and local translation of cellular and viral mRNAs, their effects on protein targeting and topogenesis, and on the regulation of protein biosynthesis in different compartments of the eukaryotic cell. Special attention is paid to new systems biology approaches, providing new cues to the study of localized translation.

Advancement of research on plant NLRs evolution, biochemical activity, structural association, and engineering.

In this review, we have included evolution of plant intracellular immune receptors, oligomeric complex formation, enzymatic action, engineering, and mechanisms of immune inspection for appropriate defense outcomes. NLR (Nucleotide binding oligomerization domain containing leucine-rich repeat) proteins are the intracellular immune receptors that recognize pathogen-derived virulence factors to confer effector-triggered immunity (ETI). Activation of plant defense by the NLRs are often conveyed through N-terminal Toll-like/ IL-1 receptor (TIR) or non-TIR (coiled-coils or CC) domains. Homodimerization or self-association property of CC/ TIR domains of plant NLRs contribute to their auto-activity and induction of in planta ectopic cell death. High resolution crystal structures of Arabidopsis thaliana RPS4TIR, L6TIR, SNC1TIR, RPP1TIR and Muscadinia rotundifolia RPV1TIR showed that interaction is mediated through one or two distinct interfaces i.e., αA and αE helices comprise AE interface and αD and αE helices were found to form DE interface. By contrast, conserved helical regions were determined for CC domains of plant NLRs. Evolutionary history of NLRs diversification has shown that paired forms were originated from NLR singletons. Plant TIRs executed NAD+ hydrolysis activity for cell death promotion. Plant NLRs were found to form large oligomeric complexes as observed in animal inflammasomes. We have also discussed different protein engineering methods includes domain shuffling, and decoy modification that increase effector recognition spectrum of plant NLRs. In summary, our review highlights structural basis of perception of the virulence factors by NLRs or NLR pairs to design novel classes of plant immune receptors.

Identification of substrate-specific inhibitors of cathepsin K through high-throughput screening.

Cathepsin K (CatK) is a cysteine protease and drug target for skeletal disorders that is known for its potent collagenase and elastase activity. The formation of oligomeric complexes of CatK in the presence of glycosaminoglycans has been associated with its collagenase activity. Inhibitors that disrupt these complexes can selectively block the collagenase activity without interfering with the other regulatory proteolytic activities of the enzyme. Here, we have developed a fluorescence polarization (FP) assay to screen 4761 compounds for substrate-specific ectosteric collagenase inhibitors of CatK. A total of 38 compounds were identified that block the collagenase activity without interfering with the hydrolysis of active site substrates such as the synthetic peptide substrate, benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, and gelatin. The identified inhibitors can be divided into two main classes, negatively charged and polyaromatic compounds which suggest the binding to different ectosteric sites. Two of the inhibitors were highly effective in preventing the bone-resorption activity of CatK in osteoclasts. Interestingly, some of the ectosteric inhibitors were capable of differentiating between the collagenase and elastase activity of CatK depending on the ectosteric site utilized by the compound. Owing to their substrate-specific selectivity, ectosteric inhibitors represent a viable alternative to side effect-prone active site-directed inhibitors.

Epsilon toxin from Clostridium perfringens induces cytotoxicity in FRT thyroid epithelial cells

Epsilon toxin (Etx) is produced by Clostridium perfringens and induces enterotoxemia in ruminants. Etx crosses the blood-brain barrier, binds to myelin structures, and kills oligodendrocytes, inducing central nervous system demyelination. In addition, Etx has a cytotoxic effect on distal and collecting kidney tubules. There are few cell lines sensitive to Etx. At present, the most sensitive in vitro model for Etx is the Madin-Darby canine kidney (MDCK) cell line, where Etx oligomerizes and forms a pore with consequent ion efflux and cell death. Although the Etx receptor has not yet been fully clarified, it is known that caveolin 1 and 2 potentiate Etx cytotoxicity and oligomerization, and more recently, the myelin and lymphocyte (MAL) protein has been implicated in Etx binding and activity. Here, we studied the effect of Etx on Fischer rat thyroid cells (FRT) and observed similar effects as those seen in MDCK cells. Etx incubated with FRT cells showed binding to the plasma membrane, and western blotting assays revealed oligomeric complex formation. Moreover, cytotoxic assays on FRT cells after Etx incubation indicated cell death at a similar level as in MDCK cells. In addition, a luminescent ATP detection assay revealed ATP depletion in FRT cells after Etx exposure. Previous studies have reported that FRT cells do not express caveolins and do not form caveolae but express MAL protein in glycolipid-enriched membrane microdomains. Our results indicate that caveolins are not directly implicated in Etx cytotoxicity, supporting the notion that the MAL protein is involved in Etx action. In addition, a cell line of thyroid origin is described for the first time as a good model to study Etx action.

LDB2 regulates the expression of DLL4 through the formation of oligomeric complexes in endothelial cells.

Delta-like ligand 4 (DLL4) expression in endothelial cells is intimately associated with angiogenic sprouting and vascular remodeling, but the precise mechanism of transcriptional regulation of DLL4 remains incompletely understood. Here, we showed that LIM-domain binding protein 2 (LDB2) plays an important role in regulating basal DLL4 and VEGF-induced DLL4 expression. Knockdown of LDB2 using siRNA enhanced endothelial sprouting and tubular network formation in vitro. Injection of ldb2-morpholino resulted in defective development of intersegmental vessels in zebrafish. Reduction or overexpression of LDB2 in endothelial cells decreased or increased DLL4 expression. LDB2 regulated DLL4 promoter activity by binding to its promoter region and the same promoter region was occupied and regulated by the LMO2/TAL1/GATA2 complex. Interestingly, LDB2 also mediated VEGF-induced DLL4 expression in endothelial cells. The regulation of DLL4 by the LDB2 complex provides a novel mechanism of DLL4 transcriptional control that may be exploited to develop therapeutics for aberrant vascular remodeling.

Membrane Protein Production in E. coli Lysates in Presence of Preassembled Nanodiscs.

Cell-free expression allows to synthesize membrane proteins in completely new formats that can relatively easily be customized for particular applications. Amphiphilic superstructures such as micelles, lipomicelles, or nanodiscs can be provided as nano-devices for the solubilization of membrane proteins. Defined empty bilayers in the form of nanodiscs offer native like environments for membrane proteins, supporting functional folding, proper oligomeric assembly as well as stability. Even very difficult and detergent-sensitive membrane proteins can be addressed by the combination of nanodisc technology with efficient cell-free expression systems as the direct co-translational insertion of nascent membrane proteins into supplied preassembled nanodiscs is possible. This chapter provides updated protocols for the synthesis of membrane proteins in presence of preassembled nanodiscs suitable for emerging applications such as screening of lipid effects on membrane protein function and the modulation of oligomeric complex formation.

Complex structure of the fission yeast SREBP-SCAP binding domains reveals an oligomeric organization.

Sterol regulatory element-binding protein (SREBP) transcription factors are master regulators of cellular lipid homeostasis in mammals and oxygen-responsive regulators of hypoxic adaptation in fungi. SREBP C-terminus binds to the WD40 domain of SREBP cleavage-activating protein (SCAP), which confers sterol regulation by controlling the ER-to-Golgi transport of the SREBP-SCAP complex and access to the activating proteases in the Golgi. Here, we biochemically and structurally show that the carboxyl terminal domains (CTD) of Sre1 and Scp1, the fission yeast SREBP and SCAP, form a functional 4:4 oligomer and Sre1-CTD forms a dimer of dimers. The crystal structure of Sre1-CTD at 3.5 Å and cryo-EM structure of the complex at 5.4 Å together with in vitro biochemical evidence elucidate three distinct regions in Sre1-CTD required for Scp1 binding, Sre1-CTD dimerization and tetrameric formation. Finally, these structurally identified domains are validated in a cellular context, demonstrating that the proper 4:4 oligomeric complex formation is required for Sre1 activation.

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis.

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones

Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone.

The cytolytic activity of vaginolysin strictly depends on cholesterol and is potentiated by human CD59.

Gardnerella vaginalis produces cytolysin vaginolysin (VLY), which has been suggested to be a contributor to bacterial vaginosis pathogenesis. VLY along with intermedilysin (ILY) from Streptococcus intermedius have been attributed to a group of cholesterol-dependent cytolysins (CDCs) whose pore-forming activity depends on human CD59 (hCD59). Here, we show that different types of cells lacking hCD59 are susceptible to VLY-mediated lysis, albeit to different extents. We analyze the effects of both hCD59 and cholesterol on VLY cytolytic activity. We show that VLY binds to cholesterol-rich membranes of non-human cells, while VLY with an impaired cholesterol recognition site retains binding to the hCD59-containing cells. We further demonstrate that cholesterol binding by VLY is sufficient to trigger the formation of oligomeric complexes on cholesterol rich-liposomes lacking hCD59. Thus, VLY may induce cell lysis following two alternative pathways. One requires only cholesterol and does not depend on hCD59. The second pathway involves hCD59 contribution similarly to ILY. Apparently, under physiological conditions VLY acts in the most effective way by accepting the assistance of hCD59.

The secrets of dermcidin action

Antimicrobial peptides (AMPs) are important effector molecules of the innate immune defense of diverse species. The majority of known AMPs are cationic therefore facilitating the initial binding of the positively charged peptides to the negatively charged bacterial membrane. Dermcidin (DCD) is constitutively expressed in eccrine sweat glands, secreted into sweat and transported to the epidermal surface where it is proteolytically processed giving rise to several truncated DCD peptides. Its processed forms such as the anionic 48mer DCD-1L and the 47mer DCD-1 possess antimicrobial activity against numerous bacteria including Staphylococcus aureus. Here, the latest knowledge regarding the mode of action of the anionic DCD-1(L) and the functional consequences of their interaction with bacterial membranes is reviewed. There is evidence that the interaction of DCD-1(L) with negatively charged bacterial phospholipids leads to Zn2+ dependent formation of oligomeric complexes in the bacterial membrane, which subsequently leads to ion channel formation resulting in membrane depolarization and bacterial cell death.

Intracellular complexes of the early-onset torsion dystonia-associated AAA+ ATPase TorsinA.

A single GAG codon deletion in the gene encoding torsinA is linked to most cases of early-onset torsion dystonia. TorsinA is an ER-localized membrane-associated ATPase from the AAA+ superfamily with an unknown biological function. We investigated the formation of oligomeric complexes of torsinA in cultured mammalian cells and found that wild type torsinA associates into a complex with a molecular weight consistent with that of a homohexamer. Interestingly, the dystonia-linked variant torsinAΔE displayed a reduced propensity to form the oligomers compared to the wild type protein. We also discovered that the deletion of the N-terminal membrane-associating region of torsinA abolished oligomer formation. Our results demonstrate that the dystonia-linked mutation in the torsinA gene produces a protein variant that is deficient in maintaining its oligomeric state and suggest that ER membrane association is required to stabilize the torsinA complex.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-743) contains supplementary material, which is available to authorized users.

DcHsp17.7, a small heat shock protein in carrot, is tissue-specifically expressed under salt stress and confers tolerance to salinity

The expression and function of DcHsp17.7, a small heat shock protein in carrot (Daucus carota L.), were examined under salt stress, which is an exacerbating environmental condition due to water shortage and irrigation. DcHsp17.7 was constitutively expressed in leaf and stem tissues under normal growth conditions. Upon exposure to 300 mM NaCl, the protein level of DcHsp17.7 increased dramatically in leaf tissue, but did not significantly change in stem tissue. Native-PAGE analysis showed tissue-specific oligomer formation. Under normal growth conditions, DcHsp17.7 was found in an approximately 240 kDa complex in both tissues. However, NaCl treatment induced an additional approximately 160 kDa complex containing DcHsp17.7. This occurred only in leaf tissue, suggesting tissue-specific oligomeric complex formation. To examine the functional mechanism of DcHsp17.7 under stress conditions, the DcHsp17.7 coding gene was introduced into Escherichia coli. Heterologous expression of DcHsp17.7 was induced by isopropyl β-d-1-thiogalactopyranoside treatment. Upon exposure to salinity, protein levels of DcHsp17.7 decreased, and the protein was not detected after 16 hours. Native-PAGE analysis showed that DcHsp17.7 was present in an approximately 250 kDa complex both before and after salt treatment. Salinity reduced bacterial cell viability; however, the transgenic E. coli expressing DcHsp17.7 exhibited a higher survival rate than control E. coli under salt stress. When the level of soluble proteins was measured under salt stress, transgenic E. coli expressing DcHsp17.7 reproducibly showed slightly higher levels than control cells. This suggests that DcHsp17.7 performs molecular chaperone activity in salt-stressed transgenic E. coli. Our results suggest that DcHsp17.7 is likely to be involved in tolerance not only to thermal stresses but also to other abiotic stresses, such as salinity.

Multimerization of Staufen1 in live cells

Transport of mRNA is an efficient mechanism to target proteins to specific regions of a cell. Although it is well documented that mRNAs are transported in ribonucleoprotein (RNP) complexes, several of the mechanisms involved in complex formation and localization are poorly understood. Staufen (Stau) 1, a double-stranded RNA-binding protein, is a well accepted marker of mRNA transport complexes. In this manuscript, we provide evidence that Stau1 self-associates in live cells using immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays. The double-stranded RNA-binding domains dsRBD3 and dsRBD4 contributed about half of the signal, suggesting that Stau1 RNA-binding activity is involved in Stau1 self-association. Protein-protein interaction also occurred, via dsRBD5 and dsRBD2, as shown by in vitro pull-down, yeast two-hybrid, and BRET assays in live cells. Interestingly, Stau1 self-association contributes to the formation of oligomeric complexes as evidenced by the coexpression of split Renilla luciferase halves covalently linked to Stau1 in a protein complementation assay (PCA) combined with a BRET assay with Stau1-YFP. Moreover, we showed that these higher-order Stau1-containing complexes carry RNAs when the RNA stain SYTO 14 was used as the energy acceptor in the PCA/BRET assay. The oligomeric composition of Stau1-containing complexes and the presence of specific mRNAs have been confirmed by biochemical approaches involving two successive immunoprecipitations of Stau1-tagged molecules followed by qRT-PCR amplification. Altogether, these results indicate that Stau1 self-associates in mRNPs via its multiple functional domains that can select mRNAs to be transported and establish protein-protein interaction.

Transmembrane Domain Interactions Control Biological Functions of Neuropilin-1

Neuropilin-1 (NRP1) is a transmembrane receptor playing a pivotal role in the control of semaphorins and VEGF signaling pathways. The exact mechanism controlling semaphorin receptor complex formation is unknown. A structural analysis and modeling of NRP1 revealed a putative dimerization GxxxG motif potentially important for NRP1 dimerization and oligomerization. Our data show that this motif mediates the dimerization of the transmembrane domain of NRP1 as demonstrated by a dimerization assay (ToxLuc assay) performed in natural membrane and FRET analysis. A synthetic peptide derived from the transmembrane segment of NRP1 abolished the inhibitory effect of Sema3A. This effect depends on the capacity of the peptide to interfere with NRP1 dimerization and the formation of oligomeric complexes. Mutation of the GxxxG dimerization motif in the transmembrane domain of NRP1 confirmed its biological importance for Sema3A signaling. Overall, our results shed light on an essential step required for semaphorin signaling and provide novel evidence for the crucial role of transmembrane domain of bitopic protein containing GxxxG motif in the formation of receptor complexes that are a prerequisite for cell signaling.

Interactions of Human Rad54 Protein with Branched DNA Molecules

The Rad54 protein plays an important role during homologous recombination in eukaryotes. The protein belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that yeast and human Rad54 (hRad54) specifically bind to Holliday junctions and promote branch migration. Here we examined the minimal DNA structural requirements for optimal hRad54 ATPase and branch migration activity. Although a 12-bp double-stranded DNA region of branched DNA is sufficient to induce ATPase activity, the minimal substrate that gave rise to optimal stimulation of the ATP hydrolysis rate consisted of two short double-stranded DNA arms, 15 bp each, combined with a 45-nucleotide single-stranded DNA branch. We showed that hRad54 binds preferentially to the open and not to the stacked conformation of branched DNA. Stoichiometric titration of hRad54 revealed formation of two types of hRad54 complexes with branched DNA substrates. The first of them, a dimer, is responsible for the ATPase activity of the protein. However, branch migration activity requires a significantly higher stoichiometry of hRad54, approximately 10 +/- 2 protein monomers/DNA molecule. This pleomorphism of hRad54 in formation of oligomeric complexes with DNA may correspond to multiple functions of the protein in homologous recombination.