ConBr lectin modulates MAPKs and Akt pathways and triggers autophagic glioma cell death by a mechanism dependent upon caspase-8 activation.

Glioblastoma multiforme is the most aggressive type of glioma, with limited treatment and poor prognosis. Despite some advances over the last decade, validation of novel and selective antiglioma agents remains a challenge in clinical pharmacology. Prior studies have shown that leguminous lectins may exert various biological effects, including antitumor properties. Accordingly, this study aimed to evaluate the mechanisms underlying the antiglioma activity of ConBr, a lectin extracted from the Canavalia brasiliensis seeds. ConBr at lower concentrations inhibited C6 glioma cell migration while higher levels promoted cell death dependent upon carbohydrate recognition domain (CRD) structure. ConBr increased p38MAPK and JNK and decreased ERK1/2 and Akt phosphorylation. Moreover, ConBr inhibited mTORC1 phosphorylation associated with accumulation of autophagic markers, such as acidic vacuoles and LC3 cleavage. Inhibition of early steps of autophagy with 3-methyl-adenine (3-MA) partially protected whereas the later autophagy inhibitor Chloroquine (CQ) had no protective effect upon ConBr cytotoxicity. ConBr also augmented caspase-3 activation without affecting mitochondrial function. Noteworthy, the caspase-8 inhibitor IETF-fmk attenuated ConBr induced autophagy and C6 glioma cell death. Finally, ConBr did not show cytotoxicity against primary astrocytes, suggesting a selective antiglioma activity. In summary, our results indicate that ConBr requires functional CRD lectin domain to exert antiglioma activity, and its cytotoxicity is associated with MAPKs and Akt pathways modulation and autophagy- and caspase-8- dependent cell death.

Structural and Functional Characterization of the Globin-Coupled Sensors of Azotobacter vinelandii and Bordetella pertussis.

Aims: Structural and functional characterization of the globin-coupled sensors (GCSs) from Azotobacter vinelandii (AvGReg) and Bordetella pertussis (BpeGReg). Results: Ultraviolet/visible and resonance Raman spectroscopies confirm the presence in AvGReg and BpeGReg of a globin domain capable of reversible gaseous ligand binding. In AvGReg, an influence of the transmitter domain on the heme proximal region of the globin domain can be seen, and k'CO is higher than for other GCSs. The O2 binding kinetics suggests the presence of an open and a closed conformation. As for BpeGReg, the fully oxygenated AvGReg show a very high diguanylate cyclase activity. The carbon monoxide rebinding to BpeGReg indicates that intra- and intermolecular interactions influence the ligand binding. The globin domains of both proteins (AvGReg globin domain and BpeGRegGb with cysteines (Cys16, 45, 114, 154) mutated to serines [BpeGReg-Gb*]) share the same GCS fold, a similar proximal but a different distal side structure. They homodimerize through a G-H helical bundle as in other GCSs. However, BpeGReg-Gb* shows also a second dimerization mode. Innovation: This article extends our knowledge on the GCS proteins and contributes to a better understanding of the GCSs role in the formation of bacterial biofilms. Conclusions:AvGReg and BpeGReg conform to the GCS family, share a similar overall structure, but they have different properties in terms of the ligand binding. In particular, AvGReg shows an open and a closed conformation that in the latter form will very tightly bind oxygen. BpeGReg has only one closed conformation. In both proteins, it is the fully oxygenated GCS form that catalyzes the production of the second messenger.

Examination of ClpB Quaternary Structure and Linkage to Nucleotide Binding.

Escherichia coli caseinolytic peptidase B (ClpB) is a molecular chaperone with the unique ability to catalyze protein disaggregation in collaboration with the KJE system of chaperones. Like many AAA+ molecular motors, ClpB assembles into hexameric rings, and this reaction is thermodynamically linked to nucleotide binding. Here we show that ClpB exists in a dynamic equilibrium of monomers, dimers, tetramers, and hexamers in the presence of both limiting and excess ATPγS. We find that ClpB monomer is only able to bind one nucleotide, whereas all 12 sites in the hexameric ring are bound by nucleotide at saturating concentrations. Interestingly, dimers and tetramers exhibit stoichiometries of ∼3 and 7, respectively, which is one fewer than the maximum number of binding sites in the formed oligomer. This observation suggests an open conformation for the intermediates based on the need for an adjacent monomer to fully form the binding pocket. We also report the protein-protein interaction constants for dimers, tetramers, and hexamers and their dependencies on nucleotide. These interaction constants make it possible to predict the concentration of hexamers present and able to bind to cochaperones and polypeptide substrates. Such information is essential for the interpretation of many in vitro studies. Finally, the strategies presented here are broadly applicable to a large number of AAA+ molecular motors that assemble upon nucleotide binding and interact with partner proteins.

Transmembrane peptides as unique tools to demonstrate the in vivo action of a cross-class GPCR heterocomplex.

Angiotensin (ANGII) and secretin (SCT) share overlapping, interdependent osmoregulatory functions in brain, where SCT peptide/receptor function is required for ANGII action, yet the molecular basis is unknown. Since receptors for these peptides (AT1aR, SCTR) are coexpressed in osmoregulatory centers, a possible mechanism is formation of a cross-class receptor heterocomplex. Here, we demonstrate such a complex and its functional importance to modulate signaling. Association of AT1aR with SCTR reduced ability of SCT to stimulate cyclic adenosine monophosphate (cAMP), with signaling augmented in presence of ANGII or constitutively active AT1aR. Several transmembrane (TM) peptides of these receptors were able to affect their conformation within complexes, reducing receptor BRET signals. AT1aR TM1 affected only formation and activity of the heterocomplex, without effect on homomers of either receptor, and reduced SCT-stimulated cAMP responses in cells expressing both receptors. This peptide was active in vivo by injection into mouse lateral ventricle, thereby suppressing water-drinking behavior after hyperosmotic shock, similar to SCTR knockouts. This supports the interpretation that active conformation of AT1aR is a key modulator of cAMP responses induced by SCT stimulation of SCTR. The SCTR/AT1aR complex is physiologically important, providing differential signaling to SCT in settings of hyperosmolality or food intake, modulated by differences in levels of ANGII.

Lateral assembly of the immunoglobulin protein SynCAM 1 controls its adhesive function and instructs synapse formation.

Synapses are specialized adhesion sites between neurons that are connected by protein complexes spanning the synaptic cleft. These trans-synaptic interactions can organize synapse formation, but their macromolecular properties and effects on synaptic morphology remain incompletely understood. Here, we demonstrate that the synaptic cell adhesion molecule SynCAM 1 self-assembles laterally via its extracellular, membrane-proximal immunoglobulin (Ig) domains 2 and 3. This cis oligomerization generates SynCAM oligomers with increased adhesive capacity and instructs the interactions of this molecule across the nascent and mature synaptic cleft. In immature neurons, cis assembly promotes the adhesive clustering of SynCAM 1 at new axo-dendritic contacts. Interfering with the lateral self-assembly of SynCAM 1 in differentiating neurons strongly impairs its synaptogenic activity. At later stages, the lateral oligomerization of SynCAM 1 restricts synaptic size, indicating that this adhesion molecule contributes to the structural organization of synapses. These results support that lateral interactions assemble SynCAM complexes within the synaptic cleft to promote synapse induction and modulate their structure. These findings provide novel insights into synapse development and the adhesive mechanisms of Ig superfamily members.

Axial and radial forces of cross-bridges depend on lattice spacing.

Nearly all mechanochemical models of the cross-bridge treat myosin as a simple linear spring arranged parallel to the contractile filaments. These single-spring models cannot account for the radial force that muscle generates (orthogonal to the long axis of the myofilaments) or the effects of changes in filament lattice spacing. We describe a more complex myosin cross-bridge model that uses multiple springs to replicate myosin's force-generating power stroke and account for the effects of lattice spacing and radial force. The four springs which comprise this model (the 4sXB) correspond to the mechanically relevant portions of myosin's structure. As occurs in vivo, the 4sXB's state-transition kinetics and force-production dynamics vary with lattice spacing. Additionally, we describe a simpler two-spring cross-bridge (2sXB) model which produces results similar to those of the 4sXB model. Unlike the 4sXB model, the 2sXB model requires no iterative techniques, making it more computationally efficient. The rate at which both multi-spring cross-bridges bind and generate force decreases as lattice spacing grows. The axial force generated by each cross-bridge as it undergoes a power stroke increases as lattice spacing grows. The radial force that a cross-bridge produces as it undergoes a power stroke varies from expansive to compressive as lattice spacing increases. Importantly, these results mirror those for intact, contracting muscle force production.

Imaging-based identification of a critical regulator of FtsZ protofilament curvature in Caulobacter.

FtsZ is an essential bacterial GTPase that polymerizes at midcell, recruits the division machinery, and may generate constrictive forces necessary for cytokinesis. However, many of the mechanistic details underlying these functions are unknown. We sought to identify FtsZ-binding proteins that influence FtsZ function in Caulobacter crescentus. Here, we present a microscopy-based screen through which we discovered two FtsZ-binding proteins, FzlA and FzlC. FzlA is conserved in α-proteobacteria and was found to be functionally critical for cell division in Caulobacter. FzlA altered FtsZ structure both in vivo and in vitro, forming stable higher-order structures that were resistant to depolymerization by MipZ, a spatial determinant of FtsZ assembly. Electron microscopy revealed that FzlA organizes FtsZ protofilaments into striking helical bundles. The degree of curvature induced by FzlA depended on the nucleotide bound to FtsZ. Induction of FtsZ curvature by FzlA carries implications for regulating FtsZ function by modulating its superstructure.

Sensing domain dynamics in protein kinase A-I{alpha} complexes by solution X-ray scattering.

The catalytic (C) and regulatory (R) subunits of protein kinase A are exceptionally dynamic proteins. Interactions between the R- and C-subunits are regulated by cAMP binding to the two cyclic nucleotide-binding domains in the R-subunit. Mammalian cells express four different isoforms of the R-subunit (RIalpha, RIbeta, RIIalpha, and RIIbeta) that all interact with the C-subunit in different ways. Here, we investigate the dynamic behavior of protein complexes between RIalpha and C-subunits using small angle x-ray scattering. We show that a single point mutation in RIalpha, R333K (which alters the cAMP-binding properties of Domain B) results in a compact shape compared with the extended shape of the wild-type R.C complex. A double mutant complex that disrupts the interaction site between the C-subunit and Domain B in RIalpha, RIalpha(AB)R333K.C(K285P), results in a broader P(r) curve that more closely resembles the P(r) profiles of wild-type complexes. These results together suggest that interactions between RIalpha Domain B and the C-subunit in the RIalpha.C complex involve large scale dynamics that can be disrupted by single point mutations in both proteins. In contrast to RIalpha.C complexes. Domain B in the RIIbeta.C heterodimer is not dynamic and is critical for both inhibition and complex formation. Our study highlights the functional differences of domain dynamics between protein kinase A isoforms, providing a framework for elucidating the global organization of each holoenzyme and the cross-talk between the R- and C-subunits.

Crystal structure of epstein-barr virus DNA polymerase processivity factor BMRF1.

The DNA polymerase processivity factor of the Epstein-Barr virus, BMRF1, associates with the polymerase catalytic subunit, BALF5, to enhance the polymerase processivity and exonuclease activities of the holoenzyme. In this study, the crystal structure of C-terminally truncated BMRF1 (BMRF1-DeltaC) was solved in an oligomeric state. The molecular structure of BMRF1-DeltaC shares structural similarity with other processivity factors, such as herpes simplex virus UL42, cytomegalovirus UL44, and human proliferating cell nuclear antigen. However, the oligomerization architectures of these proteins range from a monomer to a trimer. PAGE and mutational analyses indicated that BMRF1-DeltaC, like UL44, forms a C-shaped head-to-head dimer. DNA binding assays suggested that basic amino acid residues on the concave surface of the C-shaped dimer play an important role in interactions with DNA. The C95E mutant, which disrupts dimer formation, lacked DNA binding activity, indicating that dimer formation is required for DNA binding. These characteristics are similar to those of another dimeric viral processivity factor, UL44. Although the R87E and H141F mutants of BMRF1-DeltaC exhibited dramatically reduced polymerase processivity, they were still able to bind DNA and to dimerize. These amino acid residues are located near the dimer interface, suggesting that BMRF1-DeltaC associates with the catalytic subunit BALF5 around the dimer interface. Consequently, the monomeric form of BMRF1-DeltaC probably binds to BALF5, because the steric consequences would prevent the maintenance of the dimeric form. A distinctive feature of BMRF1-DeltaC is that the dimeric and monomeric forms might be utilized for the DNA binding and replication processes, respectively.

C-terminal elongation of growth-blocking peptide enhances its biological activity and micelle binding affinity.

Growth-blocking peptide (GBP) is a hormone-like peptide that suppresses the growth of the host armyworm. Although the 23-amino acid GBP (1-23 GBP) is expressed in nonparasitized armyworm plasma, the parasitization by wasp produces the 28-amino acid GBP (1-28 GBP) through an elongation of the C-terminal amino acid sequence. In this study, we characterized the GBP variants, which consist of various lengths of the C-terminal region, by comparing their biological activities and three-dimensional structures. The results of an injection study indicate that 1-28 GBP most strongly suppresses larval growth. NMR analysis shows that these peptides have basically the same tertiary structures and that the extension of the C-terminal region is disordered. However, the C-terminal region of 1-28 GBP undergoes a conformational transition from a random coiled state to an alpha-helical state in the presence of dodecylphosphocholine micelles. This suggests that binding of the C-terminal region would affect larval growth activity.

Structural basis for the mechanism of respiratory complex I.

Complex I plays a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation. The mechanism of this highly efficient enzyme is currently unknown. Mitochondrial complex I is a major source of reactive oxygen species, which may be one of the causes of aging. Dysfunction of complex I is implicated in many human neurodegenerative diseases. We have determined several x-ray structures of the oxidized and reduced hydrophilic domain of complex I from Thermus thermophilus at up to 3.1 A resolution. The structures reveal the mode of interaction of complex I with NADH, explaining known kinetic data and providing implications for the mechanism of reactive oxygen species production at the flavin site of complex I. Bound metals were identified in the channel at the interface with the frataxin-like subunit Nqo15, indicating possible iron-binding sites. Conformational changes upon reduction of the complex involve adjustments in the nucleotide-binding pocket, as well as small but significant shifts of several alpha-helices at the interface with the membrane domain. These shifts are likely to be driven by the reduction of nearby iron-sulfur clusters N2 and N6a/b. Cluster N2 is the electron donor to quinone and is coordinated by unique motif involving two consecutive (tandem) cysteines. An unprecedented "on/off switch" (disconnection) of coordinating bonds between the tandem cysteines and this cluster was observed upon reduction. Comparison of the structures suggests a novel mechanism of coupling between electron transfer and proton translocation, combining conformational changes and protonation/deprotonation of tandem cysteines.

[DNA and oligosaccharides stimulate oligomerization of human milk lactoferrin].

Lactoferrin (LF), the glycoprotein transferring Fe+ ions, is contained in barrier liquids, human blood and milk. LF is an acute phase protein and one of the most important factors of nonspecific defense. The protein has unique set of biological functions. Using the methods of small-angle X-ray scattering and light-scattering it was shown for the first time that addition of DNA and oligosaccharides to LF with different level of initial oligomerization leads to an enhance in the oligomerization rate. 1M NaCl stimulates almost a complete dissociation of LF oligomeric complexes obtained in the pesence of DNA, oligosaccharides, or early founded oligomerization effectors (nucleotides). It was shown that LF oligomeric complexes obtained in the presence of different oligomerization effectors have different stability. Incubation with 50 mM MgCl2 leads to complete destruction of the protein complexes formed by ATP and oligosaccharide but partially dissociate the complexes with following formation of new in the case of AMP- and d(pT)10-dependent associates, which possess higher stability i n presence of the salt. A possible role of LF oligomerization for different biological functions of the protein is discussed.

Recent advances in structure-functional studies of mitochondrial factor B.

Since the early studies on the resolution and reconstitution of the oxidative phosphorylation system from animal mitochondria, coupling factor B was recognized as an essential component of the machinery responsible for energy-driven ATP synthesis. At the phenomenological level, factor B was agreed to lie at the interface of energy transfer between the respiratory chain and the ATP synthase complex. However, biochemical characterization of the factor B polypeptide has proved difficult. It was not until 1990 that the N-terminal amino acid sequence of bovine mitochondrial factor B was reported, which followed, a decade later, by the report describing the amino acid sequence of full-length human factor B and its functional characterization. The present review summarizes the recent advances in structure-functional studies of factor B, including its recently determined crystal structure at 0.96 A resolution. Ectopic expression of human factor B in cultured animal cells has unexpectedly revealed its role in shaping mitochondrial morphology. The supramolecular assembly of ATP synthase as dimer ribbons at highly curved apices of the mitochondrial cristae was recently suggested to optimize ATP synthesis under proton-limited conditions. We propose that the binding of the ATP synthase dimers with factor B tetramers could be a means to enhance the efficiency of the terminal step of oxidative phosphorylation in animal mitochondria.

Tetrameric Orai1 is a teardrop-shaped molecule with a long, tapered cytoplasmic domain.

The Ca(2+) release-activated Ca(2+) channel is a principal regulator of intracellular Ca(2+) rise, which conducts various biological functions, including immune responses. This channel, involved in store-operated Ca(2+) influx, is believed to be composed of at least two major components. Orai1 has a putative channel pore and locates in the plasma membrane, and STIM1 is a sensor for luminal Ca(2+) store depletion in the endoplasmic reticulum membrane. Here we have purified the FLAG-fused Orai1 protein, determined its tetrameric stoichiometry, and reconstructed its three-dimensional structure at 21-A resolution from 3681 automatically selected particle images, taken with an electron microscope. This first structural depiction of a member of the Orai family shows an elongated teardrop-shape 150A in height and 95A in width. Antibody decoration and volume estimation from the amino acid sequence indicate that the widest transmembrane domain is located between the round extracellular domain and the tapered cytoplasmic domain. The cytoplasmic length of 100A is sufficient for direct association with STIM1. Orifices close to the extracellular and intracellular membrane surfaces of Orai1 seem to connect outside the molecule to large internal cavities.

Modulation of the oligomerization state of p53 by differential binding of proteins of the S100 family to p53 monomers and tetramers.

We investigated the ways S100B, S100A1, S100A2, S100A4, and S100A6 bind to the different oligomeric forms of the tumor suppressor p53 in vitro, using analytical ultracentrifugation and multiangle light scattering. It is established that members of the S100 protein family bind to the tetramerization domain (residues 325-355) of p53 when it is uncovered in the monomer, and so binding can disrupt the tetramer. We found a stoichiometry of one dimer of S100 bound to a monomer of p53. We discovered that some S100 proteins could also bind to the tetramer. S100B bound the tetramer and also disrupted the dimer by binding monomeric p53. S100A2 bound monomeric p53 as well as tetrameric, whereas S100A1 only bound monomeric p53. S100A6 bound more tightly to tetrameric than to monomeric p53. We also identified an additional binding site for S100 proteins in the transactivation domain (1-57) of p53. Based on our results and published observations in vivo, we propose a model for the binding of S100 proteins to p53 that can explain both activation and inhibition of p53-mediated transcription. Depending on the concentration of p53 and the member of the S100 family, binding can alter the balance between monomer and tetramer in either direction.

Genetically encoded Förster resonance energy transfer sensors for the conformation of the Src family kinase Lck.

The current model for regulation of the Src family kinase member Lck postulates a strict correlation between structural condensation of the kinase backbone and catalytic activity. The key regulatory tyrosine 505, when phosphorylated, interacts with the Src homology 2 domain on the same molecule, effectively suppressing tyrosine kinase activity. Dephosphorylation of Tyr(505) upon TCR engagement is supposed to lead to unfolding of the kinase structure and enhanced kinase activity. Studies on the conformation-activity relationship of Lck in living cells have not been possible to date because of the lack of tools providing spatiotemporal resolution of conformational changes. We designed a biochemically active, conformation-sensitive Förster resonance energy transfer biosensor of human Lck using the complete kinase backbone. Live cell imaging in Jurkat cells demonstrated that our biosensor performed according to Src family kinase literature. A Tyr(505) to Phe mutation opened the structure of the Lck sensor, while changing the autophosphorylation site Tyr(394) to Phe condensed the molecule. The tightly packed structure of a high-affinity YEEI tail mutant showed that under steady-state conditions the bulk of Lck molecules exist in a mean conformational configuration. Although T cell activation commenced normally, we could not detect a change in the conformational status of our Lck biosensor during T cell activation. Together with biochemical data we conclude that during T cell activation, Lck is accessible to very subtle regulatory mechanisms without the need for acute changes in Tyr(505) and Tyr(394) phosphorylation and conformational alterations.

Domain architecture of the stator complex of the A1A0-ATP synthase from Thermoplasma acidophilum.

A key structural element in the ion translocating F-, A-, and V-ATPases is the peripheral stalk, an assembly of two polypeptides that provides a structural link between the ATPase and ion channel domains. Previously, we have characterized the peripheral stalk forming subunits E and H of the A-ATPase from Thermoplasma acidophilum and demonstrated that the two polypeptides interact to form a stable heterodimer with 1:1 stoichiometry (Kish-Trier, E., Briere, L. K., Dunn, S. D., and Wilkens, S. (2008) J. Mol. Biol. 375, 673-685). To define the domain architecture of the A-ATPase peripheral stalk, we have now generated truncated versions of the E and H subunits and analyzed their ability to bind each other. The data show that the N termini of the subunits form an alpha-helical coiled-coil, approximately 80 residues in length, whereas the C-terminal residues interact to form a globular domain containingalpha- and beta-structure. We find that the isolated C-terminal domain of the E subunit exists as a dimer in solution, consistent with a recent crystal structure of the related Pyrococcus horikoshii A-ATPase E subunit (Lokanath, N. K., Matsuura, Y., Kuroishi, C., Takahashi, N., and Kunishima, N. (2007) J. Mol. Biol. 366, 933-944). However, upon the addition of a peptide comprising the C-terminal 21 residues of the H subunit (or full-length H subunit), dimeric E subunit C-terminal domain dissociates to form a 1:1 heterodimer. NMR spectroscopy was used to show that H subunit C-terminal peptide binds to E subunit C-terminal domain via the terminal alpha-helices, with little involvement of the beta-sheet region. Based on these data, we propose a structural model of the A-ATPase peripheral stalk.

Insight into the structural basis of pro- and antiapoptotic p53 modulation by ASPP proteins.

p53-dependent apoptosis is modulated by the ASPP family of proteins (apoptosis-stimulating proteins of p53; also called ankyrin repeat-, Src homology 3 domain-, and Pro-rich region-containing proteins). Its three known members, ASPP1, ASPP2, and iASPP, were previously found to interact with p53, influencing the apoptotic response of cells without affecting p53-induced cell cycle arrest. More specifically, the bona fide tumor suppressors, ASPP1 and ASPP2, bind to the core domain of p53 and stimulate transcription of apoptotic genes, whereas oncogenic iASPP also binds to the p53 core domain but inhibits p53-dependent apoptosis. Although the general interaction regions are known, details of the interfaces for each p53-ASPP complex have not been evaluated. We undertook a comprehensive biophysical characterization of ASPP-p53 complex formation and mapped the binding interfaces by NMR. We found that the interaction interface on p53 for the proapoptotic protein ASPP2 is distinct from that for the antiapoptotic iASPP. ASPP2 primarily binds to the core domain of p53, whereas iASPP predominantly interacts with a linker region adjacent to the core domain. Our detailed structural analyses of the ASPP-p53 interactions provide insight into the structural basis of the differential behavior of pro- and antiapoptotic ASPP family members.

Structural and functional analysis of the C-terminal DNA binding domain of the Salmonella typhimurium SPI-2 response regulator SsrB.

In bacterial pathogenesis, virulence gene regulation is controlled by two-component regulatory systems. In Escherichia coli, the EnvZ/OmpR two-component system is best understood as regulating expression of outer membrane proteins, but in Salmonella enterica, OmpR activates transcription of the SsrA/B two-component system located on Salmonella pathogenicity island 2 (SPI-2). The response regulator SsrB controls expression of a type III secretory system in which effectors modify the vacuolar membrane and prevent its degradation via the endocytic pathway. Vacuolar modification enables Salmonella to survive and replicate in the macrophage phagosome and disseminate to the liver and spleen to cause systemic infection. The signals that activate EnvZ and SsrA are unknown but are related to the acidic pH encountered in the vacuole. Our previous work established that SsrB binds to regions of DNA that are AT-rich, with poor sequence conservation. Although SsrB is a major virulence regulator in Salmonella, very little is known regarding how it binds DNA and activates transcription. In the present work, we solved the structure of the C-terminal DNA binding domain of SsrB (SsrB(C)) by NMR and analyzed the effect of amino acid substitutions on function. We identified residues in the DNA recognition helix (Lys(179), Met(186)) and the dimerization interface (Val(197), Leu(201)) that are important for SsrB transcriptional activation and DNA binding. An essential cysteine residue in the N-terminal receiver domain was also identified (Cys(45)), and the effect of Cys(203) on dimerization was evaluated. Our results suggest that although disulfide bond formation is not required for dimerization, dimerization occurs upon DNA binding and is required for subsequent activation of transcription. Disruption of the dimer interface by a C203E substitution reduces SsrB activity. Modification of Cys(203) or Cys(45) may be an important mode of SsrB inactivation inside the host.

Architecture of the Smc5/6 Complex of Saccharomyces cerevisiae Reveals a Unique Interaction between the Nse5-6 Subcomplex and the Hinge Regions of Smc5 and Smc6.

The evolutionarily conserved structural maintenance of chromosome (SMC) proteins forms the core structures of three multisubunit complexes as follows: cohesin, condensin, and the Smc5/6 complex. These complexes play crucial roles in different aspects of chromosomal organization, duplication, and segregation. Although the architectures of cohesin and condensin are better understood, that of the more recently identified Smc5/6 complex remains to be elucidated. We have previously shown that the Smc5/6 complex of Saccharomyces cerevisiae contains Smc5, Smc6, and six non-SMC elements (Nse1-6). In this study, we investigated the architecture of the budding yeast Smc5/6 complex employing the yeast two-hybrid assay as well as in vitro biochemical approaches using purified recombinant proteins. These analyses revealed that Smc5 and Smc6 associate with each other at their hinge regions and constitute the backbone of the complex, whereas the Nse1-6 subunits form three distinct subcomplexes/entities that interact with different regions of Smc5 and Smc6. The Nse1, -3, and -4 subunits form a stable subcomplex that binds to the head and the adjacent coiled-coil region of Smc5. Nse2 binds to the middle of the coiled-coil region of Smc5. Nse5 and Nse6 interact with each other and, as a heterodimer, bind to the hinge regions of Smc5 and Smc6. These findings provide new insights into the structures of the Smc5/6 complex and lay the foundation for further investigations into the mechanism of its functions.