In Vivo Evidence for ATPase-Dependent DNA Translocation by the Bacillus subtilis SMC Condensin Complex.

Structural maintenance of chromosomes (SMC) complexes shape the genomes of virtually all organisms, but how they function remains incompletely understood. Recent studies in bacteria and eukaryotes have led to a unifying model in which these ring-shaped ATPases act along contiguous DNA segments, processively enlarging DNA loops. In support of this model, single-molecule imaging experiments indicate that Saccharomyces cerevisiae condensin complexes can extrude DNA loops in an ATP-hydrolysis-dependent manner in vitro. Here, using time-resolved high-throughput chromosome conformation capture (Hi-C), we investigate the interplay between ATPase activity of the Bacillus subtilis SMC complex and loop formation in vivo. We show that point mutants in the SMC nucleotide-binding domain that impair but do not eliminate ATPase activity not only exhibit delays in de novo loop formation but also have reduced rates of processive loop enlargement. These data provide in vivo evidence that SMC complexes function as loop extruders.

Structural basis for the MukB-topoisomerase IV interaction and its functional implications in vivo.

Chromosome partitioning in Escherichia coli is assisted by two interacting proteins, topoisomerase (topo) IV and MukB. MukB stimulates the relaxation of negative supercoils by topo IV; to understand the mechanism of their action and to define this functional interplay, we determined the crystal structure of a minimal MukB-topo IV complex to 2.3 Å resolution. The structure shows that the so-called 'hinge' region of MukB forms a heterotetrameric assembly with a C-terminal DNA binding domain (CTD) on topo IV's ParC subunit. Biochemical studies show that the hinge stimulates topo IV by competing for a site on the CTD that normally represses activity on negatively supercoiled DNA, while complementation tests using mutants implicated in the interaction reveal that the cellular dependency on topo IV derives from a joint need for both strand passage and MukB binding. Interestingly, the configuration of the MukB·topo IV complex sterically disfavours intradimeric interactions, indicating that the proteins may form oligomeric arrays with one another, and suggesting a framework by which MukB and topo IV may collaborate during daughter chromosome disentanglement.

Structural mapping of the coiled-coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins.

The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N- and C- terminal regions pack against one another to form a globular ATPase domain. This "head" domain is connected to a central, globular, "hinge" or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50-nm coiled-coil domain of MukB, the divergent SMC protein found in γ-proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled-coil domain. We find that, in contrast to the relatively complicated coiled-coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled-coil interruptions. Near the middle of the domain is a break in coiled-coil structure in which there are three more residues on the C-terminal strand than on the N-terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled-coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled-coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans.

Escherichia coli condensin MukB stimulates topoisomerase IV activity by a direct physical interaction.

In contrast to the current state of knowledge in the field of eukaryotic chromosome segregation, relatively little is known about the mechanisms coordinating the appropriate segregation of bacterial chromosomes. In Escherichia coli, the MukB/E/F complex and topoisomerase IV (Topo IV) are both crucial players in this process. Topo IV removes DNA entanglements following the replication of the chromosome, whereas MukB, a member of the structural maintenance of chromosomes protein family, serves as a bacterial condensin. We demonstrate here a direct physical interaction between the dimerization domain of MukB and the C-terminal domain of the ParC subunit of Topo IV. In addition, we find that MukB alters the activity of Topo IV in vitro. Finally, we isolate a MukB mutant, D692A, that is deficient in its interaction with ParC and show that this mutant fails to rescue the temperature-sensitive growth phenotype of a mukB(-) strain. These results show that MukB and Topo IV are linked physically and functionally and indicate that the activities of these proteins are not limited to chromosome segregation but likely also play a key role in the control of higher-order bacterial chromosome structure.

Probing the recognition properties of the antiparallel coiled coil motif from PKN by protein grafting.

Coiled coils have long been recognized as the major constituent of many fibrous proteins and also serve as oligomerization domains in a wide variety of proteins. More recently, it has become clear that the surfaces of two-stranded coiled coils are also involved in macromolecular recognition. Indeed, the helical hairpin or intramolecular antiparallel coiled coil (ACC) can serve as a protein or nucleic acid recognition motif. Protein kinase N (PKN) interacts with the small GTPase RhoA through ACC motifs. The crystal structure of RhoA with the N-terminal ACC motif (PKN-ACC1) is unusual in that these proteins interact through two distinct surfaces. Using the ACC domain of seryl tRNA synthetase (SRS-ACC) as a scaffold for protein grafting experiments, we show that RhoA interacts with only one face of PKN-ACC1. This result highlights the potential of the SRS-ACC scaffold for protein engineering applications and provides insight into the mechanism of RhoA-mediated signal transduction through PKN.

NMR investigation of the binding between human profilin I and inositol 1,4,5-triphosphate, the soluble headgroup of phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is involved in the regulation of the actin cytoskeleton through interactions with a number of actin-binding proteins. We present here NMR titration experiments that monitor the interaction between the cytoskeletal protein profilin and inositol 1,4,5-triphosphate (IP(3)), the headgroup of PI(4,5)P(2). These experiments probe the interaction directly, at equilibrium, and with profilin in its native state. We show the binding between profilin and IP(3) can readily be observed at high concentrations, even though profilin does not bind to IP(3) under physiological conditions. Moreover, the titration data using wild-type profilin and an R88L mutant support the existence of at least three headgroup binding sites on profilin, consistent with previous experimentation with intact PI(4,5)P(2). This work suggests that various soluble inositol ligands can serve as effective probes to facilitate in vitro studies of PI-binding proteins that require membrane surfaces for high-affinity binding.

Importance of potential interhelical salt-bridges involving interior residues for coiled-coil stability and quaternary structure.

Coiled coils are formed by two or more alpha-helices that align in a parallel or an antiparallel relative orientation. Polar interactions involving residues at the interior a and d positions are important for determining the quaternary structure of coiled coils. In the model heterodimeric coiled-coil Acid-a1-Base-a1, a buried a-d' Asn-Asn interaction is sufficient to specify both a dimeric structure and an antiparallel relative helix orientation. Although the equivalent a-a' interaction is found in parallel coiled coils, there is no example of an a-d' Asn-Asn interaction in structurally characterized, naturally occurring antiparallel coiled coils. Instead, interior charged residues form interhelical salt-bridges with residues at the adjacent e or g positions. Using a model coiled-coil heterodimer, we have explored the role of a potential interhelical interaction between an Arg at an interior d position and a Glu at the adjacent g' position. Our results demonstrate that this potentially attractive interhelical Coulombic interaction has little or no influence on helix orientation. Instead, we show that burying a single Arg residue at an interior position is sufficient to specify a dimeric state at a significantly lower thermodynamic cost than burial of two interacting Asn residues.

The role of helix stabilizing residues in GCN4 basic region folding and DNA binding.

Basic region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a coiled-coil leucine zipper dimerization domain and a highly charged basic region that directly contacts DNA. The basic region is largely unfolded in the absence of DNA, but adopts a helical conformation upon DNA binding. Although a coil --> helix transition is entropically unfavorable, this conformational change positions the DNA-binding residues appropriately for sequence-specific interactions with DNA. The N-terminal residues of the GCN4 DNA-binding domain, DPAAL, make no DNA contacts and are not part of the conserved basic region, but are nonetheless important for DNA binding. Asp and Pro are often found at the N-termini of alpha-helices, and such N-capping motifs can stabilize alpha-helical structure. In the present study, we investigate whether these two residues serve to stabilize a helical conformation in the GCN4 basic region, lowering the energetic cost for DNA binding. Our results suggest that the presence of these residues contributes significantly to helical structure and to the DNA-binding ability of the basic region in the absence of the leucine zipper. Similar helix-capping motifs are found in approximately half of all bZip domains, and the implications of these findings for in vivo protein function are discussed.

A repeated coiled-coil interruption in the Escherichia coli condensin MukB.

MukB, a divergent structural maintenance of chromosomes (SMC) protein, is important for chromosome segregation and condensation in Escherichia coli and other γ-proteobacteria. MukB and canonical SMC proteins share a common five-domain structure in which globular N- and C-terminal regions combine to form an ATP-binding-cassette-like ATPase domain. This ATPase domain is connected to a central, globular dimerization domain by a long antiparallel coiled coil. The structures of both globular domains have been solved recently. In contrast, little is known about the coiled coil, in spite of its clear importance for SMC function. Recently, we identified interacting regions on the N- and C-terminal halves of the MukB coiled coil through photoaffinity cross-linking experiments. On the basis of these low-resolution experimental constraints, phylogenetic data, and coiled-coil prediction analysis, we proposed a preliminary model in which the MukB coiled coil is divided into multiple segments. Here, we use a disulfide cross-linking assay to detect paired residues on opposite strands of MukB's coiled coil. This method provides accurate register data and demonstrates the presence of at least five coiled-coil segments in this domain. Moreover, these studies show that the segments are interrupted by a repeated, unprecedented deviation from canonical coiled-coil structure. These experiments provide a sufficiently detailed view of the MukB coiled coil to allow rational manipulation of this region for the first time, opening the door for structure-function studies of this domain.

The crystal structure of the hinge domain of the Escherichia coli structural maintenance of chromosomes protein MukB.

MukB, a divergent structural maintenance of chromosomes (SMC) protein, is important for chromosomal segregation and condensation in gamma-proteobacteria. MukB and canonical SMC proteins share a characteristic five-domain structure. Globular N- and C-terminal domains interact to form an ATP-binding cassette-like ATPase or "head" domain, which is connected to a smaller dimerization or "hinge" domain by a long, antiparallel coiled coil. In addition to mediating dimerization, this hinge region has been implicated in both conformational flexibility and dynamic protein-DNA interactions. We report here the first crystallographic model of the MukB hinge domain. This model also contains approximately 20% of the coiled-coil domain, including an unusual coiled-coil deviation. These results will facilitate studies to clarify the roles of both the hinge and the coiled-coil domains in MukB function.

Identification of interacting regions within the coiled coil of the Escherichia coli structural maintenance of chromosomes protein MukB.

MukB, a divergent structural maintenance of chromosomes (SMC) protein, is important for chromosome segregation and condensation in Escherichia coli and other gamma-proteobacteria. MukB and canonical SMC proteins share a common five-domain structure in which globular N- and C-terminal regions combine to form an ABC-like ATPase domain. This ATPase domain is connected to a central, globular dimerization domain, commonly called the "hinge" domain, by a long antiparallel coiled coil. Although the ATPase and hinge domains of SMC proteins have been the subject of extensive investigation, little is known about the coiled coil, in spite of its clear importance for SMC function. This limited knowledge is primarily due to a lack of structural information. We report here the first experimental constraints on the relative alignment of the N- and C-terminal halves of the MukB coiled coil, obtained by a combination of limited proteolysis and site-directed cross-linking approaches. Using these experimental constraints, phylogenetic data, and coiled-coil prediction algorithms, we propose a pairing scheme for the discontinuous segments in the coiled coil. This structural model will not only facilitate the study of the physiological role of this unusually long and flexible antiparallel coiled coil but also help to delineate the boundaries between MukB domains.

High affinity binding to profilin by a covalently constrained, soluble mimic of phosphatidylinositol-4,5-bisphosphate micelles.

Phosphoinositide (PI) lipids are essential regulators of a wide variety of cellular functions. We present here the preparation of a multivalent analogue of a phosphatidylinositol-4,5-bisphosphate (PIP(2)) micelle containing only the polar headgroup portion of this lipid. We show that this dendrimer binds to the cytoskeletal protein profilin with an affinity indistinguishable from that of PIP(2), despite the fact that profilin discriminates between PIP(2) and its monomeric hydrolysis product inositol-1,4,5-triphosphate (IP(3)) under physiological conditions. These data demonstrate that the diacylglycerol (DAG) moiety of PIP(2) is not required for high-affinity binding and suggest that profilin uses multivalency as a key means to distinguish between the intact lipid and IP(3). The class of soluble membrane analogues described here is likely to have broad applicability in the study of protein.PI interactions.

Synthesis and characterization of covalent mimics of phosphatidylinositol-4,5-bisphosphate micelles.

There is increasing evidence that multivalency plays an important role in protein-lipid recognition and membrane targeting in biological systems. We describe here the preparation and characterization of multivalent analogues of the signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Tetherable analogues of the PIP2 headgroup were appended to polyamidoamine dendrimers via a squarate linker to afford polymers displaying four or eight headgroup moieties. This class of molecules should provide a powerful tool for the study of protein-lipid interactions.

A general method for selection and screening of coiled coils on the basis of relative helix orientation.

We have developed a method for selecting coiled coils that associate with a given relative helix orientation from a randomized pool of proteins. To select for antiparallel dimers, we have designed a model basic region-leucine zipper (bZip) heterodimer capable of binding DNA only when the coiled coil associates with an antiparallel relative helix alignment. The dimerization domain for this bZip heterodimer is the model antiparallel coiled coil Acid-a1-Base-a1 (Oakley, M. G.; Kim, P. S. Biochemistry 1998, 37, 12603), and both monomers contain the GCN4 basic region. Although the basic regions in naturally occurring bZip proteins are located N-terminal to the leucine zipper, we have attached the GCN4 basic region to the C-terminus of Acid-a1 to allow both basic regions to contact DNA in an antiparallel heterodimer. The resulting heterodimer, BR-Base-a1-Acid-a1-BR, can bind to a direct repeat of the GCN4 half-site in vivo, leading to spectinomycin resistance in the transcription interference assay of Elledge et al. (Elledge, S. J.; Sugiono, P.; Guarente, L.; Davis, R. W. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 3689). A buried interhelical polar interaction between two Asn residues in the Acid-a1-Base-a1 heterodimer is known to specify an antiparallel helix orientation. The position of one of these buried Asn residues was randomized, and bZip heterodimers containing antiparallel coiled coils were selected using the transcription interference assay. All of the selected colonies contained Asn at the randomized position, suggesting that the selection is specific for antiparallel coiled coils.

Design and characterization of a homodimeric antiparallel coiled coil.

We report the first successful design of a self-associating antiparallel coiled coil, APH. The simultaneous application of Coulombic and hydrophobic components results in a decided preference for the antiparallel alignment as judged by HPLC, sedimentation equilibrium, and chemical denaturation data. The designed peptide is of comparable stability to naturally occurring leucine zipper peptides and can be expressed in bacteria. These properties of APH suggest potential in vivo protein fusion and biomaterials applications.