Crystal structure of ScpB from Chlorobium tepidum, a protein involved in chromosome partitioning

Structural maintenance of chromosome (SMC) proteins are essential in chromosome condensation and interact with non-SMC proteins in eukaryotes and with segregation and condensation proteins (ScpA and ScpB) in prokaryotes. The highly conserved gene in Chlorobium tepidum gi 21646405 encodes ScpB (ScpB_ChTe). The high resolution crystal structure of ScpB_ChTe shows that the monomeric structure consists of two similarly shaped globular domains composed of three helices sided by beta-strands [a winged helix-turn-helix (HTH)], a motif observed in the C-terminal domain of Scc1, a functionally related eukaryotic ScpA homolog, as well as in many DNA binding proteins.     

Using DNA as a fiducial marker to study SMC complex interactions with the atomic force microscope

Atomic force microscopy can potentially provide information on protein volumes, shapes, and interactions but is susceptible to variable tip-induced artifacts. In this study, we present an atomic force microscopy approach that can measure volumes of nonglobular polypeptides such as structural maintenance of chromosomes (SMC) proteins, and use it to study the interactions that occur within and between SMC complexes. Together with the protein of interest, we coadsorb a DNA molecule and use it as a fiducial marker to account for tip-induced artifacts that affect both protein and DNA, allowing normalization of protein volumes from images taken on different days and with different tips. This approach significantly reduced the error associated with volume analysis, and allowed determination of the oligomeric states and architecture of the Bacillus subtilis SMC complex, formed by the SMC protein, and by the smaller ScpA and ScpB subunits. This work reveals that SMC and ScpB are dimers and that ScpA is a stable monomer. Moreover, whereas ScpA binds directly to SMC, ScpB only binds to SMC in the presence of ScpA. Notably, the presence of both ScpA and ScpB favored the formation of higher-order structures of SMC complexes, suggesting a role for these subunits in the organization of SMC oligomers.     

A prokaryotic condensin/cohesin-like complex can actively compact chromosomes from a single position on the nucleoid and binds to DNA as a ring-like structure

We show that Bacillus subtilis SMC (structural maintenance of chromosome protein) localizes to discrete foci in a cell cycle-dependent manner. Early in the cell cycle, SMC moves from the middle of the cell toward opposite cell poles in a rapid and dynamic manner and appears to interact with different regions on the chromosomes during the cell cycle. SMC colocalizes with its interacting partners, ScpA and ScpB, and the specific localization of SMC depends on both Scp proteins, showing that all three components of the SMC complex are required for proper localization. Cytological and biochemical experiments showed that dimeric ScpB stabilized the binding of ScpA to the SMC head domains. Purified SMC showed nonspecific binding to double-stranded DNA, independent of Scp proteins or ATP, and was retained on DNA after binding to closed DNA but not to linear DNA. The SMC head domains and hinge region did not show strong DNA binding activity, suggesting that the coiled-coil regions in SMC mediate an association with DNA and that SMC binds to DNA as a ring-like structure. The overproduction of SMC resulted in global chromosome compaction, while SMC was largely retained in bipolar foci, suggesting that the SMC complex forms condensation centers that actively affect global chromosome compaction from a defined position on the nucleoid.     

Hinge-mediated dimerization of SMC protein is essential for its dynamic interaction with DNA

Structural maintenance of chromosomes (SMC) proteins play central roles in regulating higher order chromosome dynamics from bacteria to humans. As judged by electron microscopy, the SMC homodimer from Bacillus subtilis (BsSMC) is composed of two antiparallel, coiled-coil arms with a flexible hinge. Site-directed cross-linking experiments show here that dimerization of BsSMC is mediated by a hinge-hinge interaction between self-folded monomers. This architecture is conserved in the eukaryotic SMC2-SMC4 heterodimer. Analysis of different deletion mutants of BsSMC unexpectedly reveals that the major DNA-binding activity does not reside in the catalytic ATPase domains located at the ends of a dimer. Instead, point mutations in the hinge domain that disturb dimerization of BsSMC drastically reduce its ability to interact with DNA. Proper hinge function is essential for BsSMC to recognize distinct DNA topology, and mutant proteins with altered hinge angles cross-link double-stranded DNA in a nucleotide-dependent manner. We propose that the hinge domain of SMC proteins is not a simple dimerization site, but rather it acts as an essential determinant of dynamic SMC-DNA interactions.     

Cell cycle-dependent localization of two novel prokaryotic chromosome segregation and condensation proteins in Bacillus subtilis that interact with SMC protein

Disruption of ypuG and ypuH open reading frames in Bacillus subtilis leads to temperature-sensitive slow growth, a defect in chromosome structure and formation of anucleate cells. The genes, which were named scpA and scpB, were found to be epistatic to the smc gene. Fusions of ScpA and ScpB to the fluorescent proteins YFP or CFP showed that both proteins co-localize to two or four discrete foci that were present at mid-cell in young cells, and within both cell halves, generally adjacent to chromosomal origin regions, in older cells. ScpA and ScpB foci are associated with DNA and depend on the presence of SMC and both Scps. ScpA and ScpB are associated with each other and with SMC in vivo, as determined using the FRET technique and immunoprecipitation assays. Genes similar to scpA and scpB are present in many bacteria and archaea, which suggests that their gene products form a condensation complex with SMC in most prokaryotes. The observed foci could constitute condensation factories that pull DNA away from mid-cell into both cell halves.     

Discovery of two novel families of proteins that are proposed to interact with prokaryotic SMC proteins,and characterization of the Bacillus subtilis family members ScpA and ScpB

Structural maintenance of chromosomes (SMC) proteins are present in all eukaryotes and in many prokaryotes. Eukaryotic SMC proteins form complexes with various non-SMC subunits, which affect their function, whereas the prokaryotic homologues had no known non-SMC partners and were thought to act as simple homodimers. Here we describe two novel families of proteins, widespread in archaea and (Gram-positive) bacteria, which we denote 'segregation and condensation proteins' (Scps). ScpA genes are localized next to smc genes in nearly all SMC- containing archaea, suggesting that they belong to the same operon and are thus involved in a common process in the cell. The function of ScpA was studied in Bacillus subtilis, which also harbours a well characterized smc gene. Here we show that scpA mutants display characteristic phenotypes nearly identical to those of smc mutants, including temperature- sensitive growth, production of anucleate cells, formation of aberrant nucleoids, and chromosome splitting by the so-called guillotine effect. Thus, both SMC and ScpA are required for chromosome segregation and condensation. Interestingly, mutants of another B. subtilis gene, scpB, which is localized downstream from scpA, display the same phenotypes, which indicate that ScpB is also involved in these functions. ScpB is generally present in species that also encode ScpA. The physical interaction of ScpA and SMC was proven (i) by the use of the yeast two-hybrid system and (ii) by the isolation of a complex containing both proteins from cell extracts of B. subtilis. By extension, we speculate that interaction of orthologues of the two proteins is important for chromosome segregation in many archaea and bacteria, and propose that SMC proteins generally have non-SMC protein partners that affect their function not only in eukaryotes but also in prokaryotes.     

Structural maintenance of chromosomes protein of Bacillus subtilis affects supercoiling in vivo

Structural maintenance of chromosomes (SMC) proteins are found in nearly all organisms. Members of this protein family are involved in chromosome condensation and sister chromatid cohesion. Bacillus subtilis SMC protein (BsSMC) plays a role in chromosome organization and partitioning. To better understand the function of BsSMC, we studied the effects of an smc null mutation on DNA supercoiling in vivo. We found that an smc null mutant was hypersensitive to the DNA gyrase inhibitors coumermycin A1 and norfloxacin. Furthermore, depleting cells of topoisomerase I substantially suppressed the partitioning defect of an smc null mutant. Plasmid DNA isolated from an smc null mutant was more negatively supercoiled than that from wild-type cells. In vivo cross-linking experiments indicated that BsSMC was bound to the plasmid. Our results indicate that BsSMC affects supercoiling in vivo, most likely by constraining positive supercoils, an activity which contributes to chromosome compaction and organization.     

Subcellular localization of the Bacillus subtilis structural maintenance of chromosomes (SMC) protein

The Bacillus subtilis structural maintenance of chromosomes (SMC) protein is a member of a large family of proteins involved in chromosome organization. We found that SMC is a moderately abundant protein ( approximately 1000 dimers per cell). In vivo cross-linking and immunoprecipitation assays revealed that SMC binds to many regions on the chromosome. Visualization of SMC in live cells using a fusion to the green fluorescent protein (GFP) and in fixed cells using immunofluorescence microscopy indicated that a portion of SMC localizes as discrete foci in positions similar to that of the DNA replication machinery (replisome). When visualized simultaneously, SMC and the replisome were often in similar regions of the cell but did not always co-localize. Persistence of SMC foci did not depend on ongoing replication, but did depend on ScpA and ScpB, two proteins thought to interact with SMC. Our results indicate that SMC is bound to many sites on the chromosome and a concentration of SMC is localized near replication forks, perhaps there to bind and organize newly replicated DNA.     

Dynamic assembly,localization and proteolysis of the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SMC complex

Background  

SMC proteins are key components of several protein complexes that perform vital tasks in different chromosome dynamics. Bacterial SMC forms a complex with ScpA and ScpB that is essential for chromosome arrangement and segregation. The complex localizes to discrete centres on the nucleoids that during most of the time of the cell cycle localize in a bipolar manner. The complex binds to DNA and condenses DNA in an as yet unknown manner.     

Kleisins: a superfamily of bacterial and eukaryotic SMC protein partners

We describe a superfamily of eukaryotic and prokaryotic proteins (kleisins) that includes ScpA, Scc1, Rec8, and Barren. Scc1 interacts with SMC proteins through N- and C-terminal domains to form a ring-like structure. Since these are the only domains conserved among kleisins, we suggest that ring formation with SMC proteins may define this family.     

Bimodal activation of SMC ATPase by intra- and inter-molecular interactions.

Structural maintenance of chromosomes (SMC) proteins play fundamental roles in higher-order chromosome dynamics from bacteria to humans. It has been proposed that the Bacillus subtilis SMC (BsSMC) homodimer is composed of two anti-parallel coiled-coil arms, each having an ATP-binding domain at its distal end. It remains totally unknown, however, how the two-armed structure supports ATP-dependent actions of BsSMC. By constructing a number of mutant derivatives including 'single-armed' BsSMC, we show here that the central hinge domain provides a structural flexibility that allows opening and closing of the two arms. This unique structure brings about bimodal regulation of the SMC ATPase cycle. Closing the arm can trigger ATP hydrolysis by allowing an end-end interaction within a dimer (intramolecular mode). When bound to DNA, ATP promotes a dimer-dimer interaction, which in turn activates their DNA-dependent ATPase activity (intermolecular mode). Our results reveal a novel mechanism of ATPase regulation and provide mechanistic insights into how eukaryotic SMC protein complexes could mediate diverse chromosomal functions, such as chromosome condensation and sister chromatid cohesion.     

Model for Substrate Interactions in C5a Peptidase from Streptococcus pyogenes: A 1.9 Å Crystal Structure of the Active Form of ScpA

The crystal structure of an active form of ScpA has been solved to 1.9 Å resolution. ScpA is a multidomain cell-envelope subtilase from Streptococcus pyogenes that cleaves complement component C5a. The catalytic triad of ScpA is geometrically consistent with other subtilases, clearly demonstrating that the additional activation mechanism proposed for the Streptococcus agalactiae homologue (ScpB) is not required for ScpA. The ScpA structure revealed that access to the catalytic site is restricted by variable regions in the catalytic domain (vr7, vr9, and vr11) and by the presence of the inserted protease-associated (PA) domain and the second fibronectin type III domains (Fn2). Modeling of the ScpA-C5a complex indicates that the substrate binds with carboxyl-terminal residues (65-74) extended through the active site and core residues (1-64) forming exosite-type interactions with the Fn2 domain. This is reminiscent of the two-site mechanism proposed for C5a binding to its receptor. In the nonprime region of the active site, interactions with the substrate backbone are predicted to be more similar to those observed in kexins, involving a single β-strand in the peptidase. However, in contrast to kexins, there would be diminished emphasis on side-chain interactions, with little charged character in the S3-S1 and S6-S4 subsites occupied by the side chains of residues in vr7 and vr9. Substrate binding is anticipated to be dominated by ionic interactions in two distinct regions of ScpA. On the prime side of the active site, salt bridges are predicted between P1′, P2′, and P7′ residues, and residues in the catalytic and PA domains. Remote to the active site, a larger number of ionic interactions between residues in the C5a core and the Fn2 domain are observed in the model. Thus, both PA and Fn2 domains are expected to play significant roles in substrate recognition.     

The Symmetrical Structure of Structural Maintenance of Chromosomes (SMC) and MukB Proteins: Long,Antiparallel Coiled Coils,Folded at a Flexible Hinge

Structural maintenance of chromosomes (SMC) proteins function in chromosome condensation and several other aspects of DNA processing. They are large proteins characterized by an NH₂-terminal nucleotide triphosphate (NTP)-binding domain, two long segments of coiled coil separated by a hinge, and a COOH-terminal domain. Here, we have visualized by EM the SMC protein from Bacillus subtilis (BsSMC) and MukB from Escherichia coli, which we argue is a divergent SMC protein. Both BsSMC and MukB show two thin rods with globular domains at the ends emerging from the hinge. The hinge appears to be quite flexible: the arms can open up to 180°, separating the terminal domains by 100 nm, or close to near 0°, bringing the terminal globular domains together.A surprising observation is that the ∼300–amino acid–long coiled coils are in an antiparallel arrangement. Known coiled coils are almost all parallel, and the longest antiparallel coiled coils known previously are 35–45 amino acids long. This antiparallel arrangement produces a symmetrical molecule with both an NH₂- and a COOH-terminal domain at each end. The SMC molecule therefore has two complete and identical functional domains at the ends of the long arms. The bifunctional symmetry and a possible scissoring action at the hinge should provide unique biomechanical properties to the SMC proteins.     

ATP-dependent aggregation of single-stranded DNA by a bacterial SMC homodimer.

SMC (structural maintenance of chromosomes) proteins are putative ATPases that are highly conserved among Bacteria, Archaea and Eucarya. Eukaryotic SMC proteins are implicated in a diverse range of chromosome dynamics including chromosome condensation, dosage compensation and recombinational repair. In eukaryotes, two different SMC proteins form a heterodimer, which in turn acts as the core component of a large protein complex. Despite recent progress, no ATP-dependent activity has been found in individual SMC subunits. We report here the first biochemical characterization of a bacterial SMC protein from Bacillus subtilis. Unlike eukaryotic versions, the B.subtilis SMC protein (BsSMC) is a simple homodimer with no associated subunits. It binds preferentially to single-stranded DNA (ssDNA) and has a ssDNA-stimulated ATPase activity. In the presence of ATP, BsSMC forms large nucleoprotein aggregates in a ssDNA-specific manner. Proteolytic cleavage of BsSMC is changed upon binding to ATP and ssDNA. The energy-dependent aggregation of ssDNA might represent a primitive type of chromosome condensation that occurs during segregation of bacterial chromosomes.     

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. Proteins 2015; 83:1027–1045. © 2015 Wiley Periodicals, Inc.     

Condensin and cohesin display different arm conformations with characteristic hinge angles

Structural maintenance of chromosomes (SMC) proteins play central roles in higher-order chromosome dynamics from bacteria to humans. In eukaryotes, two different SMC protein complexes, condensin and cohesin, regulate chromosome condensation and sister chromatid cohesion, respectively. Each of the complexes consists of a heterodimeric pair of SMC subunits and two or three non-SMC subunits. Previous studies have shown that a bacterial SMC homodimer has a symmetrical structure in which two long coiled-coil arms are connected by a flexible hinge. A catalytic domain with DNA- and ATP-binding activities is located at the distal end of each arm. We report here the visualization of vertebrate condensin and cohesin by electron microscopy. Both complexes display the two-armed structure characteristic of SMC proteins, but their conformations are remarkably different. The hinge of condensin is closed and the coiled-coil arms are placed close together. In contrast, the hinge of cohesin is wide open and the coiled-coils are spread apart from each other. The non-SMC subunits of both condensin and cohesin form a globular complex bound to the catalytic domains of the SMC heterodimers. We propose that the "closed" conformation of condensin and the "open" conformation of cohesin are important structural properties that contribute to their specialized biochemical and physiological functions.     

Opening closed arms: long-distance activation of SMC ATPase by hinge-DNA interactions

Structural maintenance of chromosomes (SMC) proteins form a V-shaped dimer in which a central hinge domain connects two coiled-coil arms, each having an ATP binding head domain at its distal end. Here, we show that the hinge domain plays essential roles in modulating the mechanochemical cycle of SMC proteins. An initial interaction of the hinge domain with DNA leads to opening of the arms by triggering hydrolysis of ATP bound to the head domains, which are located approximately 50 nm away from the hinge. This conformational change allows the inner surface of the hinge domain to stably interact with DNA by an ATP-independent mechanism and primes ATP-driven engagement between the liberated head domains either intramolecularly or intermolecularly. Consistently, a variety of hinge mutations drastically alter DNA binding properties of SMC proteins through distinct mechanisms. Our results suggest that SMC proteins possess an intrinsic property to change their own conformations upon binding to DNA.     

Comparison of Staphopain A (ScpA) and B (SspB) precursor activation mechanisms reveals unique secretion kinetics of proSspB (Staphopain B), and a different interaction with its cognate Staphostatin, SspC

The scp AB and ssp ABC operons of Staphylococcus aureus encode Staphopain cysteine proteases ScpA and SspB, and their respective Staphostatins ScpB and SspC, which are thought to protect against premature activation of Staphopain precursors during protein export. However, we found that the proSspB precursor was secreted and activated without detriment to S. aureus in the absence of SspC function. Our data indicate that this is feasible due to a restricted substrate specificity of mature SspB, a stable precursor structure and slow secretion kinetics. In contrast, mature ScpA had a broad substrate specificity, such that it was prone to autolytic degradation, but also was uniquely able to degrade elastin fibres. Modelling of proScpA relative to the proSspB structure identified several differences, which appear to optimize proScpA for autocatalytic activation, whereas proSspB is optimized for stability, and cannot initiate autocatalytic activation. Consequently, recombinant proSspB remained stable and unprocessed when retained in the cytoplasm of Escherichia coli , whereas proScpA initiated rapid autocatalytic activation, leading to capture of an activation intermediate by ScpB. We conclude that the status of ssp BC in S. aureus , as paralogues of the ancestral scp AB genes, facilitated a different activation mechanism, a stable proSspB isoform and modified Staphostatin function.     

A novel SMC protein complex in Schizosaccharomyces pombe contains the Rad18 DNA repair protein

In Schizosaccharomyces pombe, rad18 is an essential gene involved in the repair of DNA damage produced by ionizing radiation and in tolerance of UV-induced DNA damage. The Rad18 protein is a member of the SMC (structural maintenance of chromosomes) superfamily, and we show that, like the other SMC proteins in condensin and cohesin, Rad18 is a component of a high-molecular-weight complex. This complex contains at least six other proteins, the largest of which is Spr18, a novel SMC family member closely related to Rad18, and likely to be its heterodimeric partner. SMC proteins have ATP-binding domains at the N- and C-termini, and two extended coiled-coil domains separated by a hinge in the middle. We show that the N-terminal ATP-binding domain of Rad18 is essential for all functions, and overexpression of an N-terminal mutant has a dominant-negative effect. We have identified an important mutation (S1045A) near the C-terminus of Rad18 that separates its repair and essential roles. Potential models for the role of the Rad18-Spr18 complex during DNA repair are discussed.