A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation.

Genome segregation is a fundamental step in the life cycle of every cell. Most bacteria rely on dedicated DNA partition proteins to actively segregate chromosomes and low copy-number plasmids. Here, by employing super resolution microscopy, we establish that the ParF DNA partition protein of the ParA family assembles into a three-dimensional meshwork that uses the nucleoid as a scaffold and periodically shuttles between its poles. Whereas ParF specifies the territory for plasmid trafficking, the ParG partner protein dictates the tempo of ParF assembly cycles and plasmid segregation events by stimulating ParF adenosine triphosphate hydrolysis. Mutants in which this ParG temporal regulation is ablated show partition deficient phenotypes as a result of either altered ParF structure or dynamics and indicate that ParF nucleoid localization and dynamic relocation, although necessary, are not sufficient per se to ensure plasmid segregation. We propose a Venus flytrap model that merges the concepts of ParA polymerization and gradient formation and speculate that a transient, dynamic network of intersecting polymers that branches into the nucleoid interior is a widespread mechanism to distribute sizeable cargos within prokaryotic cells.

Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function.

UNLABELLED: Invasive fungal infections remain difficult to treat and require novel targeting strategies. The 12-kDa FK506-binding protein (FKBP12) is a ubiquitously expressed peptidyl-prolyl isomerase with considerable homology between fungal pathogens and is thus a prime candidate for future targeting efforts to generate a panfungal strategy. Despite decades of research on FKBPs, their substrates and mechanisms of action remain unclear. Here we describe structural, biochemical, and in vivo analyses of FKBP12s from the pathogenic fungi Candida albicans, Candida glabrata, and Aspergillus fumigatus Strikingly, multiple apo A. fumigatus and C. albicans FKBP12 crystal structures revealed a symmetric, intermolecular interaction involving the deep insertion of an active-site loop proline into the active-site pocket of an adjacent subunit. Such interactions have not been observed in previous FKBP structures. This finding indicates the possibility that this is a self-substrate interaction unique to the A. fumigatus and C. albicans fungal proteins that contain this central proline. Structures obtained with the proline in the cis and trans states provide more data in support of self-catalysis. Moreover, cysteine cross-linking experiments captured the interacting dimer, supporting the idea that it forms in solution. Finally, genetic studies exploring the impact of mutations altering the central proline and an adjacent residue provide evidence that any dimeric state formed in vivo, where FKBP12 concentrations are low, is transient. Taken together, these findings suggest a unique mechanism of self-substrate regulation by fungal FKBP12s, lending further novel understanding of this protein for future drug-targeting efforts. IMPORTANCE: FKBP12 is a cis-trans peptidyl-prolyl isomerase that plays key roles in cellular protein homeostasis. FKBP12s also bind the immunosuppressive drug FK506 to inhibit the phosphatase calcineurin (CaN). CaN is required for virulence of A. fumigatus, C. albicans, C. glabrata, and other deadly fungal pathogens, marking FKBP12 and CaN as potential broad-spectrum drug targets. Here we describe structures of fungal FKBP12s. Multiple apo A. fumigatus and C. albicans FKBP12 structures reveal the insertion of a proline, conspicuously conserved in these proteins, into the active sites of adjacent molecules. This suggests that these proteins might serve as their own substrates. Cysteine disulfide trapping experiments provide support for this self-interaction and hence possible intermolecular catalysis by these enzymes.

Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages.

Although recent studies have provided a wealth of information about archaeal biology, nothing is known about the molecular basis of DNA segregation in these organisms. Here, we unveil the machinery and assembly mechanism of the archaeal Sulfolobus pNOB8 partition system. This system uses three proteins: ParA; an atypical ParB adaptor; and a centromere-binding component, AspA. AspA utilizes a spreading mechanism to create a DNA superhelix onto which ParB assembles. This supercomplex links to the ParA motor, which contains a bacteria-like Walker motif. The C domain of ParB harbors structural similarity to CenpA, which dictates eukaryotic segregation. Thus, this archaeal system combines bacteria-like and eukarya-like components, which suggests the possible conservation of DNA segregation principles across the three domains of life.

Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis.

The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3',8-cyclo-7,8-dihydro-guanosine 5'-triphosphate (3',8-cH2GTP) as the product of in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, we performed biochemical characterization of 3',8-cH2GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3',8-cH2GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3',8-cH2GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3',8-cH2GTP (GTP 3',8-cyclase), and that of MoaC is to catalyze the rearrangement of 3',8-cH2GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. Thus, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.

Calcineurin orchestrates dimorphic transitions, antifungal drug responses and host-pathogen interactions of the pathogenic mucoralean fungus Mucor circinelloides.

Calcineurin plays essential roles in virulence and growth of pathogenic fungi and is a target of the natural products FK506 and Cyclosporine A. In the pathogenic mucoralean fungus Mucor circinelloides, calcineurin mutation or inhibition confers a yeast-locked phenotype indicating that calcineurin governs the dimorphic transition. Genetic analysis in this study reveals that two calcineurin A catalytic subunits (out of three) are functionally diverged. Homology modeling illustrates modes of resistance resulting from amino substitutions in the interface between each calcineurin subunit and the inhibitory drugs. In addition, we show how the dimorphic transition orchestrated by calcineurin programs different outcomes during host-pathogen interactions. For example, when macrophages phagocytose Mucor yeast, subsequent phagosomal maturation occurs, indicating host cells respond appropriately to control the pathogen. On the other hand, upon phagocytosis of spores, macrophages fail to form mature phagosomes. Cytokine production from immune cells differs following exposure to yeast versus spores (which germinate into hyphae). Thus, the morphogenic transition can be targeted as an efficient treatment option against Mucor infection. In addition, genetic analysis (including gene disruption and mutational studies) further strengthens the understanding of calcineurin and provides a foundation to develop antifungal agents targeting calcineurin to deploy against Mucor and other pathogenic fungi.

Structures of regulatory machinery reveal novel molecular mechanisms controlling B. subtilis nitrogen homeostasis.

All cells must sense and adapt to changing nutrient availability. However, detailed molecular mechanisms coordinating such regulatory pathways remain poorly understood. In Bacillus subtilis, nitrogen homeostasis is controlled by a unique circuitry composed of the regulator TnrA, which is deactivated by feedback-inhibited glutamine synthetase (GS) during nitrogen excess and stabilized by GlnK upon nitrogen depletion, and the repressor GlnR. Here we describe a complete molecular dissection of this network. TnrA and GlnR, the global nitrogen homeostatic transcription regulators, are revealed as founders of a new structural family of dimeric DNA-binding proteins with C-terminal, flexible, effector-binding sensors that modulate their dimerization. Remarkably, the TnrA sensor domains insert into GS intersubunit catalytic pores, destabilizing the TnrA dimer and causing an unprecedented GS dodecamer-to-tetradecamer conversion, which concomitantly deactivates GS. In contrast, each subunit of the GlnK trimer "templates" active TnrA dimers. Unlike TnrA, GlnR sensors mediate an autoinhibitory dimer-destabilizing interaction alleviated by GS, which acts as a GlnR chaperone. Thus, these studies unveil heretofore unseen mechanisms by which inducible sensor domains drive metabolic reprograming in the model Gram-positive bacterium B. subtilis.

Structural basis of lipid binding for the membrane-embedded tetraacyldisaccharide-1-phosphate 4'-kinase LpxK.

The membrane-bound tetraacyldisaccharide-1-phosphate 4'-kinase, LpxK, catalyzes the sixth step of the lipid A (Raetz) biosynthetic pathway and is a viable antibiotic target against emerging Gram-negative pathogens. We report the crystal structure of lipid IVA, the LpxK product, bound to the enzyme, providing a rare glimpse into interfacial catalysis and the surface scanning strategy by which many poorly understood lipid modification enzymes operate. Unlike the few previously structurally characterized proteins that bind lipid A or its precursors, LpxK binds almost exclusively to the glucosamine/phosphate moieties of the lipid molecule. Steady-state kinetic analysis of multiple point mutants of the lipid-binding pocket pinpoints critical residues involved in substrate binding, and characterization of N-terminal helix truncation mutants uncovers the role of this substructure as a hydrophobic membrane anchor. These studies make critical contributions to the limited knowledge surrounding membrane-bound enzymes that act upon lipid substrates and provide a structural template for designing small molecule inhibitors targeting this essential kinase.

Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein.

The staphylococcal multiresistance plasmids are key contributors to the alarming rise in bacterial multidrug resistance. A conserved replication initiator, RepA, encoded on these plasmids is essential for their propagation. RepA proteins consist of flexibly linked N-terminal (NTD) and C-terminal (CTD) domains. Despite their essential role in replication, the molecular basis for RepA function is unknown. Here we describe a complete structural and functional dissection of RepA proteins. Unexpectedly, both the RepA NTD and CTD show similarity to the corresponding domains of the bacterial primosome protein, DnaD. Although the RepA and DnaD NTD both contain winged helix-turn-helices, the DnaD NTD self-assembles into large scaffolds whereas the tetrameric RepA NTD binds DNA iterons using a newly described DNA binding mode. Strikingly, structural and atomic force microscopy data reveal that the NTD tetramer mediates DNA bridging, suggesting a molecular mechanism for origin handcuffing. Finally, data show that the RepA CTD interacts with the host DnaG primase, which binds the replicative helicase. Thus, these combined data reveal the molecular mechanism by which RepA mediates the specific replicon assembly of staphylococcal multiresistant plasmids.

Ligand concentration regulates the pathways of coupled protein folding and binding.

Coupled ligand binding and conformational change plays a central role in biological regulation. Ligands often regulate protein function by modulating conformational dynamics, yet the order in which binding and conformational change occurs are often hotly debated. Here we show that the "conformational selection versus induced fit" distinction on which this debate is based is a false dichotomy because the mechanism depends on ligand concentration. Using the binding of pyrophosphate (PPi) to Bacillus subtilis RNase P protein as a model, we show that coupled reactions are best understood as a change in flux between competing pathways with distinct orders of binding and conformational change. The degree of partitioning through each pathway depends strongly on PPi concentration, with ligand binding redistributing the conformational ensemble toward the folded state by both increasing folding rates and decreasing unfolding rates. These results indicate that ligand binding induces marked and varied changes in protein conformational dynamics, and that the order of binding and conformational change is ligand concentration dependent.

The molecular mechanisms of allosteric mutations impairing MepR repressor function in multidrug-resistant strains of Staphylococcus aureus.

UNLABELLED: Overexpression of the Staphylococcus aureus multidrug efflux pump MepA confers resistance to a wide variety of antimicrobials. mepA expression is controlled by MarR family member MepR, which represses mepA and autorepresses its own production. Mutations in mepR are a primary cause of mepA overexpression in clinical isolates of multidrug-resistant S. aureus. Here, we report crystal structures of three multidrug-resistant MepR variants, which contain the single-amino-acid substitution A103V, F27L, or Q18P, and wild-type MepR in its DNA-bound conformation. Although each mutation impairs MepR function by decreasing its DNA binding affinity, none is located in the DNA binding domain. Rather, all are found in the linker region connecting the dimerization and DNA binding domains. Specifically, the A103V substitution impinges on F27, which resolves potential steric clashes via displacement of the DNA binding winged-helix-turn-helix motifs that lead to a 27-fold reduction in DNA binding affinity. The F27L substitution forces F104 into an alternative rotamer, which kinks helix 5, thereby interfering with the positioning of the DNA binding domains and decreasing mepR operator affinity by 35-fold. The Q18P mutation affects the MepR structure and function most significantly by either creating kinks in the middle of helix 1 or completely unfolding its C terminus. In addition, helix 5 of Q18P is either bent or completely dissected into two smaller helices. Consequently, DNA binding is diminished by 2,000-fold. Our structural studies reveal heretofore-unobserved allosteric mechanisms that affect repressor function of a MarR family member and result in multidrug-resistant Staphylococcus aureus. IMPORTANCE: Staphylococcus aureus is a major health threat to immunocompromised patients. S. aureus multidrug-resistant variants that overexpress the multidrug efflux pump mepA emerge frequently due to point mutations in MarR family member MepR, the mepA transcription repressor. Significantly, the majority of MepR mutations identified in these S. aureus clinical isolates are found not in the DNA binding domain but rather in a linker region, connecting the dimerization and DNA binding domains. The location of these mutants underscores the critical importance of a properly functioning allosteric mechanism that regulates MepR function. Understanding the dysregulation of such allosteric MepR mutants underlies this study. The high-resolution structures of three such allosteric MepR mutants reveal unpredictable conformational consequences, all of which preclude cognate DNA binding, while biochemical studies emphasize their debilitating effects on DNA binding affinity. Hence, mutations in the linker region of MepR and their structural consequences are key generators of multidrug-resistant Staphylococcus aureus.

SlmA forms a higher-order structure on DNA that inhibits cytokinetic Z-ring formation over the nucleoid.

The spatial and temporal control of Filamenting temperature sensitive mutant Z (FtsZ) Z-ring formation is crucial for proper cell division in bacteria. In Escherichia coli, the synthetic lethal with a defective Min system (SlmA) protein helps mediate nucleoid occlusion, which prevents chromosome fragmentation by binding FtsZ and inhibiting Z-ring formation over the nucleoid. However, to perform its function, SlmA must be bound to the nucleoid. To deduce the basis for this chromosomal requirement, we performed biochemical, cellular, and structural studies. Strikingly, structures show that SlmA dramatically distorts DNA, allowing it to bind as an orientated dimer-of-dimers. Biochemical data indicate that SlmA dimer-of-dimers can spread along the DNA. Combined structural and biochemical data suggest that this DNA-activated SlmA oligomerization would prevent FtsZ protofilament propagation and bundling. Bioinformatic analyses localize SlmA DNA sites near membrane-tethered chromosomal regions, and cellular studies show that SlmA inhibits FtsZ reservoirs from forming membrane-tethered Z rings. Thus, our combined data indicate that SlmA DNA helps block Z-ring formation over chromosomal DNA by forming higher-order protein-nucleic acid complexes that disable FtsZ filaments from coalescing into proper structures needed for Z-ring creation.

Structures of the Escherichia coli transcription activator and regulator of diauxie, XylR: an AraC DNA-binding family member with a LacI/GalR ligand-binding domain.

Escherichia coli can rapidly switch to the metabolism of l-arabinose and d-xylose in the absence of its preferred carbon source, glucose, in a process called carbon catabolite repression. Transcription of the genes required for l-arabinose and d-xylose consumption is regulated by the sugar-responsive transcription factors, AraC and XylR. E. coli represents a promising candidate for biofuel production through the metabolism of hemicellulose, which is composed of d-xylose and l-arabinose. Understanding the l-arabinose/d-xylose regulatory network is key for such biocatalyst development. Unlike AraC, which is a well-studied protein, little is known about XylR. To gain insight into XylR function, we performed biochemical and structural studies. XylR contains a C-terminal AraC-like domain. However, its N-terminal d-xylose-binding domain contains a periplasmic-binding protein (PBP) fold with structural homology to LacI/GalR transcription regulators. Like LacI/GalR proteins, the XylR PBP domain mediates dimerization. However, unlike LacI/GalR proteins, which dimerize in a parallel, side-to-side manner, XylR PBP dimers are antiparallel. Strikingly, d-xylose binding to this domain results in a helix to strand transition at the dimer interface that reorients both DNA-binding domains, allowing them to bind and loop distant operator sites. Thus, the combined data reveal the ligand-induced activation mechanism of a new family of DNA-binding proteins.

Molecular basis for a protein-mediated DNA-bridging mechanism that functions in condensation of the E. coli chromosome.

The E. coli chromosome is condensed into insulated regions termed macrodomains (MDs), which are essential for genomic packaging. How chromosomal MDs are specifically organized and compacted is unknown. Here, we report studies revealing the molecular basis for Terminus-containing (Ter) chromosome condensation by the Ter-specific factor MatP. MatP contains a tripartite fold with a four-helix bundle DNA-binding motif, ribbon-helix-helix and C-terminal coiled-coil. Strikingly, MatP-matS structures show that the MatP coiled-coils form bridged tetramers that flexibly link distant matS sites. Atomic force microscopy and electron microscopy studies demonstrate that MatP alone loops DNA. Mutation of key coiled-coil residues destroys looping and causes a loss of Ter condensation in vivo. Thus, these data reveal the molecular basis for a protein-mediated DNA-bridging mechanism that mediates condensation of a large chromosomal domain in enterobacteria.

Implications for proteasome nuclear localization revealed by the structure of the nuclear proteasome tether protein Cut8.

Degradation of nuclear proteins by the 26S proteasome is essential for cell viability. In yeast, the nuclear envelope protein Cut8 mediates nuclear proteasomal sequestration by an uncharacterized mechanism. Here we describe structures of Schizosaccharomyces pombe Cut8, which shows that it contains a unique, modular fold composed of an extended N-terminal, lysine-rich segment that when ubiquitinated binds the proteasome, a dimer domain followed by a six-helix bundle connected to a flexible C tail. The Cut8 six-helix bundle shows structural similarity to 14-3-3 phosphoprotein-binding domains, and binding assays show that this domain is necessary and sufficient for liposome and cholesterol binding. Moreover, specific mutations in the 14-3-3 regions corresponding to putative cholesterol recognition/interaction amino acid consensus motifs abrogate cholesterol binding. In vivo studies confirmed that the 14-3-3 region is necessary for Cut8 membrane localization and that dimerization is critical for its function. Thus, the data reveal the Cut8 organization at the nuclear envelope. Reconstruction of Cut8 evolution suggests that it was present in the last common ancestor of extant eukaryotes and accordingly that nuclear proteasomal sequestration is an ancestral eukaryotic feature. The importance of Cut8 for cell viability and its absence in humans suggests it as a possible target for the development of specific chemotherapeutics against invasive fungal infections.