Dynamics of interdomain rotation facilitates FtsZ filament assembly.

FtsZ, the tubulin homolog essential for bacterial cell division, assembles as Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. It is unclear how FtsZ achieves its kinetic polarity that drives treadmilling. To obtain insights into fundamental features of FtsZ assembly dynamics independent of peptidoglycan synthesis, we carried out the structural and biochemical characterization of FtsZ from the cell wall-less bacteria, Spiroplasma melliferum (SmFtsZ). Interestingly the structures of SmFtsZ, determined in GDP and GMPPNP bound form, were captured as domain swapped dimers. SmFtsZ was found to be a slower GTPase and has higher critical concentration (CC) for polymerization compared to Escherichia coli FtsZ (EcFtsZ). In FtsZs, a conformational switch from R-state (close) to T-state (open) favors polymerization. We identified a residue, Phe224, located at the interdomain cleft of SmFtsZ, which is crucial for R- to T-state transition. The mutation F224M in SmFtsZ cleft resulted in higher GTPase activity and lower CC, whereas the corresponding M225F in EcFtsZ resulted in cell division defects in E. coli. Our results demonstrate that relative rotation of the domains is a rate-limiting step of polymerization and that the dynamics of the interdomain rotation is important for the assembly of FtsZ filament. Our structural analysis of interdomain interactions suggests that this step is plausibly triggered upon addition of a GTP-bound monomer to the filament through interaction of the preformed N-terminal domain (NTD). Hence, the addition of monomers to the NTD-exposed end of filament is slower in comparison to the C-terminal domain (CTD) end, thus explaining kinetic polarity. In summary, the study sheds light on the molecular mechanisms underlying FtsZ assembly dynamics and highlights the importance of interdomain interactions, conformational changes, and specific residues in regulating FtsZ polymerization, which are crucial for bacterial cell division.

Mechanism of GTPase activation of a prokaryotic small Ras-like GTPase MglA by an asymmetrically interacting MglB dimer.

Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one cell pole to the other in response to extracellular signals. MglA dynamics is regulated by MglB, which functions both as a GTPase activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. With an aim to dissect the asymmetric role of the two MglB protomers in the dual GAP and GEF activities, we generated a functional MglAB complex by coexpressing MglB with a linked construct of MglA and MglB. This strategy enabled us to generate mutations of individual MglB protomers (MglB1 or MglB2 linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB1, but not MglB2, stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal ß-strand of MglB1 and ß0 of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB2, which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB2 protomer in the GAP activity of MglB is limited to restricting the conformation of MglA active site loops. The direct demonstration of the allosteric mechanism of GEF action provides us new insights into the regulation of small Ras-like GTPases, a feature potentially present in many uncharacterized GEFs.

Getting bacterial cells into shape.

The conformational state of a structural protein in bacteria can vary, depending on the concentration level of potassium ions or the nucleotide bound to it.

A mechanism of salt bridge-mediated resistance to FtsZ inhibitor PC190723 revealed by a cell-based screen.

Bacterial cell division proteins, especially the tubulin homologue FtsZ, have emerged as strong targets for developing new antibiotics. Here, we have utilized the fission yeast heterologous expression system to develop a cell-based assay to screen for small molecules that directly and specifically target the bacterial cell division protein FtsZ. The strategy also allows for simultaneous assessment of the toxicity of the drugs to eukaryotic yeast cells. As a proof-of-concept of the utility of this assay, we demonstrate the effect of the inhibitors sanguinarine, berberine, and PC190723 on FtsZ. Though sanguinarine and berberine affect FtsZ polymerization, they exert a toxic effect on the cells. Further, using this assay system, we show that PC190723 affects Helicobacter pylori FtsZ function and gain new insights into the molecular determinants of resistance to PC190723. On the basis of sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt bridge interactions between the central H7 helix and ß-strands S9 and S10 mediates resistance to PC190723 in FtsZ. The single-step in vivo cell-based assay using fission yeast enabled us to dissect the contribution of sequence-specific features of FtsZ and cell permeability effects associated with bacterial cell envelopes. Thus, our assay serves as a potent tool to rapidly identify novel compounds targeting polymeric bacterial cytoskeletal proteins like FtsZ to understand how they alter polymerization dynamics and address resistance determinants in targets.

Characterization of heterologously expressed Fibril, a shape and motility determining cytoskeletal protein of the helical bacterium Spiroplasma.

Fibril is a constitutive filament-forming cytoskeletal protein of unidentified fold, exclusive to members of genus Spiroplasma. It is hypothesized to undergo conformational changes necessary to bring about Spiroplasma motility through changes in cell helicity. However, the mechanism driving conformational changes in Fibril remains unknown. We expressed Fibril from S. citri in E. coli for its purification and characterization. Sodium dodecyl sulfate solubilized Fibril filaments and facilitated purification by affinity chromatography. An alternative protocol for obtaining enriched insoluble Fibril filaments was standardized using density gradient centrifugation. Electron microscopy of Fibril purified by these protocols revealed filament bundles. Probable domain boundaries of Fibril protein were identified based on mass spectrometric analysis of proteolytic fragments. Presence of α-helical and ß-sheet signatures in FT-IR measurements suggests that Fibril filaments consist of an assembly of folded globular domains, and not a ß-strand-based aggregation like amyloid fibrils.

Filament organization of the bacterial actin MreB is dependent on the nucleotide state.

MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod-shaped bacteria. Despite belonging to the actin family, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we provide insights into the effect of nucleotide state on membrane binding of Spiroplasma citri MreB5 (ScMreB5). Filaments of ScMreB5WT and an ATPase-deficient mutant, ScMreB5E134A, assemble independently of the nucleotide state. However, capture of the filament dynamics revealed that efficient filament formation and organization through lateral interactions are affected in ScMreB5E134A. Hence, the catalytic glutamate functions as a switch, (a) by sensing the ATP-bound state for filament assembly and (b) by assisting hydrolysis, thereby potentially triggering disassembly, as observed in other actins. Glu134 mutation and the bound nucleotide exhibit an allosteric effect on membrane binding, as observed from the differential liposome binding. We suggest that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis among actins has been repurposed in MreBs for modulating filament organization on the membrane.

Dual specificity of a prokaryotic GTPase-activating protein (GAP) to two small Ras-like GTPases in Myxococcus xanthus.

Two small Ras-like GTPases, MglA and SofG, work in synchrony to drive cell polarity and motility in the soil bacterium, Myxococcus xanthus. While MglA regulates two types of motility in Myxococcus and drives cell polarity reversals, SofG regulates social motility enabled by the type IV pili (T4P) machinery. In order to understand the molecular basis of how multiple GTPases act concertedly, we initiated biochemical studies on SofG. A construct of SofG (SofG∆60 ) was purified as a homogenous monomer and could bind to GDP and GTP. Intrinsic GTP hydrolysis by SofG∆60 was negligible. Earlier work from the laboratory revealed that MglB functions both as a GTPase-activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. Biochemical assays of SofG∆60 established that MglB interacts with GTP-bound SofG∆60 and acts as a GAP for SofG∆60 . Interaction of MglB with SofG∆60 in the GDP-bound conformation was not observed, thereby suggesting that MglB might not act as a GEF for SofG∆60 . The existence of a common GAP for both SofG and MglA could potentially contribute to concerted regulation of their GTPase activities, and mediate crosstalk between the two GTPases involved in motility of M. xanthus. Sequence analysis revealed the features for a SofG-like subclass of prokaryotic small Ras-like GTPases that enable MglB to act as a dual-specificity GAP.

Exploring Spiroplasma Biology: Opportunities and Challenges.

Spiroplasmas are cell-wall-deficient helical bacteria belonging to the class Mollicutes. Their ability to maintain a helical shape in the absence of cell wall and their motility in the absence of external appendages have attracted attention from the scientific community for a long time. In this review we compare and contrast motility, shape determination and cytokinesis mechanisms of Spiroplasma with those of other Mollicutes and cell-walled bacteria. The current models for rod-shape determination and cytokinesis in cell-walled bacteria propose a prominent role for the cell wall synthesis machinery. These models also involve the cooperation of the actin-like protein MreB and FtsZ, the bacterial homolog of tubulin. However the exact role of the cytoskeletal proteins is still under much debate. Spiroplasma possess MreBs, exhibit a rod-shape dependent helical morphology, and divide by an FtsZ-dependent mechanism. Hence, spiroplasmas represent model organisms for deciphering the roles of MreBs and FtsZ in fundamental mechanisms of non-spherical shape determination and cytokinesis in bacteria, in the absence of a cell wall. Identification of components implicated in these processes and deciphering their functions would require genetic experiments. Challenges in genetic manipulations in spiroplasmas are a major bottleneck in understanding their biology. We discuss advancements in genome sequencing, gene editing technologies, super-resolution microscopy and electron cryomicroscopy and tomography, which can be employed for addressing long-standing questions related to Spiroplasma biology.

MreB5 Is a Determinant of Rod-to-Helical Transition in the Cell-Wall-less Bacterium Spiroplasma.

In most rod-shaped bacteria, the spatial coordination of cell wall synthesis machinery by MreBs is the main theme for shape determination and maintenance in cell-walled bacteria [1-9]. However, how rod or spiral shapes are achieved and maintained in cell-wall-less bacteria is currently unknown. Spiroplasma, a helical Mollicute that lacks cell wall synthesis genes, encodes five MreB paralogs and a unique cytoskeletal protein fibril [10, 11]. Here, we show that MreB5, one of the five MreB paralogs, contributes to cell elongation and is essential for the transition from rod-to-helical shape in Spiroplasma. Comparative genomic and proteomic characterization of a helical and motile wild-type Spiroplasma strain and a non-helical, non-motile natural variant helped delineate the specific roles of MreB5. Moreover, complementation of the non-helical strain with MreB5 restored its helical shape and motility by a kink-based mechanism described for Spiroplasma [12]. Earlier studies had proposed that length changes in fibril filaments are responsible for the change in handedness of the helical cell and kink propagation during motility [13]. Through structural and biochemical characterization, we identify that MreB5 exists as antiparallel double protofilaments that interact with fibril and the membrane, and thus potentially assists in kink propagation. In summary, our study provides direct experimental evidence for MreB in maintaining cell length, helical shape, and motility-revealing the role of MreB in sculpting the cell in the absence of a cell wall.

A Distinct Motif in a Prokaryotic Small Ras-Like GTPase Highlights Unifying Features of Walker B Motifs in P-Loop NTPases.

A hallmark of the catalytically essential Walker B motif of P-loop NTPases is the presence of an acidic residue (aspartate/glutamate) for efficient Mg2+ coordination. Although the Walker B motif has been identified in well-studied examples of P-loop NTPases, its identity is ambiguous in many families, for example, in the prokaryotic small Ras-like GTPase family of MglA. MglA, belonging to TRAFAC class of P-loop NTPases, possesses a threonine at the position equivalent to Walker B aspartate in eukaryotic Ras-like GTPases. To resolve the identity of the Walker B residue in MglA, we carried out a comprehensive analysis of Mg2+ coordination on P-loop NTPase structures. Atoms in the octahedral coordination of Mg2+ and their interactions comprise a network including water molecules, Walker A, Walker B and switch motifs of P-loop NTPases. Based on the conserved geometry of Mg2+ coordination, we confirm that a conserved aspartate functions as the Walker B residue of MglA, and validate it through mutagenesis and biochemical characterization. Location of the newly identified aspartate is spatially equivalent to the Walker B residue of the ASCE division of P-loop NTPases. Furthermore, similar to the allosteric regulation of the Walker B aspartate conformation in MglA, we identify protein families in which large conformational changes involving Walker B motif potentially function as allosteric regulators. The study unravels conserved features of Mg2+ coordination among divergent families of P-loop NTPases, especially between ancient Ras-like GTPases and ASCE family of ATPases. The conserved geometric features provide a foundation for design of nucleotide-hydrolyzing enzymes.

Genetic suppression of defective profilin by attenuated Myosin II reveals a potential role for Myosin II in actin dynamics in vivo in fission yeast.

The actin cytoskeleton plays a variety of roles in eukaryotic cell physiology, ranging from cell polarity and migration to cytokinesis. Key to the function of the actin cytoskeleton is the mechanisms that control its assembly, stability, and turnover. Through genetic analyses in Schizosaccharomyces pombe, we found that myo2-S1 (myo2-G515D), a Myosin II mutant allele, was capable of rescuing lethality caused by partial defects in actin nucleation/stability caused, for example, through compromised function of the actin-binding protein Cdc3-profilin. The mutation in myo2-S1 affects the activation loop of Myosin II, which is involved in physical interaction with subdomain 1 of actin and in stimulating the ATPase activity of Myosin. Consistently, actomyosin rings in myo2-S1 cell ghosts were unstable and severely compromised in contraction on ATP addition. These studies strongly suggest a role for Myo2 in actin cytoskeletal disassembly and turnover in vivo, and that compromise of this activity leads to genetic suppression of mutants defective in actin filament assembly/stability at the division site.

Allosteric regulation of a prokaryotic small Ras-like GTPase contributes to cell polarity oscillations in bacterial motility.

Mutual gliding motility A (MglA), a small Ras-like GTPase; Mutual gliding motility B (MglB), its GTPase activating protein (GAP); and Required for Motility Response Regulator (RomR), a protein that contains a response regulator receiver domain, are major components of a GTPase-dependent biochemical oscillator that drives cell polarity reversals in the bacterium Myxococcus xanthus. We report the crystal structure of a complex of M. xanthus MglA and MglB, which reveals that the C-terminal helix (Ct-helix) from one protomer of the dimeric MglB binds to a pocket distal to the active site of MglA. MglB increases the GTPase activity of MglA by reorientation of key catalytic residues of MglA (a GAP function) combined with allosteric regulation of nucleotide exchange by the Ct-helix (a guanine nucleotide exchange factor [GEF] function). The dual GAP-GEF activities of MglB accelerate the rate of GTP hydrolysis over multiple enzymatic cycles. Consistent with its GAP and GEF activities, MglB interacts with MglA bound to either GTP or GDP. The regulation is essential for cell polarity, because deletion of the Ct-helix causes bipolar localization of MglA, MglB, and RomR, thereby causing reversal defects in M. xanthus. A bioinformatics analysis reveals the presence of Ct-helix in homologues of MglB in other bacterial phyla, suggestive of the prevalence of the allosteric mechanism among other prokaryotic small Ras-like GTPases.

Mechanistic insights into enzymatic catalysis by trehalase from the insect gut endosymbiont Enterobacter cloacae.

Energy metabolism in the diamondback moth Plutella xylostella is facilitated by trehalase, an enzyme which assists in trehalose hydrolysis, from the predominant gut bacterium Enterobacter cloacae. We report the biochemical and structural characterization of recombinant trehalase from E. cloacae (Px_EclTre). Px_EclTre showed KM of 1.47 (±0.05) mm, kcat of 6254.72 min-1 and Vmax 0.2 (±0.002) mm·min-1 at 55 °C and acidic pH. Crystal structures of Px_EclTre were determined in the ligand-free form and bound to the inhibitor Validoxylamine A. The crystal structure of the ligand-free form, unavailable until now for any other bacterial trehalases, enabled us to delineate the conformational changes accompanying ligand binding in trehalases. Multiple salt bridges were identified that potentially facilitated closure of a hood over the substrate-binding site. A cluster of five tryptophans lined the -1 substrate-binding subsite, interacted with crucial active site residues and contributed to both trehalase activity and stability. The importance of these residues in enzyme activity was further validated by mutagenesis studies. Many of these identified residues form part of signature motifs and other conserved sequences in trehalases. The structure analysis thus led to the assignment of the functional role to these conserved residues. This information can be further explored for the design of effective inhibitors against trehalases.

Steric hindrance in the upper 50†kDa domain of the motor Myo2p leads to cytokinesis defects in fission yeast.

Cytokinesis in many eukaryotes requires a contractile actomyosin ring that is placed at the division site. In fission yeast, which is an attractive organism for the study of cytokinesis, actomyosin ring assembly and contraction requires the myosin II heavy chain Myo2p. Although myo2-E1, a temperature-sensitive mutant defective in the upper 50 kDa domain of Myo2p, has been studied extensively, the molecular basis of the cytokinesis defect is not understood. Here, we isolate myo2-E1-Sup2, an intragenic suppressor that contains the original mutation in myo2-E1 (G345R) and a second mutation in the upper 50 kDa domain (Y297C). Unlike myo2-E1-Sup1, a previously characterized myo2-E1 suppressor, myo2-E1-Sup2 reverses actomyosin ring contraction defects in vitro and in vivo Structural analysis of available myosin motor domain conformations suggests that a steric clash in myo2-E1, which is caused by the replacement of a glycine with a bulky arginine, is relieved in myo2-E1-Sup2 by mutation of a tyrosine to a smaller cysteine. Our work provides insight into the function of the upper 50 kDa domain of Myo2p, informs a molecular basis for the cytokinesis defect in myo2-E1, and may be relevant to the understanding of certain cardiomyopathies.

The nucleoid as a scaffold for the assembly of bacterial signaling complexes.

The FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays.

Structure and Dynamics of Actin-Like Cytomotive Filaments in Plasmid Segregation.

One of the well-known functions of the bacterial cytoskeleton is plasmid segregation. Type II plasmid segregation systems, among the best characterized with respect to the mechanism of action, possess an actin-like cytomotive filament as the motor component. This chapter describes the essential components of the plasmid segregation machinery and their mechanism of action, concentrating on the actin-like protein family of the bacterial cytoskeleton. The structures of the actin-like filaments depend on their nucleotide state and these in turn contribute to the dynamics of the filaments. The components that link the filaments to the plasmid DNA also regulate filament dynamics. The modulation of the dynamics facilitates the cytomotive filament to function as a mitotic spindle with a minimal number of components.

Motor Activity Dependent and Independent Functions of Myosin II Contribute to Actomyosin Ring Assembly and Contraction in Schizosaccharomyces pombe.

Cytokinesis depends on a contractile actomyosin ring in many eukaryotes [1-3]. Myosin II is a key component of the actomyosin ring, although whether it functions as a motor or as an actin cross-linker to exert its essential role is disputed [1, 4, 5]. In Schizosaccharomyces pombe, the myo2-E1 mutation affects the upper 50 kDa sub-domain of the myosin II heavy chain, and cells carrying this lethal mutation are defective in actomyosin ring assembly at the non-permissive temperature [6, 7]. myo2-E1 also affects actomyosin ring contraction when rings isolated from permissive temperature-grown cells are incubated with ATP [8]. Here we report isolation of a compensatory suppressor mutation in the lower 50 kDa sub-domain (myo2-E1-Sup1) that reverses the inability of myo2-E1 to form colonies at the restrictive temperature. myo2-E1-Sup1 is capable of assembling normal actomyosin rings, although rings isolated from myo2-E1-Sup1 are defective in ATP-dependent contraction in vitro. Furthermore, the product of myo2-E1-Sup1 does not translocate actin filaments in motility assays in vitro. Superimposition of myo2-E1 and myo2-E1-Sup1 on available rigor and blebbistatin-bound myosin II structures suggests that myo2-E1-Sup1 may represent a novel actin translocation-defective allele. Actomyosin ring contraction and viability of myo2-E1-Sup1 cells depend on the late cytokinetic S. pombe myosin II isoform, Myp2p, a non-essential protein that is normally dispensable for actomyosin ring assembly and contraction. Our work reveals that Myo2p may function in two different and essential modes during cytokinesis: a motor activity-independent form that can promote actomyosin ring assembly and a motor activity-dependent form that supports ring contraction.

Nup358 binds to AGO proteins through its SUMO-interacting motifs and promotes the association of target mRNA with miRISC.

MicroRNA (miRNA)-guided mRNA repression, mediated by the miRNA-induced silencing complex (miRISC), is an important component of post-transcriptional gene silencing. However, how miRISC identifies the target mRNA in vivo is not well understood. Here, we show that the nucleoporin Nup358 plays an important role in this process. Nup358 localizes to the nuclear pore complex and to the cytoplasmic annulate lamellae (AL), and these structures dynamically associate with two mRNP granules: processing bodies (P bodies) and stress granules (SGs). Nup358 depletion disrupts P bodies and concomitantly impairs the miRNA pathway. Furthermore, Nup358 interacts with AGO and GW182 proteins and promotes the association of target mRNA with miRISC A well-characterized SUMO-interacting motif (SIM) in Nup358 is sufficient for Nup358 to directly bind to AGO proteins. Moreover, AGO and PIWI proteins interact with SIMs derived from other SUMO-binding proteins. Our study indicates that Nup358-AGO interaction is important for miRNA-mediated gene silencing and identifies SIM as a new interacting motif for the AGO family of proteins. The findings also support a model wherein the coupling of miRISC with the target mRNA could occur at AL, specialized domains within the ER, and at the nuclear envelope.

Bacterial Actins and Their Interactors.

Bacterial actins polymerize in the presence of nucleotide (preferably ATP), form a common arrangement of monomeric interfaces within a protofilament, and undergo ATP hydrolysis-dependent change in stability of the filament-all of which contribute to performing their respective functions. The relative stability of the filament in the ADP-bound form compared to that of ATP and the rate of addition of monomers at the two ends decide the filament dynamics. One of the major differences between eukaryotic actin and bacterial actins is the variety in protofilament arrangements and dynamics exhibited by the latter. The filament structure and the polymerization dynamics enable them to perform various functions such as shape determination in rod-shaped bacteria (MreB), cell division (FtsA), plasmid segregation (ParM family of actin-like proteins), and organelle positioning (MamK). Though the architecture and dynamics of a few representative filaments have been studied, information on the effect of interacting partners on bacterial actin filament dynamics is not very well known. The chapter reviews some of the structural and functional aspects of bacterial actins, with special focus on the effect that interacting partners exert on the dynamics of bacterial actins, and how these assist them to carry out the functions within the bacterial cell.

Structure of the ParM filament at 8.5Å resolution.

The actin-like protein ParM forms the cytomotive filament of the ParMRC system, a type II plasmid segregation system encoded by Escherichia coli R1 plasmid. We report an 8.5Å resolution reconstruction of the ParM filament, obtained using cryo-electron microscopy. Fitting of the 3D density reconstruction with monomeric crystal structures of ParM provides insights into dynamic instability of ParM filaments. The structural analysis suggests that a ParM conformation, corresponding to a metastable state, is held within the filament by intrafilament contacts. This filament conformation of ParM can be attained only from the ATP-bound state, and induces a change in conformation of the bound nucleotide. The structural analysis also provides a rationale for the observed stimulation of hydrolysis upon polymerisation into the filament.