THOUSAND-GRAIN WEIGHT 6, which is an IAA-glucose hydrolase, preferentially recognizes the structure of the indole ring.

An indole-3-acetic acid (IAA)-glucose hydrolase, THOUSAND-GRAIN WEIGHT 6 (TGW6), negatively regulates the grain weight in rice. TGW6 has been used as a target for breeding increased rice yield. Moreover, the activity of TGW6 has been thought to involve auxin homeostasis, yet the details of this putative TGW6 activity remain unclear. Here, we show the three-dimensional structure and substrate preference of TGW6 using X-ray crystallography, thermal shift assays and fluorine nuclear magnetic resonance (19F NMR). The crystal structure of TGW6 was determined at 2.6 Å resolution and exhibited a six-bladed ß-propeller structure. Thermal shift assays revealed that TGW6 preferably interacted with indole compounds among the tested substrates, enzyme products and their analogs. Further analysis using 19F NMR with 1,134 fluorinated fragments emphasized the importance of indole fragments in recognition by TGW6. Finally, docking simulation analyses of the substrate and related fragments in the presence of TGW6 supported the interaction specificity for indole compounds. Herein, we describe the structure and substrate preference of TGW6 for interacting with indole fragments during substrate recognition. Uncovering the molecular details of TGW6 activity will stimulate the use of this enzyme for increasing crop yields and contributes to functional studies of IAA glycoconjugate hydrolases in auxin homeostasis.

Probing the substrate binding modes and catalytic mechanisms of BLEG-1, a promiscuous B3 metallo-ß-lactamase with glyoxalase II properties.

BLEG-1 from Bacillus lehensis G1 is an evolutionary divergent B3 metallo-ß-lactamase (MBL) that exhibited both ß-lactamase and glyoxalase II (GLXII) activities. Sequence, phylogeny, biochemical and structural relatedness of BLEG-1 to B3 MBL and GLXII suggested BLEG-1 might be an intermediate in the evolutionary path of B3 MBL from GLXII. The unique active site cavity of BLEG-1 that recognizes both ß-lactam antibiotics and S-D-lactoylglutathione (SLG) had been postulated as the key factor for its dual activity. In this study, dynamic ensembles of BLEG-1 and its substrate complexes divulged conformational plasticity and binding modes of structurally distinct substrates to the enzyme, providing better insights into its structure-to-function relationship and enzymatic promiscuity. Our results highlight the flexible nature of the active site pocket of BLEG-1, which is governed by concerted loop motions involving loop7+α3+loop8 and loop12 around the catalytic core, thereby moulding the binding pocket and facilitate interactions of BLEG-1 with both ampicillin and SLG. The distribution of (i) predominantly hydrophobic amino acids in the N-terminal domain, and (ii) flexible amino acids with polar and/or charged side chains in both N- and C-termini provide additional advantages to BLEG-1 in confining the aromatic group of ampicillin, and polar groups of SLG, respectively. The importance of these residues for substrates binding was further confirmed by the reduction in MBL and GLXII activities upon alanine substitutions of Ile-10, Phe-57, Arg-94, Leu-95, and Arg-159. Based on molecular dynamics simulation, mutational, and biochemical data presented herein, the catalytic mechanisms of BLEG-1 toward the hydrolysis of ß-lactams and SLG were proposed.

Structures of a FtsZ single protofilament and a double-helical tube in complex with a monobody.

FtsZ polymerizes into protofilaments to form the Z-ring that acts as a scaffold for accessory proteins during cell division. Structures of FtsZ have been previously solved, but detailed mechanistic insights are lacking. Here, we determine the cryoEM structure of a single protofilament of FtsZ from Klebsiella pneumoniae (KpFtsZ) in a polymerization-preferred conformation. We also develop a monobody (Mb) that binds to KpFtsZ and FtsZ from Escherichia coli without affecting their GTPase activity. Crystal structures of the FtsZ-Mb complexes reveal the Mb binding mode, while addition of Mb in vivo inhibits cell division. A cryoEM structure of a double-helical tube of KpFtsZ-Mb at 2.7 Å resolution shows two parallel protofilaments. Our present study highlights the physiological roles of the conformational changes of FtsZ in treadmilling that regulate cell division.

The apiosyltransferase celery UGT94AX1 catalyzes the biosynthesis of the flavone glycoside apiin.

Apiose is a unique branched-chain pentose found in plant glycosides and a key component of the cell wall polysaccharide pectin and other specialized metabolites. More than 1,200 plant-specialized metabolites contain apiose residues, represented by apiin, a distinctive flavone glycoside found in celery (Apium graveolens) and parsley (Petroselinum crispum) in the family Apiaceae. The physiological functions of apiin remain obscure, partly due to our lack of knowledge on apiosyltransferase during apiin biosynthesis. Here, we identified UGT94AX1 as an A. graveolens apiosyltransferase (AgApiT) responsible for catalyzing the last sugar modification step in apiin biosynthesis. AgApiT showed strict substrate specificity for the sugar donor, UDP-apiose, and moderate specificity for acceptor substrates, thereby producing various apiose-containing flavone glycosides in celery. Homology modeling of AgApiT with UDP-apiose, followed by site-directed mutagenesis experiments, identified unique Ile139, Phe140, and Leu356 residues in AgApiT, which are seemingly crucial for the recognition of UDP-apiose in the sugar donor pocket. Sequence comparison and molecular phylogenetic analysis of celery glycosyltransferases suggested that AgApiT is the sole apiosyltransferase-encoding gene in the celery genome. Identification of this plant apiosyltransferase gene will enhance our understanding of the physioecological functions of apiose and apiose-containing compounds.

Structural and Antibacterial Characterization of a New Benzamide FtsZ Inhibitor with Superior Bactericidal Activity and In Vivo Efficacy Against Multidrug-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant (MDR) bacterial pathogen of acute clinical significance. Resistance to current standard-of-care antibiotics, such as vancomycin and linezolid, among nosocomial and community-acquired MRSA clinical isolates is on the rise. This threat to global public health highlights the need to develop new antibiotics for the treatment of MRSA infections. Here, we describe a new benzamide FtsZ inhibitor (TXH9179) with superior antistaphylococcal activity relative to earlier-generation benzamides like PC190723 and TXA707. TXH9179 was found to be 4-fold more potent than TXA707 against a library of 55 methicillin-sensitive S. aureus (MSSA) and MRSA clinical isolates, including MRSA isolates resistant to vancomycin and linezolid. TXH9179 was also associated with a lower frequency of resistance relative to TXA707 in all but one of the MSSA and MRSA isolates examined, with the observed resistance being due to mutations in the ftsZ gene. TXH9179 induced changes in MRSA cell morphology, cell division, and FtsZ localization are fully consistent with its actions as a FtsZ inhibitor. Crystallographic studies demonstrate the direct interaction of TXH9179 with S. aureus FtsZ (SaFtsZ), while delineating the key molecular contacts that drive complex formation. TXH9179 was not associated with any mammalian cytotoxicity, even at a concentration 10-fold greater than that producing antistaphylococcal activity. In serum, the carboxamide prodrug of TXH9179 (TXH1033) is rapidly hydrolyzed to TXH9179 by serum acetylcholinesterases. Significantly, both intravenously and orally administered TXH1033 exhibited enhanced in vivo efficacy relative to the carboxamide prodrug of TXA707 (TXA709) in treating a mouse model of systemic (peritonitis) MRSA infection. Viewed as a whole, our results highlight TXH9179 as a promising new benzamide FtsZ inhibitor worthy of further development.

Real-time monitoring of polyacrylamide gel electrophoresis by the shadowgraph technique.

Polyacrylamide gel electrophoresis (PAGE) with sodium dodecyl sulphate (SDS) and Coomassie brilliant blue (CBB) staining is widely used in protein research and requires time for electrophoresis, staining and destaining. Because the protein bands electrophoresed in the gel are invisible in most cases, the results cannot be observed until the whole process is complete. In this study, shadowgraph was applied to detect biomolecules such as proteins during electrophoresis. A simple optical system and camera-enabled real-time monitoring of migration and separation of individual protein bands in polyacrylamide gels without staining. The visibility was high enough that it was possible to visualize substances other than proteins, such as DNA. This method provides protein profiles instantly in the early stage of electrophoresis. The elimination of the staining and destaining steps will help save researchers' time. The method is also environmentally friendly and will help reduce the generation of waste solutions containing synthetic dyes.

Procedure of the overexpression, purification and crystallization of BLEG-1, a bifunctional and evolutionary divergent B3 metallo-ß-lactamase, for structure-function studies.

Metallo-ß-lactamases (MBLs) are class B ß-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of ß-lactam antibiotics, thus conferring antibiotics resistance to bacterial pathogens. The attempt to structurally characterize BLEG-1, an evolutionary divergent B3 metallo-ß-lactamase (MBL) with dual activity from Bacillus lehensis G1, led to the optimization of its purification, post-purification and crystallization processes for X-ray diffraction purpose. The workflow, conditions used and dataset obtained from each stage of the processes are presented herein. The optimization workflow has enabled the obtainment of purified, active BLEG-1 in high yield for its activity assays, crystallization and structure determination via X-ray diffraction. This is the first step to gain a better insight into its dual activity and evolutionary divergence from a structural perspective. The complete research article, including BLEG-1 dual activity analysis, is published in the International Journal of Molecular Sciences (Au et al., 2021). • The method was optimized to increase the stability of BLEG-1 in purification, post-purification and crystallization processes. • Protein crystallization using the optimized conditions presented herein is able to produce and regenerate BLEG-1 protein crystals of medium-size, which is an advantage in X-ray diffraction. • The method can be used for relevant homologs and variants of BLEG-1 for structure-function and mechanistic studies of such proteins.

C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers.

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-ß2 (Kapß2) at 1:1 ratio. The nuclear magnetic resonances of Kapß2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapß2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.

Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-ß-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence.

Metallo-ß-lactamases (MBLs) are class B ß-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of ß-lactam antibiotics. A previous study on BLEG-1 (formerly called Bleg1_2437), a hypothetical protein from Bacillus lehensis G1, revealed sequence similarity and activity to B3 subclass MBLs, despite its evolutionary divergence from these enzymes. Its relatedness to glyoxalase II (GLXII) raises the possibility of its enzymatic promiscuity and unique structural features compared to other MBLs and GLXIIs. This present study highlights that BLEG-1 possessed both MBL and GLXII activities with similar catalytic efficiencies. Its crystal structure revealed highly similar active site configuration to YcbL and GloB GLXIIs from Salmonella enterica, and L1 B3 MBL from Stenotrophomonas maltophilia. However, different from GLXIIs, BLEG-1 has an insertion of an active-site loop, forming a binding cavity similar to B3 MBL at the N-terminal region. We propose that BLEG-1 could possibly have evolved from GLXII and adopted MBL activity through this insertion.

Expression, purification and crystallization of TGW6, which limits grain weight in rice.

Rice is the staple food for over half the world's population. Genes associated with rice yield include THOUSAND GRAIN WEIGHT 6 (TGW6), which negatively regulates the number of endosperm cells as well as grain weight. The 1-bp deletion allele of tgw6 cloned from the Indian landrace rice cultivar Kasalath, which has lost function, enhances both grain size and yield. TGW6 has been utilized as a target for breeding and genome editing to increase the yield of rice. In the present study, we describe an improved heterologous expression system of TGW6 in Escherichia coli to enable purification of the recombinant protein. The best expression was achieved using codon optimized TGW6 with a 30 amino acid truncation at the N-terminus (Δ30TGW6) in the Rosetta-gami 2(DE3) host strain. Furthermore, we found that calcium ions were critical for the purification of stable Δ30TGW6. Crystals of Δ30TGW6 were obtained using the sitting-drop vapor-diffusion method at 283 K, which diffracted X-rays to at least 2.6 Å resolution. Herein, we established an efficient procedure for the production and purification of TGW6 in sufficient quantities for structural and functional studies. Detailed information concerning the molecular mechanism of TGW6 will enable the design of more efficient ways to control the activity of the enzyme.

Structure-function studies of ultrahigh molecular weight isoprenes provide key insights into their biosynthesis.

Some plant trans-1,4-prenyltransferases (TPTs) produce ultrahigh molecular weight trans-1,4-polyisoprene (TPI) with a molecular weight of over 1.0 million. Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear. Here, we identified three novel Eucommia ulmoides TPT isoforms-EuTPT1, 3, and 5, which synthesized TPI in vitro without other components. Crystal structure analysis of EuTPT3 revealed a dimeric architecture with a central hydrophobic tunnel. Mutation of Cys94 and Ala95 on the central hydrophobic tunnel no longer synthesizd TPI, indicating that Cys94 and Ala95 were essential for forming the dimeric architecture of ultralong-chain TPTs and TPI biosynthesis. A spatiotemporal analysis of the physiological function of TPI in E. ulmoides suggested that it is involved in seed development and maturation. Thus, our analysis provides functional and mechanistic insights into TPI biosynthesis and uncovers biological roles of TPI in plants.

Dynamic Assembly/Disassembly of Staphylococcus aureus FtsZ Visualized by High-Speed Atomic Force Microscopy.

FtsZ is a key protein in bacterial cell division and is assembled into filamentous architectures. FtsZ filaments are thought to regulate bacterial cell division and have been investigated using many types of imaging techniques such as atomic force microscopy (AFM), but the time scale of the method was too long to trace the filament formation process. Development of high-speed AFM enables us to achieve sub-second time resolution and visualize the formation and dissociation process of FtsZ filaments. The analysis of the growth and dissociation rates of the C-terminal truncated FtsZ (FtsZt) filaments indicate the net growth and dissociation of FtsZt filaments in the growth and dissociation conditions, respectively. We also analyzed the curvatures of the full-length FtsZ (FtsZf) and FtsZt filaments, and the comparative analysis indicated the straight-shape preference of the FtsZt filaments than those of FtsZf. These findings provide insights into the fundamental dynamic behavior of FtsZ protofilaments and bacterial cell division.

Mechanism of karyopherin-ß2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X).

Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid-liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-ß2 (Kapß2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapß2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapß2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapß2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapß2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapß2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.

Insertion loop-mediated folding propagation governs efficient maturation of hyperthermophilic Tk-subtilisin at high temperatures.

The serine protease Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1-IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk-subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk-subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur. Alanine substitution of the aspartate residue in IS3 disturbed the intraloop hydrogen-bonding network, as evidenced by crystallographic analysis, resulting in compromised folding at high temperatures. Taking into account the high conservation of IS3 across hyperthermophilic homologues, we propose that the presence of IS3 is important for folding of hyperthermophilic subtilisins in high-temperature environments.

Effect of nuclear import receptors on liquid-liquid phase separation.

Low-complexity (LC) sequences, regions that are predominantly made up of limited amino acids, are often observed in eukaryotic nuclear proteins. The role of these LC sequences has remained unclear for decades. Recent studies have shown that LC sequences are important in the formation of membrane-less organelles via liquid-liquid phase separation (LLPS). The RNA binding protein, fused in sarcoma (FUS), is the most widely studied of the proteins that undergo LLPS. It forms droplets, fibers, or hydrogels using its LC sequences. The N-terminal LC sequence of FUS is made up of Ser, Tyr, Gly, and Gln, which form a labile cross-ß polymer core while the C-terminal Arg-Gly-Gly repeats accelerate LLPS. Normally, FUS localizes to the nucleus via the nuclear import receptor karyopherin ß2 (Kapß2) with the help of its C-terminal proline-tyrosine nuclear localization signal (PY-NLS). Recent findings revealed that Kapß2 blocks FUS mediated LLPS, suggesting that Kapß2 is not only a transport protein but also a chaperone which regulates LLPS during the formation of membrane-less organelles. In this review, we discuss the effects of the nuclear import receptors on LLPS.

Hybrid Rubisco with Complete Replacement of Rice Rubisco Small Subunits by Sorghum Counterparts Confers C4 Plant-like High Catalytic Activity.

Photosynthetic rate at the present atmospheric condition is limited by the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) because of its extremely low catalytic rate (kcat) and poor affinity for CO2 (Kc) and specificity for CO2 (Sc/o). Rubisco in C4 plants generally shows higher kcat than that in C3 plants. Rubisco consists of eight large subunits and eight small subunits (RbcS). Previously, the chimeric incorporation of sorghum C4-type RbcS significantly increased the kcat of Rubisco in a C3 plant, rice. In this study, we knocked out rice RbcS multigene family using the CRISPR-Cas9 technology and completely replaced rice RbcS with sorghum RbcS in rice Rubisco. Obtained hybrid Rubisco showed almost C4 plant-like catalytic properties, i.e., higher kcat, higher Kc, and lower Sc/o. Transgenic lines expressing the hybrid Rubisco accumulated reduced levels of Rubisco, whereas they showed slightly but significantly higher photosynthetic capacity and similar biomass production under high CO2 condition compared with wild-type rice. High-resolution crystal structural analysis of the wild-type Rubisco and hybrid Rubisco revealed the structural differences around the central pore of Rubisco and the ßC-ßD hairpin in RbcS. We propose that such differences, particularly in the ßC-ßD hairpin, may impact the flexibility of Rubisco catalytic site and change its catalytic properties.

Crystal structure of a GH1 ß-glucosidase from Hamamotoa singularis.

A GH1 ß-glucosidase from the fungus Hamamotoa singularis (HsBglA) has high transgalactosylation activity and efficiently converts lactose to galactooligosaccharides. Consequently, HsBglA is among the most widely used enzymes for industrial galactooligosaccharide production. Here, we present the first crystal structures of HsBglA with and without 4'-galactosyllactose, a tri-galactooligosaccharide, at 3.0 and 2.1 Å resolutions, respectively. These structures reveal details of the structural elements that define the catalytic activity and substrate binding of HsBglA, and provide a possible interpretation for its high catalytic potency for transgalactosylation reaction.

Affinity shift of ATP upon glycerol binding to a glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1.

Glycerol kinase (GK) is a key enzyme of glycerol metabolism. It participates in glycolysis and lipid membrane biosynthesis. A hexamer of GK from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1(Tk-GK) was identified as a substrate-binding form of the enzyme. Here, the X-ray crystal structure analysis and the biochemical analysis was done and the relationships between its unique oligomer structure and substrate binding affinity were investigated. Wild type GK and mutant K271E GK, which disrupts the hexamer formation interface, were crystallized with and without their substrates and analyzed at 2.19-3.05 Å resolution. In the absence of glycerol, Tk-GK was a dimer in solution. In the presence of its glycerol substrate, however, it became a hexamer consisting of three symmetrical dimers about the threefold axis. Through glycerol binding, all Tk-GK molecules in the hexamer were in closed form as a result of domain-motion. The closed form of Tk-GK had tenfold higher ATP affinity than the open form of Tk-GK. The hexamer structure stabilized the closed conformation and enhanced ATP binding affinity when the GK was bound to glycerol. This molecular mechanism is quite simple activity regulation mechanism among known GKs.

Crystal structures of the cell-division protein FtsZ from Klebsiella pneumoniae and Escherichia coli.

FtsZ, a tubulin-like GTPase, is essential for bacterial cell division. In the presence of GTP, FtsZ polymerizes into filamentous structures, which are key to generating force in cell division. However, the structural basis for the molecular mechanism underlying FtsZ function remains to be elucidated. In this study, crystal structures of the enzymatic domains of FtsZ from Klebsiella pneumoniae (KpFtsZ) and Escherichia coli (EcFtsZ) were determined at 1.75 and 2.50 Šresolution, respectively. Both FtsZs form straight protofilaments in the crystals, and the two structures adopted relaxed (R) conformations. The T3 loop, which is involved in GTP/GDP binding and FtsZ assembly/disassembly, adopted a unique open conformation in KpFtsZ, while the T3 loop of EcFtsZ was partially disordered. The crystal structure of EcFtsZ can explain the results from previous functional analyses using EcFtsZ mutants.

Structure-Guided Design of a Fluorescent Probe for the Visualization of FtsZ in Clinically Important Gram-Positive and Gram-Negative Bacterial Pathogens.

Addressing the growing problem of antibiotic resistance requires the development of new drugs with novel antibacterial targets. FtsZ has been identified as an appealing new target for antibacterial agents. Here, we describe the structure-guided design of a new fluorescent probe (BOFP) in which a BODIPY fluorophore has been conjugated to an oxazole-benzamide FtsZ inhibitor. Crystallographic studies have enabled us to identify the optimal position for tethering the fluorophore that facilitates the high-affinity FtsZ binding of BOFP. Fluorescence anisotropy studies demonstrate that BOFP binds the FtsZ proteins from the Gram-positive pathogens Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae with Kd values of 0.6-4.6 µM. Significantly, BOFP binds the FtsZ proteins from the Gram-negative pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii with an even higher affinity (Kd = 0.2-0.8 µM). Fluorescence microscopy studies reveal that BOFP can effectively label FtsZ in all the above Gram-positive and Gram-negative pathogens. In addition, BOFP is effective at monitoring the impact of non-fluorescent inhibitors on FtsZ localization in these target pathogens. Viewed as a whole, our results highlight the utility of BOFP as a powerful tool for identifying new broad-spectrum FtsZ inhibitors and understanding their mechanisms of action.