FtsZ filament structures in different nucleotide states reveal the mechanism of assembly dynamics.

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogs and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP γ-phosphate and Mg2+ at the subunit interface, a K+ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signaling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

The crystal structure of human XPG, the xeroderma pigmentosum group G endonuclease, provides insight into nucleotide excision DNA repair.

Nucleotide excision repair (NER) is an essential pathway to remove bulky lesions affecting one strand of DNA. Defects in components of this repair system are at the ground of genetic diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS). The XP complementation group G (XPG) endonuclease cleaves the damaged DNA strand on the 3' side of the lesion coordinated with DNA re-synthesis. Here, we determined crystal structures of the XPG nuclease domain in the absence and presence of DNA. The overall fold exhibits similarities to other flap endonucleases but XPG harbors a dynamic helical arch that is uniquely oriented and defines a gateway. DNA binding through a helix-2-turn-helix motif, assisted by one flanking α-helix on each side, shows high plasticity, which is likely relevant for DNA scanning. A positively-charged canyon defined by the hydrophobic wedge and ß-pin motifs provides an additional DNA-binding surface. Mutational analysis identifies helical arch residues that play critical roles in XPG function. A model for XPG participation in NER is proposed. Our structures and biochemical data represent a valuable tool to understand the atomic ground of XP and CS, and constitute a starting point for potential therapeutic applications.

Structural Characterization of TssL from Acinetobacter baumannii: a Key Component of the Type VI Secretion System.

The type VI secretion system (T6SS) is a complex molecular nanomachine used by Gram-negative bacteria to deliver diverse effectors into adjacent cells. A membrane complex (MC) anchors this transport system to the bacterial cell wall. One of the proteins forming the MC is TssL, a cytoplasmic protein bound to the inner membrane through a single transmembrane helix. Here, we report the structure of the cytoplasmic N-terminal region of TssL from Acinetobacter baumannii, a bacterium encoding in a single locus a secretion system that is a special case among other T6SSs. The protein structure, consisting of two antiparallel alpha-helical bundles connected by a short loop, reveals several interesting particularities compared with homologous proteins from other organisms. In addition, we demonstrate the structural significance of residues Asp98 and Glu99, which are strongly conserved among T6SS-encoding Gram-negative bacteria. Mutations in these two residues strongly impact protein dynamics, expression, and functionality. Our results improve our understanding of the T6SS of A. baumannii, which remains largely understudied compared with that of other pathogens.IMPORTANCE Several Acinetobacter species carry one functional type VI secretion system (T6SS). The T6SS is encoded in a single locus containing 16 conserved genes, most of which code for proteins essential to T6SS activity. One of these key components is TssL, a cytoplasmic protein bound to the inner membrane. Despite its importance and its particular characteristics, the structure of T6SS in A. baumannii remains understudied. Here, we present structural, in silico, and in vivo studies of TssL, highlighting the importance of two well-conserved residues and improving our understanding of this secretion system in this bacterium.

Crystal structure of the 14-subunit RNA polymerase I.

Protein biosynthesis depends on the availability of ribosomes, which in turn relies on ribosomal RNA production. In eukaryotes, this process is carried out by RNA polymerase I (Pol I), a 14-subunit enzyme, the activity of which is a major determinant of cell growth. Here we present the crystal structure of Pol I from Saccharomyces cerevisiae at 3.0 Å resolution. The Pol I structure shows a compact core with a wide DNA-binding cleft and a tightly anchored stalk. An extended loop mimics the DNA backbone in the cleft and may be involved in regulating Pol I transcription. Subunit A12.2 extends from the A190 jaw to the active site and inserts a transcription elongation factor TFIIS-like zinc ribbon into the nucleotide triphosphate entry pore, providing insight into the role of A12.2 in RNA cleavage and Pol I insensitivity to α-amanitin. The A49-A34.5 heterodimer embraces subunit A135 through extended arms, thereby contacting and potentially regulating subunit A12.2.

Combining Crystallography and Hydrogen-Deuterium Exchange to Study Galectin-Ligand Complexes.

The physiological significance arising from translating information stored in glycans into cellular effects explains the interest in structurally defining lectin-carbohydrate recognition. The relatively small set of adhesion/growth-regulatory galectins in chicken makes this system attractive to study the origins of specificity and divergence. Cell binding by using glycosylation mutants reveals binding of the N-terminal domain of chicken galectin-8 (CG-8N) to α-2,3-sialylated and galactose-terminated glycan chains. Cocrystals with lactose and its 3'-sialylated derivative disclose Arg58 as a key contact for the carboxylic acid and differences in loop lengths to the three homodimeric chicken galectins. Monitoring hydrogen-deuterium exchange by mass spectrometry revealed an effective reduction of deuteration after ligand binding within the contact area. In addition, evidence for changes in solvent accessibility of amide protons beyond this site was obtained. Their detection, which highlights the sensor capacity of this technique, encourages systematic studies on galectins and beyond.

Solving the RNA polymerase I structural puzzle.

Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Šresolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution.

The cryo-EM structure of trypanosome 3-methylcrotonyl-CoA carboxylase provides mechanistic and dynamic insights into its enzymatic function.

3-Methylcrotonyl-CoA carboxylase (MCC) catalyzes the two-step, biotin-dependent production of 3-methylglutaconyl-CoA, an essential intermediate in leucine catabolism. Given the critical metabolic role of MCC, deficiencies in this enzyme lead to organic aciduria, while its overexpression is linked to tumor development. MCC is a dodecameric enzyme composed of six copies of each α- and ß-subunit. We present the cryo-EM structure of the endogenous MCC holoenzyme from Trypanosoma brucei in a non-filamentous state at 2.4 Å resolution. Biotin is covalently bound to the biotin carboxyl carrier protein domain of α-subunits and positioned in a non-canonical pocket near the active site of neighboring ß-subunit dimers. Moreover, flexibility of key residues at α-subunit interfaces and loops enables pivoting of α-subunit trimers to partly reduce the distance between α- and ß-subunit active sites, required for MCC catalysis. Our results provide a structural framework to understand the enzymatic mechanism of eukaryotic MCCs and to assist drug discovery against trypanosome infections.

Fine-tuning of prototype chicken galectins: structure of CG-2 and structure-activity correlations.

The comparatively small number of members of the family of adhesion/growth-regulatory galectins in chicken predestines this system as an attractive model to study the divergence of these lectins after gene duplication. Expression profiling of the three homodimeric (prototype) chicken galectins (CG-1A, CG-1B and CG-2) has raised evidence of distinct functionalities, explaining the interest in a detailed crystallographic analysis of CG-2. As revealed here, marked differences are found in the ligand-binding site and in the contact pattern within the homodimer interface, underlying a characteristic orientation of the two subunits. Notably, a distinctive trimer of dimers that is unique in all galectin crystal structures reported to date forms the core unit of the crystallographic assembly. Combination with spectroscopic and thermodynamic measurements, and comparisons with CG-1A and CG-1B, identify differential changes in the circular-dichroism spectra in the presence of lactose, reflecting the far-reaching impact of the ligand on hydrodynamic behaviour, and inter-galectin differences in both the entropy and the enthalpy of binding. This structural information is a salient step to complete the analysis of the full set of galectins from this model organism.

The binding of triclosan to SmeT, the repressor of the multidrug efflux pump SmeDEF, induces antibiotic resistance in Stenotrophomonas maltophilia.

The wide utilization of biocides poses a concern on the impact of these compounds on natural bacterial populations. Furthermore, it has been demonstrated that biocides can select, at least in laboratory experiments, antibiotic resistant bacteria. This situation has raised concerns, not just on scientists and clinicians, but also on regulatory agencies, which are demanding studies on the impact that the utilization of biocides may have on the development on resistance and consequently on the treatment of infectious diseases and on human health. In the present article, we explored the possibility that the widely used biocide triclosan might induce antibiotic resistance using as a model the opportunistic pathogen Stenotrophomonas maltophilia. Biochemical, functional and structural studies were performed, focusing on SmeDEF, the most relevant antibiotic- and triclosan-removing multidrug efflux pump of S. maltophilia. Expression of smeDEF is regulated by the repressor SmeT. Triclosan released SmeT from its operator and induces the expression of smeDEF, thus reducing the susceptibility of S. maltophilia to antibiotics in the presence of the biocide. The structure of SmeT bound to triclosan is described. Two molecules of triclosan were found to bind to one subunit of the SmeT homodimer. The binding of the biocide stabilizes the N terminal domain of both subunits in a conformation unable to bind DNA. To our knowledge this is the first crystal structure obtained for a transcriptional regulator bound to triclosan. This work provides the molecular basis for understanding the mechanisms allowing the induction of phenotypic resistance to antibiotics by triclosan.

Conserved GTPase mechanism in bacterial FtsZ and archaeal tubulin filaments.

Self-assembling protein filaments are at the heart of cell function. Among them, tubulin-like proteins are essential for cell division, DNA segregation and cytoskeletal functions across the domains of life. FtsZ and tubulin share their core structures, a characteristic nucleotide-binding pocket and similar protofilament architecture. GTP hydrolysis between consecutive subunits drives their assembly dynamics. Two recent studies provide previously missing, filament atomic structures of bacterial FtsZ and a recently discovered archaeal tubulin in their nucleotide triphosphate-bound states. Both filament structures reveal strikingly conserved interfacial GTPase active sites, with Mg2+ and K+ /Na+ cations and an NxDxxD/E triad of catalytic residues, probably inherited from the common ancestor of FtsZs and tubulins. Moreover, both proteins exhibit nucleotide-regulated subunit association mediated by interfacial water bridges, as well as polymerization-induced structural changes, likely enabling related dynamic assembly mechanisms.

Small molecule MMRi62 targets MDM4 for degradation and induces leukemic cell apoptosis regardless of p53 status.

MDM2 and MDM4 proteins are key negative regulators of tumor suppressor p53. MDM2 and MDM4 interact via their RING domains and form a heterodimer polyubiquitin E3 ligase essential for p53 degradation. MDM4 also forms heterodimer E3 ligases with MDM2 isoforms that lack p53-binding domains, which regulate p53 and MDM4 stability. We are working to identify small-molecule inhibitors targeting the RING domain of MDM2-MDM4 (MMRi) that can inactivate the total oncogenic activity of MDM2-MDM4 heterodimers. Here, we describe the identification and characterization of MMRi62 as an MDM4-degrader and apoptosis inducer in leukemia cells. Biochemically, in our experiments, MMRi62 bound to preformed RING domain heterodimers altered the substrate preference toward MDM4 ubiquitination and promoted MDM2-dependent MDM4 degradation in cells. This MDM4-degrader activity of MMRi62 was found to be associated with potent apoptosis induction in leukemia cells. Interestingly, MMRi62 effectively induced apoptosis in p53 mutant, multidrug-resistant leukemia cells and patient samples in addition to p53 wild-type cells. In contrast, MMRi67 as a RING heterodimer disruptor and an enzymatic inhibitor of the MDM2-MDM4 E3 complex lacked MDM4-degrader activity and failed to induce apoptosis in these cells. In summary, this study identifies MMRi62 as a novel MDM2-MDM4-targeting agent and suggests that small molecules capable of promoting MDM4 degradation may be a viable new approach to killing leukemia cells bearing non-functional p53 by apoptosis.

Structural Characterization of Rat Galectin-5, an N-Tailed Monomeric Proto-Type-like Galectin.

Galectins are multi-purpose effectors acting via interactions with distinct counterreceptors based on protein-glycan/protein recognition. These processes are emerging to involve several regions on the protein so that the availability of a detailed structural characterization of a full-length galectin is essential. We report here the first crystallographic information on the N-terminal extension of the carbohydrate recognition domain of rat galectin-5, which is precisely described as an N-tailed proto-type-like galectin. In the ligand-free protein, the three amino-acid stretch from Ser2 to Ser5 is revealed to form an extra ß-strand (F0), and the residues from Thr6 to Asn12 are part of a loop protruding from strands S1 and F0. In the ligand-bound structure, amino acids Ser2-Tyr10 switch position and are aligned to the edge of the ß-sandwich. Interestingly, the signal profile in our glycan array screening shows the sugar-binding site to preferentially accommodate the histo-blood-group B (type 2) tetrasaccharide and N-acetyllactosamine-based di- and oligomers. The crystal structures revealed the characteristically preformed structural organization around the central Trp77 of the CRD with involvement of the sequence signature's amino acids in binding. Ligand binding was also characterized calorimetrically. The presented data shows that the N-terminal extension can adopt an ordered structure and shapes the hypothesis that a ligand-induced shift in the equilibrium between flexible and ordered conformers potentially acts as a molecular switch, enabling new contacts in this region.

Targeting the FtsZ Allosteric Binding Site with a Novel Fluorescence Polarization Screen, Cytological and Structural Approaches for Antibacterial Discovery.

Bacterial resistance to antibiotics makes previously manageable infections again disabling and lethal, highlighting the need for new antibacterial strategies. In this regard, inhibition of the bacterial division process by targeting key protein FtsZ has been recognized as an attractive approach for discovering new antibiotics. Binding of small molecules to the cleft between the N-terminal guanosine triphosphate (GTP)-binding and the C-terminal subdomains allosterically impairs the FtsZ function, eventually inhibiting bacterial division. Nonetheless, the lack of appropriate chemical tools to develop a binding screen against this site has hampered the discovery of FtsZ antibacterial inhibitors. Herein, we describe the first competitive binding assay to identify FtsZ allosteric ligands interacting with the interdomain cleft, based on the use of specific high-affinity fluorescent probes. This novel assay, together with phenotypic profiling and X-ray crystallographic insights, enables the identification and characterization of FtsZ inhibitors of bacterial division aiming at the discovery of more effective antibacterials.

Nucleotide-induced folding of cell division protein FtsZ from Staphylococcus aureus.

The essential bacterial division protein FtsZ uses GTP binding and hydrolysis to assemble into dynamic filaments that treadmill around the Z-ring, guiding septal wall synthesis and cell division. FtsZ is a structural homolog of tubulin and a target for discovering new antibiotics. Here, using FtsZ from the pathogen S. aureus (SaFtsZ), we reveal that, prior to assembly, FtsZ monomers require nucleotide binding for folding; this is possibly relevant to other mesophilic FtsZs. Apo-SaFtsZ is essentially unfolded, as assessed by nuclear magnetic resonance and circular dichroism. Binding of GTP (≥ 1 mm) dramatically shifts the equilibrium toward the active folded protein. Supportingly, SaFtsZ refolded with GDP crystallizes in a native structure. Apo-SaFtsZ also folds with 3.4 m glycerol, enabling high-affinity GTP binding (KD 20 nm determined by isothermal titration calorimetry) similar to thermophilic stable FtsZ. Other stabilizing agents that enhance nucleotide binding include ethylene glycol, trimethylamine N-oxide, and several bacterial osmolytes. High salt stabilizes SaFtsZ without bound nucleotide in an inactive twisted conformation. We identified a cavity behind the SaFtsZ-GDP nucleotide-binding pocket that harbors different small compounds, which is available for extended nucleotide-replacing inhibitors. Furthermore, we devised a competition assay to detect any inhibitors that overlap the nucleotide site of SaFtsZ, or Escherichia coli FtsZ, employing osmolyte-stabilized apo-FtsZs and the specific fluorescence anisotropy change in mant-GTP upon dissociation from the protein. This robust assay provides a basis to screening for high-affinity GTP-replacing ligands, which combined with structural studies and phenotypic profiling should facilitate development of a next generation of FtsZ-targeting antibacterial inhibitors.

Engineering protein assemblies with allosteric control via monomer fold-switching.

The macromolecular machines of life use allosteric control to self-assemble, dissociate and change shape in response to signals. Despite enormous interest, the design of nanoscale allosteric assemblies has proven tremendously challenging. Here we present a proof of concept of allosteric assembly in which an engineered fold switch on the protein monomer triggers or blocks assembly. Our design is based on the hyper-stable, naturally monomeric protein CI2, a paradigm of simple two-state folding, and the toroidal arrangement with 6-fold symmetry that it only adopts in crystalline form. We engineer CI2 to enable a switch between the native and an alternate, latent fold that self-assembles onto hexagonal toroidal particles by exposing a favorable inter-monomer interface. The assembly is controlled on demand via the competing effects of temperature and a designed short peptide. These findings unveil a remarkable potential for structural metamorphosis in proteins and demonstrate key principles for engineering protein-based nanomachinery.

Chicken GRIFIN: Structural characterization in crystals and in solution.

Despite its natural abundance in lenses of vertebrates the physiological function(s) of the galectin-related inter-fiber protein (GRIFIN) is (are) still unclear. The same holds true for the significance of the unique interspecies (fish/birds vs mammals) variability in the capacity to bind lactose. In solution, ultracentrifugation and small angle X-ray scattering (at concentrations up to 9 mg/mL) characterize the protein as compact and stable homodimer without evidence for aggregation. The crystal structure of chicken (C-)GRIFIN at seven pH values from 4.2 to 8.5 is reported, revealing compelling stability. Binding of lactose despite the Arg71Val deviation from the sequence signature of galectins matched the otherwise canonical contact pattern with thermodynamics of an enthalpically driven process. Upon lactose accommodation, the side chain of Arg50 is shifted for hydrogen bonding to the 3-hydroxyl of glucose. No evidence for a further ligand-dependent structural alteration was obtained in solution by measuring hydrogen/deuterium exchange mass spectrometrically in peptic fingerprints. The introduction of the Asn48Lys mutation, characteristic for mammalian GRIFINs that have lost lectin activity, lets labeled C-GRIFIN maintain capacity to stain tissue sections. Binding is no longer inhibitable by lactose, as seen for the wild-type protein. These results establish the basis for detailed structure-activity considerations and are a step to complete the structural description of all seven members of the galectin network in chicken.

The structure of VgrG1 from Pseudomonas aeruginosa, the needle tip of the bacterial type VI secretion system.

The type VI secretion system (T6SS) is a mechanism that is commonly used by pathogenic bacteria to infect host cells and for survival in competitive environments. This system assembles on a core baseplate and elongates like a phage puncturing device; it is thought to penetrate the target membrane and deliver effectors into the host or competing bacteria. Valine-glycine repeat protein G1 (VgrG1) forms the spike at the tip of the elongating tube formed by haemolysin co-regulated protein 1 (Hcp1); it is structurally similar to the T4 phage (gp27)3-(gp5)3 puncturing complex. Here, the crystal structure of full-length VgrG1 from Pseudomonas aeruginosa is reported at a resolution of 2.0 Å, which through a trimeric arrangement generates a needle-like shape composed of two main parts, the head and the spike, connected via a small neck region. The structure reveals several remarkable structural features pointing to the possible roles of the two main segments of VgrG1: the head as a scaffold cargo domain and the ß-roll spike with implications in the cell-membrane puncturing process and as a carrier of cognate toxins.

Crystal Structure of Hcp from Acinetobacter baumannii: A Component of the Type VI Secretion System.

The type VI secretion system (T6SS) is a bacterial macromolecular machine widely distributed in Gram-negative bacteria, which transports effector proteins into eukaryotic host cells or other bacteria. Membrane complexes and a central tubular structure, which resembles the tail of contractile bacteriophages, compose the T6SS. One of the proteins forming this tube is the hemolysin co-regulated protein (Hcp), which acts as virulence factor, as transporter of effectors and as a chaperone. In this study, we present the structure of Hcp from Acinetobacter baumannii, together with functional and oligomerization studies. The structure of this protein exhibits a tight ß barrel formed by two ß sheets and flanked at one side by a short α-helix. Six Hcp molecules associate to form a donut-shaped hexamer, as observed in both the crystal structure and solution. These results emphasize the importance of this oligomerization state in this family of proteins, despite the low similarity of sequence among them. The structure presented in this study is the first one for a protein forming part of a functional T6SS from A. baumannii. These results will help us to understand the mechanism and function of this secretion system in this opportunistic nosocomial pathogen.

Preliminary X-ray crystallographic analysis of an engineered variant of human chimera-type galectin-3 with a shortened N-terminal domain.

How lectins translate sugar-encoded information into cellular effects not only depends on glycan recognition. Other domains of the protein can contribute to the functional profile of a lectin. Human galectin-3 (Gal-3), an adhesion/growth-regulatory galectin, is composed of three different domains and is thus called a chimera-type protein. In addition to the carbohydrate-recognition domain, this lectin encompasses an N-terminal domain consisting of a peptide harbouring two phosphorylation sites and nine non-triple-helical collagen-like repeats. This region plays an as yet structurally undefined role in Gal-3 aggregation and ligand recognition. To date, crystallization of full-length Gal-3 has not been achieved. With the aim of providing structural insights into this modular organization, a Gal-3 variant was crystallized maintaining the terminal peptide and three of the nine collagen-like repeats. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 94.04, b = 97.96, c = 236.20 Å, and diffracted to a resolution of 3.3 Å.