Visualizing the subcellular localization of RHOB-GTP and GTPase-Effector complexes using a split-GFP/nanobody labelling assay.

Small GTPases are highly regulated proteins that control essential signaling pathways through the activity of their effector proteins. Among the RHOA subfamily, RHOB regulates peculiar functions that could be associated with the control of the endocytic trafficking of signaling proteins. Here, we used an optimized assay based on tripartite split-GFP complementation to localize GTPase-effector complexes with high-resolution. The detection of RHOB interaction with the Rhotekin Rho binding domain (RBD) that specifically recognizes the active GTP-bound GTPase, is performed in vitro by the concomitant addition of recombinant GFP1-9 and a GFP nanobody. Analysis of RHOB-RBD complexes localization profiles combined with immunostaining and live cell imaging indicated a serum-dependent reorganization of the endosomal and membrane pool of active RHOB. We further applied this technology to the detection of RHO-effector complexes that highlighted their subcellular localization with high resolution among the different cellular compartments.

Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells.

Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.

Phosphopantetheinyl transferase binding and inhibition by amidino-urea and hydroxypyrimidinethione compounds.

Owing to their role in activating enzymes essential for bacterial viability and pathogenicity, phosphopantetheinyl transferases represent novel and attractive drug targets. In this work, we examined the inhibitory effect of the aminido-urea 8918 compound against the phosphopantetheinyl transferases PptAb from Mycobacterium abscessus and PcpS from Pseudomonas aeruginosa, two pathogenic bacteria associated with cystic fibrosis and bronchiectasis, respectively. Compound 8918 exhibits inhibitory activity against PptAb but displays no activity against PcpS in vitro, while no antimicrobial activity against Mycobacterium abscessus or Pseudomonas aeruginosa could be detected. X-ray crystallographic analysis of 8918 bound to PptAb-CoA alone and in complex with an acyl carrier protein domain in addition to the crystal structure of PcpS in complex with CoA revealed the structural basis for the inhibition mechanism of PptAb by 8918 and its ineffectiveness against PcpS. Finally, in crystallo screening of potent inhibitors from the National Cancer Institute library identified a hydroxypyrimidinethione derivative that binds PptAb. Both compounds could serve as scaffolds for the future development of phosphopantetheinyl transferases inhibitors.

Molecular Basis for Extender Unit Specificity of Mycobacterial Polyketide Synthases.

Mycobacterium tuberculosis is the causative agent of the tuberculosis disease, which claims more human lives each year than any other bacterial pathogen. M. tuberculosis and other mycobacterial pathogens have developed a range of unique features that enhance their virulence and promote their survival in the human host. Among these features lies the particular cell envelope with high lipid content, which plays a substantial role in mycobacterial pathogenicity. Several envelope components of M. tuberculosis and other mycobacteria, e.g., mycolic acids, phthiocerol dimycocerosates, and phenolic glycolipids, belong to the "family" of polyketides, secondary metabolites synthesized by fascinating versatile enzymes-polyketide synthases. These megasynthases consist of multiple catalytic domains, among which the acyltransferase domain plays a key role in selecting and transferring the substrates required for polyketide extension. Here, we present three new crystal structures of acyltransferase domains of mycobacterial polyketide synthases and, for one of them, provide evidence for the identification of residues determining extender unit specificity. Unravelling the molecular basis for such specificity is of high importance considering the role played by extender units for the final structure of key mycobacterial components. This work provides major advances for the use of mycobacterial polyketide synthases as potential therapeutic targets and, more generally, contributes to the prediction and bioengineering of polyketide synthases with desired specificity.

Conformational flexibility of coenzyme A and its impact on the post-translational modification of acyl carrier proteins by 4'-phosphopantetheinyl transferases.

One central question surrounding the biosynthesis of fatty acids and polyketide-derived natural products is how the 4'-phosphopantetheinyl transferase (PPTase) interrogates the essential acyl carrier protein (ACP) domain to fulfill the initial activation step. The triggering factor of this study was the lack of structural information on PPTases at physiological pH, which could bias our comprehension of the mechanism of action of these important enzymes. Structural and functional studies on the family II PPTase PptAb of Mycobacterium abscessus show that pH has a profound effect on the coordination of metal ions and on the conformation of endogenously bound coenzyme A (CoA). The observed conformational flexibility of CoA at physiological pH is accompanied by a disordered 4'-phosphopantetheine (Ppant) moiety. Finally, structural and dynamical information on an isolated mycobacterial ACP domain, in its apo form and in complex with the activator PptAb, suggests an alternate mechanism for the post-translational modification of modular megasynthases.

A Targeted Protein Degradation Cell-Based Screening for Nanobodies Selective toward the Cellular RHOB GTP-Bound Conformation.

The selective downregulation of activated intracellular proteins is a key challenge in cell biology. RHO small GTPases switch between a guanosine diphosphate (GDP)-bound and a guanosine triphosphate (GTP)-bound state that drives downstream signaling. At present, no tool is available to study endogenous RHO-GTPinduced conformational changes in live cells. Here, we established a cell-based screen to selectively degrade RHOB-GTP using F-box-intracellular single-domain antibody fusion. We identified one intracellular antibody (intrabody) that shows selective targeting of endogenous RHOB-GTP mediated by interactions between the CDR3 loop of the domain antibody and the GTP-binding pocket of RHOB. Our results suggest that, while RHOB is highly regulated at the expression level, only the GTP-bound pool, but not its global expression, mediates RHOB functions in genomic instability and in cell invasion. The F-box/intrabody-targeted protein degradation represents a unique approach to knock down the active form of small GTPases or other proteins with multiple cellular activities.

Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology.

Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite ¼ split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein-protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.

High-Throughput Isolation of Soluble Protein Domains Using a Bipartite Split-GFP Complementation System.

The identification of soluble, folded domains of proteins is a recurring task in modern molecular biology. We detail a protocol for identifying compact soluble protein domains using a self-assembling two-part split-GFP comprised of a detector fragment (GFP ß-strands 1 through 10, or GFP1-10) and a tagging fragment (GFP ß-strand 11, or GFP11). The assay is performed in E. coli cells and in cell extracts. A selection step insures the protein fragments are in frame and contain no stop codons, while an inverse PCR is used to enrich protein fragment libraries containing a specific target sequence.

High-Throughput Protein-Protein Interaction Assays Using Tripartite Split-GFP Complementation.

Most cellular processes are driven by complex protein-protein interaction networks. Identifying key players and characterizing their interactions at the cellular and molecular level is of key importance to understand biochemical mechanisms that control cellular responses. Here, we detail a protocol for monitoring protein-protein interactions in E. coli cells or in cell extracts using a tripartite split-GFP system comprised of a protein interaction detector fragment (GFP ß-strands 1 through 9 or GFP1-9) and small tagging fragments of GFP ß-strands 10 (GFP10) and 11 (GFP11). Interaction of bait and prey proteins fused to GFP10 and GFP11 tether the small GFP fragments, allowing self-association with GFP1-9. In this scenario, fluorescence intensity of the reconstituted GFP is correlated with the strength of interaction.

Insights into Substrate Modification by Dehydratases from Type I Polyketide Synthases.

Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine. The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids. Given the insolubility of the natural C28-C30 fatty acyl substrate of the PpsC dehydratase (DH) domain, we investigated its structure-function relationships in the presence of shorter surrogate substrates. Since most enzymes belonging to the (R)-specific enoyl hydratase/hydroxyacyl dehydratase family conduct the reverse hydration reaction in vitro, we have determined the X-ray structures of the PpsC DH domain, both unliganded (apo) and in complex with trans-but-2-enoyl-CoA or trans-dodec-2-enoyl-CoA derivatives. This study provides for the first time a snapshot of dehydratase-ligand interactions following a hydration reaction. Our structural analysis allowed us to identify residues essential for substrate binding and activity. The structural comparison of the two complexes also sheds light on the need for long acyl chains for this dehydratase to carry out its function, consistent with both its in vitro catalytic behavior and the physiological role of the PpsC enzyme.

A new protein-protein interaction sensor based on tripartite split-GFP association.

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

Detection of soluble co-factor dependent protein expression in vivo: application to the 4'-phosphopantetheinyl transferase PptT from Mycobacterium tuberculosis.

The need for early-on diagnostic tools to assess the folding and solubility of expressed protein constructs in vivo is of great interest when dealing with recalcitrant proteins. In this paper, we took advantage of the picomolar sensitivity of the bipartite GFP1-10/GFP11 system to investigate the solubility of the Mycobacterium tuberculosis 4'-phosphopantetheinyl transferase PptT, an enzyme essential for the viability of the tubercle bacillus. In vivo and in vitro complementation assays clearly showed the improved solubility of the full-length PptT compared to its N- and C-terminally truncated counterparts. However, initial attempts to purify the full-length enzyme overexpressed in Escherichia coli cells were hampered by aggregation issues overtime that caused the protein to precipitate within hours. The fact that the naturally occurring Coenzyme A and Mg(2+), essentials for PptT to carry out its function, could play a role in stabilizing the enzyme was confirmed using DSF experiments. In vitro activity assays were performed using the ACP substrate from the type I polyketide synthase PpsC from M. tuberculosis, a 2188 amino-acid enzyme that plays a major role in the virulence and pathogenicity of this microbial pathogen. We selected the most soluble and compact ACP fragment (2042-2188), identified by genetic selection of in-frame fragments from random library experiments, to monitor the transfer of the P-pant moiety from Coenzyme A onto a conserved serine residue of this ACP domain.

Biochemical and structural study of the atypical acyltransferase domain from the mycobacterial polyketide synthase Pks13.

Pks13 is a type I polyketide synthase involved in the final biosynthesis step of mycolic acids, virulence factors, and essential components of the Mycobacterium tuberculosis envelope. Here, we report the biochemical and structural characterization of a 52-kDa fragment containing the acyltransferase domain of Pks13. This fragment retains the ability to load atypical extender units, unusually long chain acyl-CoA with a predilection for carboxylated substrates. High resolution crystal structures were determined for the apo, palmitoylated, and carboxypalmitoylated forms. Structural conservation with type I polyketide synthases and related fatty-acid synthases also extends to the interdomain connections. Subtle changes could be identified both in the active site and in the upstream and downstream linkers in line with the organization displayed by this singular polyketide synthase. More importantly, the crystallographic analysis illustrated for the first time how a long saturated chain can fit in the core structure of an acyltransferase domain through a dedicated channel. The structures also revealed the unexpected binding of a 12-mer peptide that might provide insight into domain-domain interaction.

Experimental mapping of soluble protein domains using a hierarchical approach.

Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes.

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1-10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon(®) resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length 'breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit.

The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis.

Protein production in Escherichia coli involves high-level expression in a culture, followed by harvesting of the cells and finally their disruption, or lysis, to release the expressed proteins. We compare three high-throughput chemical lysis methods to sonication, using a robotic platform and methodologies developed in our laboratory [1]. Under the same expression conditions, all lysis methods varied in the degree of released soluble proteins. With a set of 96 test proteins, we used our split GFP to quantify the soluble and insoluble protein fractions after lysis. Both the amount of soluble protein and the percentage recovered in the soluble fraction using SoluLyse were well correlated with sonication. Two other methods, Bugbuster and lysozyme, did not correlate well with sonication. Considering the effects of lysis methods on protein solubility is especially important when accurate protein solubility measurements are needed, for example, when testing adjuvants, growth media, temperature, or when establishing the effects of truncation or sequence variation on protein stability.

Functional and structural characterization of a thiol peroxidase from Mycobacterium tuberculosis.

A thiol peroxidase (Tpx) from Mycobacterium tuberculosis was functionally analyzed. The enzyme shows NADPH-linked peroxidase activity using a thioredoxin-thioredoxin reductase system as electron donor, and anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. It reduces H2O2, t-butyl hydroperoxide, and cumene hydroperoxide, and is inhibited by sulfhydryl reagents. Mutational studies revealed that the peroxidatic (Cys60) and resolving (Cys93) cysteine residues are critical amino acids for catalytic activity. The X-ray structure determined to a resolution of 1.75 A shows a thioredoxin fold similar to that of other peroxiredoxin family members. Superposition with structural homologues in oxidized and reduced forms indicates that the M. tuberculosis Tpx is a member of the atypical two-Cys peroxiredoxin family. In addition, the short distance that separates the Calpha atoms of Cys60 and Cys93 and the location of these cysteine residues in unstructured regions may indicate that the M. tuberculosis enzyme is oxidized, though the side-chain of Cys60 is poorly visible. It is solely in the reduced Streptococcus pneumoniae Tpx structure that both residues are part of two distinct helical segments. The M. tuberculosis Tpx is dimeric both in solution and in the crystal structure. Amino acid residues from both monomers delineate the active site pocket.

Engineering and characterization of a superfolder green fluorescent protein.

Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.

Crystal structure of a putative pyridoxine 5'-phosphate oxidase (Rv2607) from Mycobacterium tuberculosis.

The three-dimensional structure of Rv2607, a putative pyridoxine 5'-phosphate oxidase (PNPOx) from Mycobacterium tuberculosis, has been determined by X-ray crystallography to 2.5 A resolution. Rv2607 has a core domain similar to known PNPOx structures with a flavin mononucleotide (FMN) cofactor. Electron density for two FMN at the dimer interface is weak despite the bright yellow color of the protein solution and crystal. The shape and size of the putative binding pocket is markedly different from that of members of the PNPOx family, which may indicate some significant changes in the FMN binding mode of this protein relative to members of the family.

Recent advances in GFP folding reporter and split-GFP solubility reporter technologies. Application to improving the folding and solubility of recalcitrant proteins from Mycobacterium tuberculosis.

We have improved our green fluorescent protein (GFP) folding reporter technology [Waldo et al., (1999) Nat. Biotechnol. 17, 691-695] to evolve recalcitrant proteins from Mycobacterium tuberculosis. The target protein is inserted into the scaffolding of the GFP, eliminating false-positive artifacts caused by expression of truncated protein variants from internal cryptic ribosome binding sites in the target RNA. In parallel, we have developed a new quantitative fluorescent protein tagging and detection system based on micro-domains of GFP. This split-GFP system, which works both in vivo and in vitro, is amenable to high-throughput assays of protein expression and solubility [Cabantous et al., (2005) Nat. Biotechnol. 23, 102-107]. Together, the GFP folding reporter and split-GFP technologies offer a comprehensive system for manipulating and improving protein folding and solubility.