Computational Prediction of Structure, Function, and Interaction of Myzus persicae (Green Peach Aphid) Salivary Effector Proteins.

Similar to plant pathogens, phloem-feeding insects such as aphids deliver effector proteins inside their hosts that act to promote host susceptibility and enable feeding and infestation. Despite exciting progress toward identifying and characterizing effector proteins from these insects, their functions remain largely unknown. The recent groundbreaking development in protein structure prediction algorithms, combined with the availability of proteomics and transcriptomic datasets for agriculturally important pests, provides new opportunities to explore the structural and functional diversity of effector repertoires. In this study, we sought to gain insight into the infection strategy used by the Myzus persicae (green peach aphid) by predicting and analyzing the structures of a set of 71 effector candidate proteins. We used two protein structure prediction methods, AlphaFold and OmegaFold, that produced mutually consistent results. We observed a wide continuous spectrum of structures among the effector candidates, from disordered proteins to globular enzymes. We made use of the structural information and state-of-the-art computational methods to predict M. persicae effector protein properties, including function and interaction with host plant proteins. Overall, our investigation provides novel insights into prediction of structure, function, and interaction of M. persicae effector proteins and will guide the necessary experimental characterization to address new hypotheses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Rhs NADase effectors and their immunity proteins are exchangeable mediators of inter-bacterial competition in Serratia.

Many bacterial species use Type VI secretion systems (T6SSs) to deliver anti-bacterial effector proteins into neighbouring bacterial cells, representing an important mechanism of inter-bacterial competition. Specific immunity proteins protect bacteria from the toxic action of their own effectors, whilst orphan immunity proteins without a cognate effector may provide protection against incoming effectors from non-self competitors. T6SS-dependent Rhs effectors contain a variable C-terminal toxin domain (CT), with the cognate immunity protein encoded immediately downstream of the effector. Here, we demonstrate that Rhs1 effectors from two strains of Serratia marcescens, the model strain Db10 and clinical isolate SJC1036, possess distinct CTs which both display NAD(P)+ glycohydrolase activity but belong to different subgroups of NADase from each other and other T6SS-associated NADases. Comparative structural analysis identifies conserved functions required for NADase activity and reveals that unrelated NADase immunity proteins utilise a common mechanism of effector inhibition. By replicating a natural recombination event, we show successful functional exchange of CTs and demonstrate that Db10 encodes an orphan immunity protein which provides protection against T6SS-delivered SJC1036 NADase. Our findings highlight the flexible use of Rhs effectors and orphan immunity proteins during inter-strain competition and the repeated adoption of NADase toxins as weapons against bacterial cells.

The structure of a tautomerase superfamily member linked to the type VI secretion system of Acinetobacter baumannii.

Bacteria exploit specialized secretion systems to assist in competition for resources, in collaboration and in communication. Here, a protocol for the recombinant production, purification and crystallization of a protein linked to the Acinetobacter baumannii type VI secretion system is provided. A high-resolution structure of this trimeric protein is reported, revealing the characteristic dual ß-α-ß subunit fold typical of longer subunit members of the tautomerase superfamily. The protein does not appear to be toxic to bacteria or yeast under the conditions tested. The possible biological role of this protein is discussed.

Delineating the activity of the potent nicotinic acetylcholine receptor agonists (+)-anatoxin-a and (-)-hosieine-A.

The affinity and thermodynamic parameters for the interactions of two naturally occurring neurotoxins, (+)-anatoxin-a and (-)-hosieine-A, with acetylcholine-binding protein were investigated using a fluorescence-quenching assay and isothermal titration calorimetry. The crystal structures of their complexes with acetylcholine-binding protein from Aplysia californica (AcAChBP) were determined and reveal details of molecular recognition in the orthosteric binding site. Comparisons treating AcAChBP as a surrogate for human α4ß2 and α7 nicotinic acetylcholine receptors (nAChRs) suggest that the molecular features involved in ligand recognition and affinity for the protein targets are conserved. The ligands exploit interactions with similar residues as the archetypal nAChR agonist nicotine, but with greater affinity. (-)-Hosieine-A in particular has a high affinity for AcAChBP driven by a favorable entropic contribution to binding. The ligand affinities help to rationalize the potent biological activity of these alkaloids. The structural data, together with comparisons with related molecules, suggest that there may be opportunities to extend the hosieine-A scaffold to incorporate new interactions with the complementary side of the orthosteric binding site. Such a strategy may guide the design of new entities to target human α4ß2 nAChR that may have therapeutic benefit.

Interactions between 2'-fluoro-(carbamoylpyridinyl)deschloroepibatidine analogues and acetylcholine-binding protein inform on potent antagonist activity against nicotinic receptors.

Low-nanomolar binding constants were recorded for a series of six 2'-fluoro-(carbamoylpyridinyl)deschloroepibatidine analogues with acetylcholine-binding protein (AChBP). The crystal structures of three complexes with AChBP reveal details of molecular recognition in the orthosteric binding site and imply how the other three ligands bind. Comparisons exploiting AChBP as a surrogate for α4ß2 and α7 nicotinic acetylcholine receptors (nAChRs) suggest that the key interactions are conserved. The ligands interact with the same residues as the archetypal nAChR agonist nicotine yet display greater affinity, thereby rationalizing their in vivo activity as potent antagonists of nicotine-induced antinociception. An oxyanion-binding site is formed on the periphery of the AChBP orthosteric site by Lys42, Asp94, Glu170 and Glu210. These residues are highly conserved in the human α4, ß2 and α7 nAChR sequences. However, specific sequence differences are discussed that could contribute to nAChR subtype selectivity and in addition may represent a point of allosteric modulation. The ability to engage with this peripheral site may explain, in part, the function of a subset of ligands to act as agonists of α7 nAChR.

The thermodynamic profile and molecular interactions of a C(9)-cytisine derivative-binding acetylcholine-binding protein from Aplysia californica.

Cytisine, a natural product with high affinity for clinically relevant nicotinic acetylcholine receptors (nAChRs), is used as a smoking-cessation agent. The compound displays an excellent clinical profile and hence there is an interest in derivatives that may be further improved or find use in the treatment of other conditions. Here, the binding of a cytisine derivative modified by the addition of a 3-(hydroxypropyl) moiety (ligand 4) to Aplysia californica acetylcholine-binding protein (AcAChBP), a surrogate for nAChR orthosteric binding sites, was investigated. Isothermal titration calorimetry revealed that the favorable binding of cytisine and its derivative to AcAChBP is driven by the enthalpic contribution, which dominates an unfavorable entropic component. Although ligand 4 had a less unfavorable entropic contribution compared with cytisine, the affinity for AcAChBP was significantly diminished owing to the magnitude of the reduction in the enthalpic component. The high-resolution crystal structure of the AcAChBP-4 complex indicated close similarities in the protein-ligand interactions involving the parts of 4 common to cytisine. The point of difference, the 3-(hydroxypropyl) substituent, appears to influence the conformation of the Met133 side chain and helps to form an ordered solvent structure at the edge of the orthosteric binding site.

A Structural Rationale for N-Methylbicuculline Acting as a Promiscuous Competitive Antagonist of Inhibitory Pentameric Ligand-Gated Ion Channels.

Bicuculline, a valued chemical tool in neurosciences research, is a competitive antagonist of specific GABAA receptors and affects other pentameric ligand-gated ion channels including the glycine, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors. We used a fluorescence-quenching assay and isothermal titration calorimetry to record low-micromolar dissociation constants for N-methylbicuculline interacting with acetylcholine-binding protein and an engineered version called glycine-binding protein (GBP), which provides a surrogate for the heteromeric interface of the extracellular domain of the glycine receptor (GlyR). The 2.4 Šresolution crystal structure of the GBP:N-methylbicuculline complex, sequence and structural alignments reveal similarities and differences between GlyR and the GABAA receptor-bicuculline interactions. N-methylbicuculline displays a similar conformation in different structures, but adopts distinct orientations enforced by interactions and steric blocks with key residues and plasticity in the binding sites. These features explain the promiscuous activity of bicuculline against the principal inhibitory pentameric ligand-gated ion channels in the CNS.

Engineering a surrogate human heteromeric α/ß glycine receptor orthosteric site exploiting the structural homology and stability of acetylcholine-binding protein.

Protein-engineering methods have been exploited to produce a surrogate system for the extracellular neurotransmitter-binding site of a heteromeric human ligand-gated ion channel, the glycine receptor. This approach circumvents two major issues: the inherent experimental difficulties in working with a membrane-bound ion channel and the complication that a heteromeric assembly is necessary to create a key, physiologically relevant binding site. Residues that form the orthosteric site in a highly stable ortholog, acetylcholine-binding protein, were selected for substitution. Recombinant proteins were prepared and characterized in stepwise fashion exploiting a range of biophysical techniques, including X-ray crystallography, married to the use of selected chemical probes. The decision making and development of the surrogate, which is termed a glycine-binding protein, are described, and comparisons are provided with wild-type and homomeric systems that establish features of molecular recognition in the binding site and the confidence that the system is suited for use in early-stage drug discovery targeting a heteromeric α/ß glycine receptor.

Burkholderia pseudomallei d-alanine-d-alanine ligase; detailed characterisation and assessment of a potential antibiotic drug target.

Burkholderia pseudomallei is a serious, difficult to treat Gram-negative pathogen and an increase in the occurrence of drug-resistant strains has been detected. We have directed efforts to identify and to evaluate potential drug targets relevant to treatment of infection by B. pseudomallei. We have selected and characterised the essential enzyme d-alanine-d-alanine ligase (BpDdl), required for the ATP-assisted biosynthesis of a peptidoglycan precursor. A recombinant supply of protein supported high-resolution crystallographic and biophysical studies with ligands (AMP and AMP+d-Ala-d-Ala), and comparisons with orthologues enzymes suggest a ligand-induced conformational change occurring that might be relevant to the catalytic cycle. The detailed biochemical characterisation of the enzyme, development and optimisation of ligand binding assays supported the search for novel inhibitors by screening of selected compound libraries. In a similar manner to that observed previously in other studies, we note a paucity of hits that are worth follow-up and then in combination with a computational analysis of the active site, we conclude that this ligase represents a difficult target for drug discovery. Nevertheless, our reagents, protocols and data can underpin future efforts exploiting more diverse chemical libraries and structure-based approaches.

The structure of lipopolysaccharide transport protein B (LptB) from Burkholderia pseudomallei.

The thick outer membrane (OM) of Gram-negative bacteria performs an important protective role against hostile environments, supports cell integrity, and contributes to surface adhesion and in some cases also to virulence. A major component of the OM is lipopolysaccharide (LPS), a complex glycolipid attached to a core containing fatty-acyl chains. The assembly and transport of lipid A, the membrane anchor for LPS, to the OM begins when a heteromeric LptB2FG protein complex extracts lipid A from the outer leaflet of the inner membrane. This process requires energy, and upon hydrolysis of ATP one component of the heteromeric assembly, LptB, triggers a conformational change in LptFG in support of lipid A transport. A structure of LptB from the intracellular pathogen Burkholderia pseudomallei is reported here. LptB forms a dimer that displays a relatively fixed structure irrespective of whether it is in complex with LptFG or in isolation. Highly conserved sequence and structural features are discussed that allow LptB to fuel the transport of lipid A.

An assessment of three human methylenetetrahydrofolate dehydrogenase/cyclohydrolase-ligand complexes following further refinement.

The enzymes involved in folate metabolism are key drug targets for cell-growth modulation, and accurate crystallographic structures provide templates to be exploited for structure-based ligand design. In this context, three ternary complex structures of human methylenetetrahydrofolate dehydrogenase/cyclohydrolase have been published [Schmidt et al. (2000), Biochemistry, 39, 6325-6335] and potentially represent starting points for the development of new antifolate inhibitors. However, an inspection of the models and the deposited data revealed deficiencies and raised questions about the validity of the structures. A number of inconsistencies relating to the publication were also identified. Additional refinement was carried out with the deposited data, seeking to improve the models and to then validate the complex structures or correct the record. In one case, the inclusion of the inhibitor in the structure was supported and alterations to the model allowed details of enzyme-ligand interactions to be described that had not previously been discussed. For one weak inhibitor, the data suggested that the ligand may adopt two poses in the binding site, both with few interactions with the enzyme. In the third case, that of a potent inhibitor, inconsistencies were noted in the assignment of the chemical structure and there was no evidence to support the inclusion of the ligand in the active site.

Amino acid substitutions in the human homomeric ß3 GABAA receptor that enable activation by GABA.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels that mediate synaptic inhibition throughout the central nervous system. The α1ß2γ2 receptor is the major subtype in the brain; GABA binds at the ß2(+)α1(-) interface. The structure of the homomeric ß3 GABAAR, which is not activated by GABA, has been solved. Recently, four additional heteromeric structures were reported, highlighting key residues required for agonist binding. Here, we used a protein engineering method, taking advantage of knowledge of the key binding residues, to create a ß3(+)α1(-) heteromeric interface in the homomeric human ß3 GABAAR that enables GABA-mediated activation. Substitutions were made in the complementary side of the orthosteric binding site in loop D (Y87F and Q89R), loop E (G152T), and loop G (N66D and A70T). The Q89R and G152T combination enabled low-potency activation by GABA and potentiation by propofol but impaired direct activation by higher propofol concentrations. At higher concentrations, GABA inhibited gating of ß3 GABAAR variants containing Y87F, Q89R, and G152T. Reversion of Phe87 to tyrosine abolished GABA's inhibitory effect and partially recovered direct activation by propofol. This tyrosine is conserved in homomeric GABAARs and in the Erwinia chrysanthemi ligand-gated ion channel and may be essential for the absence of an inhibitory effect of GABA on homomeric channels. This work demonstrated that only two substitutions, Q89R and G152T, in ß3 GABAAR are sufficient to reconstitute GABA-mediated activation and suggests that Tyr87 prevents inhibitory effects of GABA.

Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens.

The Gram-negative bacterium Serratia marcescens secretes many proteins that are involved in extracellular chitin degradation. This so-called chitinolytic machinery includes three types of chitinase enzymes and a lytic polysaccharide monooxygenase. An operon has been identified in S. marcescens, chiWXYZ, that is thought to be involved in the secretion of the chitinolytic machinery. Genetic evidence points to the ChiX protein being a key player in the secretion mechanism, since deletion of the chiX gene in S. marcescens led to a mutant strain blocked for secretion of all members of the chitinolytic machinery. In this work, a detailed structural and biochemical characterisation of ChiX is presented. The high-resolution crystal structure of ChiX reveals the protein to be a member of the LAS family of peptidases. ChiX is shown to be a zinc-containing metalloenzyme, and in vitro assays demonstrate that ChiX is an l-Ala d-Glu endopeptidase that cleaves the cross-links in bacterial peptidoglycan. This catalytic activity is shown to be intimately linked with the secretion of the chitinolytic machinery, since substitution of the ChiX Asp-120 residue results in a variant protein that is both unable to digest peptidoglycan and cannot rescue the phenoytype of a chiX mutant strain.

Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit Trypanosoma brucei Pteridine Reductase in Support of Early-Stage Drug Discovery.

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 µM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.

An Improved Model of the Trypanosoma brucei CTP Synthetase Glutaminase Domain-Acivicin Complex.

The natural product acivicin inhibits the glutaminase activity of cytidine triphosphate (CTP) synthetase and is a potent lead compound for drug discovery in the area of neglected tropical diseases, specifically trypanosomaisis. A 2.1-Å-resolution crystal structure of the acivicin adduct with the glutaminase domain from Trypanosoma brucei CTP synthetase has been deposited in the RCSB Protein Data Bank (PDB) and provides a template for structure-based approaches to design new inhibitors. However, our assessment of that data identified deficiencies in the model. We now report an improved and corrected inhibitor structure with changes to the chirality at one position, the orientation and covalent structure of the isoxazoline moiety, and the location of a chloride ion in an oxyanion binding site that is exploited during catalysis. The model is now in agreement with established chemical principles and allows an accurate description of molecular recognition of the ligand and the mode of binding in a potentially valuable drug target.

Open and compressed conformations of Francisella tularensis ClpP.

Caseinolytic proteases are large oligomeric assemblies responsible for maintaining protein homeostasis in bacteria and in so doing influence a wide range of biological processes. The functional assembly involves three chaperones together with the oligomeric caseinolytic protease catalytic subunit P (ClpP). This protease represents a potential target for therapeutic intervention in pathogenic bacteria. Here, we detail an efficient protocol for production of recombinant ClpP from Francisella tularensis, and the structural characterization of three crystal forms which grow under similar conditions. One crystal form reveals a compressed state of the ClpP tetradecamer and two forms an open state. A comparison of the two types of structure infers that differences at the enzyme active site result from a conformational change involving a highly localized disorder-order transition of a ß-strand α-helix combination. This transition occurs at a subunit-subunit interface. Our study may now underpin future efforts in a structure-based approach to target ClpP for inhibitor or activator development. Proteins 2016; 85:188-194. © 2016 Wiley Periodicals, Inc.

EssC: domain structures inform on the elusive translocation channel in the Type VII secretion system.

The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1-D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein.

Membrane interactions and self-association of components of the Ess/Type VII secretion system of Staphylococcus aureus.

The Ess/Type VII protein secretion system, essential for virulence of pathogenic Staphylococcus aureus, is dependent upon the four core membrane proteins EssA, EssB, EssC and EsaA. Here, we use crosslinking and blue native PAGE analysis to show that the EssB, EssC and EsaA proteins individually form homomeric complexes. Surprisingly, these components appear unable to interact with each other, or with the EssA protein. We further show that two high molecular weight multimers of EssC detected in whole cells are not dependent upon the presence of EsxA, EsxB or any other Ess component for their assembly.

Characterization of 2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl Ureido Based Inhibitors of Trypanosoma brucei FolD and Testing for Antiparasitic Activity.

The bifunctional enzyme N(5),N(10)-methylenetetrahydrofolate dehydrogenase/cyclo hydrolase (FolD) is essential for growth in Trypanosomatidae. We sought to develop inhibitors of Trypanosoma brucei FolD (TbFolD) as potential antiparasitic agents. Compound 2 was synthesized, and the molecular structure was unequivocally assigned through X-ray crystallography of the intermediate compound 3. Compound 2 showed an IC50 of 2.2 µM, against TbFolD and displayed antiparasitic activity against T. brucei (IC50 49 µM). Using compound 2, we were able to obtain the first X-ray structure of TbFolD in the presence of NADP(+) and the inhibitor, which then guided the rational design of a new series of potent TbFolD inhibitors.

Structures of Pseudomonas aeruginosa ß-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form.

Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa ß-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.