Ching-I Anderson Wang

Crystal Structures of Flax Rust Avirulence Proteins AvrL567-A and -D Reveal Details of the Structural Basis for Flax Disease Resistance Specificity

The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-Å resolution, respectively. The structures of both proteins are very similar and reveal a β-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

Semienzymatic cyclization of disulfide-rich peptides using Sortase A.

Background: Sortase A (SrtA) is a transpeptidase capable of catalyzing the formation of amide bonds. Results: SrtA was used to backbone-cyclize disulfide-rich peptides, including kalata B1, α-conotoxin Vc1.1, and SFTI-1. Conclusion: SrtA-mediated cyclization is applicable to small disulfide-rich peptides. Significance: SrtA-mediated cyclization is an alternative to native chemical ligation for the cyclization of small peptides of therapeutic interest. Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.

Emerging opportunities for allosteric modulation of G-protein coupled receptors.

Their ubiquitous nature, wide cellular distribution and versatile molecular recognition and signalling help make G-protein binding receptors (GPCRs) the most important class of membrane proteins in clinical medicine, accounting for ∼40% of all current therapeutics. A large percentage of current drugs target the endogenous ligand binding (orthosteric) site, which are structurally and evolutionarily conserved, particularly among members of the same GPCR subfamily. With the recent advances in GPCR X-ray crystallography, new opportunities for developing novel subtype selective drugs have emerged. Given the increasing recognition that the extracellular surface conformation changes in response to ligand binding, it is likely that all GPCRs possess an allosteric site(s) capable of regulating GPCR signalling. Allosteric sites are less structurally conserved than their corresponding orthosteric site and thus provide new opportunities for the development of more selective drugs. Constitutive oligomerisation (dimerisation) identified in many of the GPCRs investigated, adds another dimension to the structural and functional complexity of GPCRs. In this review, we compare 60 crystal structures of nine GPCR subtypes (rhodopsin, ß₂-AR, ß₁-AR, A(2a)-AR, CXCR4, D₃R, H₁R, M₂R, M₃R) across four subfamilies of Class A GPCRs, and discuss mechanisms involved in receptor activation and potential allosteric binding sites across the highly variable extracellular surface of these GPCRs. This analysis has identified a new extracellular salt bridge (ESB-2) that might be exploited in the design of allosteric modulators.

The atypical alpha-conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor

Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. α-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of α-conotoxins may serve as a platform for development of new therapies. Previously characterized α-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel α-conotoxin LtIA, which lacks this highly conserved motif but potently blocked α3β2 nAChRs with a 9.8 nm IC50 value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing α-conotoxin MII. Nevertheless, pre-block of α3β2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR β subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for α3β2 versus α3β4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC50 value on α3β2(F119Q) versus wild type nAChRs, whereas T59K and V111I β2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the α3β2 nAChR that includes β2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.

Chemical engineering and structural and pharmacological characterization of the α-scorpion toxin OD1.

Scorpion α-toxins are invaluable pharmacological tools for studying voltage-gated sodium channels, but few structure-function studies have been undertaken due to their challenging synthesis. To address this deficiency, we report a chemical engineering strategy based upon native chemical ligation. The chemical synthesis of α-toxin OD1 was achieved by chemical ligation of three unprotected peptide segments. A high resolution X-ray structure (1.8 Å) of synthetic OD1 showed the typical βαββ α-toxin fold and revealed important conformational differences in the pharmacophore region when compared with other α-toxin structures. Pharmacological analysis of synthetic OD1 revealed potent α-toxin activity (inhibition of fast inactivation) at Nav1.7, as well as Nav1.4 and Nav1.6. In addition, OD1 also produced potent β-toxin activity at Nav1.4 and Nav1.6 (shift of channel activation in the hyperpolarizing direction), indicating that OD1 might interact at more than one site with Nav1.4 and Nav1.6. Investigation of nine OD1 mutants revealed that three residues in the reverse turn contributed significantly to selectivity, with the triple OD1 mutant (D9K, D10P, K11H) being 40-fold more selective for Nav1.7 over Nav1.6, while OD1 K11V was 5-fold more selective for Nav1.6 than Nav1.7. This switch in selectivity highlights the importance of the reverse turn for engineering α-toxins with altered selectivity at Nav subtypes.

Vicinal Disulfide Constrained Cyclic Peptidomimetics: a Turn Mimetic Scaffold Targeting the Norepinephrine Transporter

Loopy peptides: Peptide turn mimetics of a clinically relevant norepinephrine reuptake inhibitor were developed employing a high-throughput synthesis approach to generate peptide thioesters, with subsequent cyclization through native chemical ligation. The vicinal disulfide constrained cyclic peptidomimetics (see scheme) show high structural and functional similarity to the parent peptide, though with superior metabolic stability. Copyright

Emerging structure-function relationships defining monoamine NSS transporter substrate and ligand affinity.

Monoamine transporters are a group of transmembrane neurotransmitter sodium symporter (NSS) transporters that play a crucial role in regulating biogenic monoamine concentrations at peripheral and central synapses. Given the key role played by serotonin, dopamine and noradrenaline in addictive and disease states, structure-function studies have been conducted to help guide the development of improved central nervous system therapeutics. Extensive pharmacological, immunological and biochemical studies, in conjunction with three-dimensional homology modeling, have been performed to structurally and functionally characterise the monoamine transporter substrate permeation pathway, substrate selectivity, and binding sites for ions, substrates and inhibitors at the molecular level. However, only recently has it been possible to start to construct an accurate molecular interaction network for the monoamine transporters and their corresponding substrates and inhibitors. Crystal structures of Aquifex aeolicus leucine transporter (LeuT(Aa)), a homologous protein to monoamine transporters that has been experimentally demonstrated to share similar structural folds with monoamine transporters, have been determined in complex with amino acids and inhibitors. The molecular interactions of leucine and tricyclic antidepressants (TCA) has supported many of the predictions based on the mutational studies. Models constructed from LeuT(Aa) are now allowing a rational approach to further clarify the molecular determinants of NSS transporter-ligand complexes, and potentially the ability to better manipulate drug specificity and affinity. In this review, we compare the structure-function relationships of other SLC6 NSS family transporters with monoamine transporters, and discuss possible mechanisms involved in substrate binding and transport, and modes of inhibition by TCAs.

Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor

Background: Mechanistic insight into allosteric modulation of GPCRs can facilitate antagonist design. Results: Extracellular surface residues (ECS) of the α1B-adrenoceptor at the base of extracellular loop 3 interact with the allosteric antagonist TIA. Conclusion: The identified ECS pharmacophore provides the first structural constraints for allosteric antagonist design at α1-adrenoceptors. Significance: Binding to the ECS of a GPCR can allosterically inhibit agonist signaling. The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β1-adrenergic receptor crystal structure, we modeled the α1B-adrenoceptor (α1B-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α1B-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α1-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

Stabilization of the Cysteine-Rich Conotoxin MrIA by Using a 1,2,3-Triazole as a Disulfide Bond Mimetic

The design of disulfide bond mimetics is an important strategy for optimising cysteine-rich peptides in drug development. Mimetics of the drug lead conotoxin MrIA, in which one disulfide bond is selectively replaced of by a 1,4-disubstituted-1,2,3-triazole bridge, are described. Sequential copper-catalyzed azide-alkyne cycloaddition (CuAAC; click reaction) followed by disulfide formation resulted in the regioselective syntheses of triazole-disulfide hybrid MrIA analogues. Mimetics with a triazole replacing the Cys4-Cys13 disulfide bond retained tertiary structure and full in vitro and in vivo activity as norepinephrine reuptake inhibitors. Importantly, these mimetics are resistant to reduction in the presence of glutathione, thus resulting in improved plasma stability and increased suitability for drug development.

Conotoxin engineering: dual pharmacophoric noradrenaline transport inhibitor/integrin binding peptide with improved stability.

A dual-pharmacophoric peptide was engineered by grafting the integrin binding RGD motif between the C- and N-termini of a disulfide-rich noradrenaline transporter inhibiting χ-conotoxin resulting in a stable backbone cyclized peptide. The construct maintained two independent biological activities and showed increased plasma stability with no adverse effects observed following administration to rats, highlighting the potential value of pharmacophore grafting into constrained peptide scaffolds.