Dean R. Madden

Intracellular Delivery of Peptidyl Ligands by Reversible Cyclization: Discovery of a PDZ Domain Inhibitor that Rescues CFTR Activity.

A general strategy was developed for the intracellular delivery of linear peptidyl ligands through fusion to a cell-penetrating peptide and cyclization of the fusion peptides via a disulfide bond. The resulting cyclic peptides are cell permeable and have improved proteolytic stability. Once inside the cell, the disulfide bond is reduced to produce linear biologically active peptides. This strategy was applied to generate a cell-permeable peptide substrate for real-time detection of intracellular caspase activities during apoptosis and an inhibitor for the CFTR-associated ligand (CAL) PDZ domain as a potential treatment for cystic fibrosis.

A Compact Structure of Cytochrome c Trapped in a Lysine-Ligated State: Loop Refolding and Functional Implications of a Conformational Switch

It has been suggested that the alkaline form of cytochrome c (cyt c) regulates function of this protein as an electron carrier in oxidative phosphorylation and as a peroxidase that reacts with cardiolipin (CL) during apoptosis. In this form, Met80, the native ligand to the heme iron, is replaced by a Lys. While it has become clear that the structure of cyt c changes, the extent and sequence of conformational rearrangements associated with this ligand replacement remain a subject of debate. Herein we report a high-resolution crystal structure of a Lys73-ligated cyt c conformation that reveals intricate change in the heme environment upon this switch in the heme iron ligation. The structure is surprisingly compact, and the heme coordination loop refolds into a β-hairpin with a turn formed by the highly conserved residues Pro76 and Gly77. Repositioning of residue 78 modifies the intraprotein hydrogen-bonding network and, together with adjustments of residues 52 and 74, increases the volume of the heme pocket to allow for insertion of one of the CL acyl moieties next to Asn52. Derivatization of Cys78 with maleimide creates a solution mimic of the Lys-ligated cyt c that has enhanced peroxidase activity, adding support for a role of the Lys-ligated cyt c in the apoptotic mechanism. Experiments with the heme peptide microperoxidase-8 and engineered model proteins provide a thermodynamic rationale for the switch to Lys ligation upon perturbations in the protein scaffold.

Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

Background: PDZ-peptide binding specificities establish a complex network of protein-protein interactions in the cell. Results: Crystal structures of multiple PDZ-peptide complexes reveal distinct mechanisms for accommodating C-terminal ligand side chains. Conclusion: A residue in the PDZ “XΦ1GΦ2” signature sequence co-determines peptide carboxylate and C-terminal side-chain binding. Significance: Understanding the stereochemical determinants of peptide binding leads to an improved ability to predict PDZ interaction specificity. PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P0), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P0 Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

Signature motifs identify an acinetobacter cif virulence factor with epoxide hydrolase activity.

Background: Pathogens target airway clearance mechanisms to facilitate infection. Results: Sequence analysis reveals an Acinetobacter epoxide hydrolase (EH) that triggers loss of the cystic fibrosis transmembrane conductance regulator (CFTR). Conclusion: Homologous EH virulence factors found in a variety of opportunistic pathogens can impair CFTR, a key element of host airway defenses. Significance: EH virulence factors are potential therapeutic targets. Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii (“aCif”). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog (“aCifR”) and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

Stereochemical Preferences Modulate Affinity and Selectivity among Five PDZ Domains that Bind CFTR: Comparative Structural and Sequence Analyses.

PDZ domain interactions are involved in signaling and trafficking pathways that coordinate crucial cellular processes. Alignment-based PDZ binding motifs identify the few most favorable residues at certain positions along the peptide backbone. However, sequences that bind the CAL (CFTR-associated ligand) PDZ domain reveal only a degenerate motif that overpredicts the true number of high-affinity interactors. Here, we combine extended peptide-array motif analysis with biochemical techniques to show that non-motif modulator residues influence CAL binding. The crystallographic structures of 13 CAL:peptide complexes reveal defined, but accommodating stereochemical environments at non-motif positions, which are reflected in modulator preferences uncovered by multisequence substitutional arrays. These preferences facilitate the identification of high-affinity CAL binding sequences and differentially affect CAL and NHERF PDZ binding. As a result, they also help determine the specificity of a PDZ domain network that regulates the trafficking of CFTR at the apical membrane.

Tissue-specific control of CFTR endocytosis by Dab2 Cargo recruitment as a therapeutic target

Clathrin-mediated endocytosis dynamically regulates cell membrane abundance of CFTR and plays an essential role in CFTR-dependent Cl- conductance in fluid-transporting epithelia. It requires two closely related, but distinct processes: assembly of the clathrin coat and recruitment of cargo proteins for endocytosis. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor responsible for optimal clathrin coat formation. Disabled-2 (Dab2) is a clathrin associated sorting protein (CLASP) that also mediates clathrin assembly and cargo selection. Both of these complexes have clearly been shown to play roles in CFTR endocytosis in cells that endogenously express the channel. However, their precise functions exhibit cell-specific differences. While Dab2 appears to play a central role in CFTR recruitment to the clathrin coat in airway epithelial cells, it does not play a direct role in CFTR endocytosis in intestinal epithelial cells. Here, we review our current understanding of the role of Dab2 in CFTR endocytosis in different tissues. Next, we present new data demonstrating the role of Dab2 in endocytosis of the most commonly mutated CFTR gene product, ∆F508-CFTR, in human airwy epithelial cells. Finally we discuss the potential therapeutic implications of targeting the functional interaction between ∆F508-CFTR and Dab2.

The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9

Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9s interaction with CAL. Mutation of SLC26A9s PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.

Inhibiting an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa Protects CFTR

Opportunistic pathogens exploit diverse strategies to sabotage host defenses. Pseudomonas aeruginosa secretes the CFTR inhibitory factor Cif and thus triggers loss of CFTR, an ion channel required for airway mucociliary defense. However, the mechanism of action of Cif has remained unclear. It catalyzes epoxide hydrolysis, but there is no known role for natural epoxides in CFTR regulation. It was demonstrated that the hydrolase activity of Cif is strictly required for its effects on CFTR. A small-molecule inhibitor that protects this key component of the mucociliary defense system was also uncovered. These results provide a basis for targeting the distinctive virulence chemistry of Cif and suggest an unanticipated role of physiological epoxides in intracellular protein trafficking.

Visualizing the Mechanism of Epoxide Hydrolysis by the Bacterial Virulence Enzyme Cif

The CFTR inhibitory factor (Cif) is an epoxide hydrolase (EH) virulence factor secreted by the bacterium Pseudomonas aeruginosa. Sequence alignments reveal a pattern of Cif-like substitutions that proved to be characteristic of a new subfamily of bacterial EHs. At the same time, crystallographic and mutagenetic data suggest that EH activity is required for virulence and that Cifs active site remains generally compatible with a canonical two-step EH mechanism. A hallmark of this mechanism is the formation of a covalent hydroxyalkyl-enzyme intermediate by nucleophilic attack. In several well-studied EHs, this intermediate has been captured at near stoichiometric levels, presumably reflecting rate-limiting hydrolysis. Here we show by mass spectrometry that only minimal levels of the expected intermediate can be trapped with WT Cif. In contrast, substantial amounts of intermediate are recovered from an active-site mutant (Cif-E153Q) that selectively targets the second, hydrolytic release step. Utilizing Cif-E153Q and a previously reported nucleophile mutant (Cif-D129S), we then captured Cif in the substrate-bound, hydroxyalkyl-intermediate, and product-bound states for 1,2-epoxyhexane, yielding the first crystallographic snapshots of an EH at these key stages along the reaction coordinate. Taken together, our data illuminate the proposed two-step hydrolytic mechanism of a new class of bacterial virulence factor. They also suggest that the failure of WT Cif to accumulate a covalent hydroxyalkyl-enzyme intermediate reflects an active-site chemistry in which hydrolysis is no longer the rate-limiting step, a noncanonical kinetic regime that may explain similar observations with a number of other EHs.

Chemically Modified Peptide Scaffolds Target the CFTR-Associated Ligand PDZ Domain.

PDZ domains are protein-protein interaction modules that coordinate multiple signaling and trafficking pathways in the cell and that include active therapeutic targets for diseases such as cancer, cystic fibrosis, and addiction. Our previous work characterized a PDZ interaction that restricts the apical membrane half-life of the cystic fibrosis transmembrane conductance regulator (CFTR). Using iterative cycles of peptide-array and solution-binding analysis, we targeted the PDZ domain of the CFTR-Associated Ligand (CAL), and showed that an engineered peptide inhibitor rescues cell-surface expression of the most common CFTR disease mutation ΔF508. Here, we present a series of scaffolds containing chemically modifiable side chains at all non-motif positions along the CAL PDZ domain binding cleft. Concordant equilibrium dissociation constants were determined in parallel by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance techniques, confirming robust affinity for each scaffold and revealing an enthalpically driven mode of inhibitor binding. Structural studies demonstrate a conserved binding mode for each peptide, opening the possibility of combinatorial modification. Finally, we diversified one of our peptide scaffolds with halogenated substituents that yielded modest increases in binding affinity. Overall, this work validates our approach and provides a stereochemical foundation for further CAL inhibitor design and screening.