Patrick R. Cushing

Disease mutations in RUNX1 and RUNX2 create nonfunctional, dominant-negative, or hypomorphic alleles

Monoallelic RUNX1 mutations cause familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML). Sporadic mono‐ and biallelic mutations are found at high frequencies in AML M0, in radiation‐associated and therapy‐related myelodysplastic syndrome and AML, and in isolated cases of AML M2, M5a, M3 relapse, and chronic myelogenous leukemia in blast phase. Mutations in RUNX2 cause the inherited skeletal disorder cleidocranial dysplasia (CCD). Most hematopoietic missense mutations in Runx1 involve DNA‐contacting residues in the Runt domain, whereas the majority of CCD mutations in Runx2 are predicted to impair CBFβ binding or the Runt domain structure. We introduced different classes of missense mutations into Runx1 and characterized their effects on DNA and CBFβ binding by the Runt domain, and on Runx1 function in vivo. Mutations involving DNA‐contacting residues severely inactivate Runx1 function, whereas mutations that affect CBFβ binding but not DNA binding result in hypomorphic alleles. We conclude that hypomorphic RUNX2 alleles can cause CCD, whereas hematopoietic disease requires more severely inactivating RUNX1 mutations.

The Relative Binding Affinities of PDZ Partners for CFTR: A Biochemical Basis for Efficient Endocytic Recycling †

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis. Its expression and functional interactions in the apical membrane are regulated by several PDZ (PSD-95, discs large, zonula occludens-1) proteins, which mediate protein-protein interactions, typically by binding C-terminal recognition motifs. In particular, the CFTR-associated ligand (CAL) limits cell-surface levels of the most common disease-associated mutant DeltaF508-CFTR. CAL also mediates degradation of wild-type CFTR, targeting it to lysosomes following endocytosis. Nevertheless, wild-type CFTR survives numerous cycles of uptake and recycling. In doing so, how does it repeatedly avoid CAL-mediated degradation? One mechanism may involve competition between CAL and other PDZ proteins including Na (+)/H (+) exchanger-3 regulatory factors 1 and 2 (NHERF1 and NHERF2), which functionally stabilize cell-surface CFTR. Thus, to understand the biochemical basis of WT-CFTR persistence, we need to know the relative affinities of these partners. However, no quantitative binding data are available for CAL or the individual NHERF2 PDZ domains, and published estimates for the NHERF1 PDZ domains conflict. Here we demonstrate that the affinity of the CAL PDZ domain for the CFTR C-terminus is much weaker than those of NHERF1 and NHERF2 domains, enabling wild-type CFTR to avoid premature entrapment in the lysosomal pathway. At the same time, CALs affinity is evidently sufficient to capture and degrade more rapidly cycling mutants, such as DeltaF508-CFTR. The relatively weak affinity of the CAL:CFTR interaction may provide a pharmacological window for stabilizing rescued DeltaF508-CFTR in patients with cystic fibrosis.

Computational Design of a PDZ Domain Peptide Inhibitor that Rescues CFTR Activity

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis (CF). The most prevalent CFTR mutation, ΔF508, blocks folding in the endoplasmic reticulum. Recent work has shown that some ΔF508-CFTR channel activity can be recovered by pharmaceutical modulators (“potentiators” and “correctors”), but ΔF508-CFTR can still be rapidly degraded via a lysosomal pathway involving the CFTR-associated ligand (CAL), which binds CFTR via a PDZ interaction domain. We present a study that goes from theory, to new structure-based computational design algorithms, to computational predictions, to biochemical testing and ultimately to epithelial-cell validation of novel, effective CAL PDZ inhibitors (called “stabilizers”) that rescue ΔF508-CFTR activity. To design the “stabilizers”, we extended our structural ensemble-based computational protein redesign algorithm to encompass protein-protein and protein-peptide interactions. The computational predictions achieved high accuracy: all of the top-predicted peptide inhibitors bound well to CAL. Furthermore, when compared to state-of-the-art CAL inhibitors, our design methodology achieved higher affinity and increased binding efficiency. The designed inhibitor with the highest affinity for CAL (kCAL01) binds six-fold more tightly than the previous best hexamer (iCAL35), and 170-fold more tightly than the CFTR C-terminus. We show that kCAL01 has physiological activity and can rescue chloride efflux in CF patient-derived airway epithelial cells. Since stabilizers address a different cellular CF defect from potentiators and correctors, our inhibitors provide an additional therapeutic pathway that can be used in conjunction with current methods.

A Stabilizing Influence: CAL PDZ Inhibition Extends the Half‐Life of ΔF508‐CFTR

From synthesis to degradation, membrane proteins navigate interwoven networks that control their localization and activity within the cell. At branchpoints within these networks, protein-protein interactions often determine the flux of individual proteins through specific pathways and thus offer targets for therapeutic modulation. The PDZ (PSD-95, Dlg, and ZO-1) proteins constitute a major family of trafficking regulators. Characterized by the presence of eponymous protein-protein interaction domains (PPIDs), PDZ proteins generally bind the C-termini of their partners and help choreograph their movements throughout the cell. The targets of PDZ regulation include the cystic fibrosis transmembrane conductance regulator (CFTR), the chloride channel mutated in patients with cystic fibrosis (CF)[1].

Hybrid organic-inorganic inhibitors of a PDZ interaction that regulates the endocytic fate of CFTR.

Together strong: Cooperative binding of organic (see picture, red) and inorganic fragments provides a strategy for the potent inhibition of protein-protein interactions. By targeting specific Lewis basic side chains in peripheral regions of the binding site for coordination to a rhodium(II) center, the affinity of otherwise weak ligands is improved.

Engineering Peptide Inhibitors To Overcome PDZ Binding Promiscuity

Protein-protein interaction domains (PPIDs) are key elements in assembling functional protein complexes and controlling cellular activities. A major class of PPIDs is mediated by PDZ (for PSD-95, Dlg, ZO-1) domains[1–3], widespread scaffolding modules essential for regulating the localization and activity of numerous cellular effector proteins. Among the diverse protein interaction domains, PDZ domains are highly conserved in organisms from bacteria to humans[4]. They usually bind the C-terminus of their ligands.

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.

Crystallization and preliminary diffraction analysis of the CAL PDZ domain in complex with a selective peptide inhibitor

Cystic fibrosis (CF) is associated with loss-of-function mutations in the CF transmembrane conductance regulator (CFTR), which regulates epithelial fluid and ion homeostasis. The CFTR cytoplasmic C-terminus interacts with a number of PDZ (PSD-95/Dlg/ZO-1) proteins that modulate its intracellular trafficking and chloride-channel activity. Among these, the CFTR-associated ligand (CAL) has a negative effect on apical-membrane expression levels of the most common disease-associated mutant ΔF508-CFTR, making CAL a candidate target for the treatment of CF. A selective peptide inhibitor of the CAL PDZ domain (iCAL36) has recently been developed and shown to stabilize apical expression of ΔF508-CFTR, enhancing net chloride-channel activity, both alone and in combination with the folding corrector corr-4a. As a basis for structural studies of the CAL-iCAL36 interaction, a purification protocol has been developed that increases the oligomeric homogeneity of the protein. Here, the cocrystallization of the complex in space group P2(1)2(1)2(1), with unit-cell parameters a = 35.9, b = 47.7, c = 97.3 Å, is reported. The crystals diffracted to 1.4 Å resolution. Based on the calculated Matthews coefficient (1.96 Å(3) Da(-1)), it appears that the asymmetric unit contains two complexes.

Small molecule inhibition of the Na(+)/H(+) exchange regulatory factor 1 and parathyroid hormone 1 receptor interaction.

We have identified a series of small molecules that bind to the canonical peptide binding groove of the PDZ1 domain of NHERF1 and effectively compete with the association of the C-terminus of the parathyroid hormone 1 receptor (PTH1R). Employing nuclear magnetic resonance and molecular modeling, we characterize the mode of binding that involves the GYGF loop important for the association of the C-terminus of PTH1R. We demonstrate that the common core of the small molecules binds to the PDZ1 domain of NHERF1 and displaces a 15N-labeled peptide corresponding to the C-terminus of PTH1R. The small size (molecular weight of 192) of this core scaffold makes it an excellent candidate for further elaboration in the development of an inhibitor for this important protein–protein interaction.

Design of protein-protein interactions with a novel ensemble-based scoring algorithm

Protein-protein interactions (PPIs) are vital for cell signaling, protein trafficking and localization, gene expression, and many other biological functions. Rational modification of PPI targets provides a mechanism to understand their function and importance. However, PPI systems often have many more degrees of freedom and flexibility than the small-molecule binding sites typically targeted by protein design algorithms. To handle these challenging design systems, we have built upon the computational protein design algorithm K* [8,19] to develop a new design algorithm to study protein-protein and protein-peptide interactions. We validated our algorithm through the design and experimental testing of novel peptide inhibitors. Previously, K* required that a complete partition function be computed for one member of the designed protein complex. While this requirement is generally obtainable for active-site designs, PPI systems are often much larger, precluding the exact determination of the partition function. We have developed proofs that show that the new K* algorithm combinatorially prunes the protein sequence and conformation space and guarantees that a provably-accurate e-approximation to the K* score can be computed. These new proofs yield new algorithms to better model large protein systems, which have been integrated into the K* code base. K* computationally searches for sequence mutations that will optimize the affinity of a given protein complex. The algorithm scores a single protein complex sequence by computing Boltzmann-weighted partition functions over structural molecular ensembles and taking a ratio of the partition functions to find provably-accurate e-approximations to the K* score, which predicts the binding constant. The K* algorithm uses several provable methods to guarantee that it finds the gap-free optimal sequences for the designed protein complex. The algorithm allows for flexible minimization during the conformational search while still maintaining provable guarantees by using the minimization-aware dead-end elimination criterion, minDEE. Further pruning conditions are applied to fully explore the sequence and conformation space. To demonstrate the ability of K* to design protein-peptide interactions, we applied the ensemble-based design algorithm to the CFTR-associated ligand, CAL, which binds to the C-terminus of CFTR, the chloride channel mutated in human patients with cystic fibrosis. K* was retrospectively used to search over a set of peptide ligands that can inhibit the CAL-CFTR interaction, and K* successfully enriched for peptide inhibitors of CAL. We then used K* to prospectively design novel inhibitor peptides. The top-ranked K*-designed peptide inhibitors were experimentally validated in the wet lab and, remarkably, all bound with µM affinity. The top inhibitor bound with seven-fold higher affinity than the best hexamer peptide inhibitor previously available and with 331- fold higher affinity than the CFTR C-terminus.