The cystic fibrosis transmembrane conductance regulator (CFTR)1 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 diseaseassociated mutant ΔF508-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, CAL's affinity is evidently sufficient to capture and degrade more rapidly cycling mutants, such as ΔF508-CFTR. The relatively weak affinity of the CAL:CFTR interaction may provide a pharmacological window for stabilizing rescued ΔF508-CFTR in patients with cystic fibrosis.CFTR is a cAMP-activated, ATP-gated chloride channel. It plays a central role in maintaining fluid and ion homeostasis in epithelial tissues and is mutated in patients with cystic fibrosis (CF) (1). Although CFTR is subject to rapid endocytosis (2), this appears to be coupled with a highly efficient constitutive recycling mechanism (e.g refs. 3,4). As a result, mature CFTR exhibits a long functional half-life (5,6), requiring individual molecules to cycle through the endocytic pathway dozens or even hundreds of times. † This work was supported in part by grants from the Cystic Fibrosis Foundation (MADDEN06P0 and STANTO97R0) and the NIH (grants P20-RR018787 from the Institutional Development Award (IDeA) Program of the NCRR and R01-DK075309 from NIDDK). P.B. was supported by the Deutsche Forschungsgemeinschaft (DFG grant VO 885/3-1). 1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; PDZ, PSD-95, discs large, zonula occludens-1; CAL, CFTR-Associated Ligand; NHERF1, Na + /H + Exchanger-3 Regulatory Factor-1; NHERF2, Na + /H + Exchanger-3 Regulatory Factor-2; CF, cystic fibrosis; DTT, dithiothreitol; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; SPR, surface-plasmon resonance; ITC, isothermal titration calorimetry; FP, fluorescence polarizatio...

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.

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 CBFb binding or the Runt domain structure. We introduced different classes of missense mutations into Runx1 and characterized their effects on DNA and CBFb binding by the Runt domain, and on Runx1 function in vivo. Mutations involving DNA-contacting residues severely inactivate Runx1 function, whereas mutations that affect CBFb 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.

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 "X⌽ 1 G⌽ 2 " 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.

Summary 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 new CAL:peptide complexes reveal defined, but accommodating stereochemical environments at non-motif positions, which are reflected in modulator preferences uncovered by multi-sequence substitutional arrays. These preferences facilitate the identification of new 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.