Andrea Piserchio

Targeting CAL as a Negative Regulator of ΔF508-CFTR Cell-Surface Expression AN RNA INTERFERENCE AND STRUCTURE-BASED MUTAGENETIC APPROACH

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated ΔF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL·CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.

NMR Structural Analysis of α-Bungarotoxin and Its Complex with the Principal α-Neurotoxin-binding Sequence on the α7 Subunit of a Neuronal Nicotinic Acetylcholine Receptor

We report a new, higher resolution NMR structure of α-bungarotoxin that defines the structure-determining disulfide core and β-sheet regions. We further report the NMR structure of the stoichiometric complex formed between α-bungarotoxin and a recombinantly expressed 19-mer peptide (178IPGKRTESFYECCKEPYPD196) derived from the α7 subunit of the chick neuronal nicotinic acetylcholine receptor. A comparison of these two structures reveals binding-induced stabilization of the flexible tip of finger II in α-bungarotoxin. The conformational rearrangements in the toxin create an extensive binding surface involving both sides of the α7 19-mer hairpin-like structure. At the contact zone, Ala7, Ser9, and Ile11 in finger I and Arg36, Lys38, Val39, and Val40 in finger II of α-bungarotoxin interface with Phe186, Tyr187, Glu188, and Tyr194 in the α7 19-mer underscoring the importance of receptor aromatic residues as critical neurotoxin-binding determinants. Superimposing the structure of the complex onto that of the acetylcholine-binding protein (1I9B), a soluble homologue of the extracellular domain of the α7 receptor, places α-bungarotoxin at the peripheral surface of the inter-subunit interface occluding the agonist-binding site. The disulfide-rich core of α-bungarotoxin is suggested to be tilted in the direction of the membrane surface with finger II extending into the proposed ligand-binding cavity.

Solution NMR insights into docking interactions involving inactive ERK2.

The mitogen-activated protein (MAP) kinase ERK2 contains recruitment sites that engage canonical and noncanonical motifs found in a variety of upstream kinases, regulating phosphatases and downstream targets. Interactions involving two of these sites, the D-recruitment site (DRS) and the F-recruitment site (FRS), have been shown to play a key role in signal transduction by ERK/MAP kinases. The dynamic nature of these recruitment events makes NMR uniquely suited to provide significant insight into these interactions. While NMR studies of kinases in general have been greatly hindered by their large size and complex dynamic behavior leading to the suboptimal performance of standard methodologies, we have overcome these difficulties for inactive full-length ERK2 and obtained an acceptable level of backbone resonance assignments. This allowed a detailed investigation of the structural perturbations that accompany interactions involving both canonical and noncanonical recruitment events. No crystallographic information exists for the latter. We found that the chemical shift perturbations in inactive ERK2, indicative of structural changes in the presence of canonical and noncanonical motifs, are not restricted to the recruitment sites but also involve the linker that connects the N- and C-lobes and, in most cases, a gatekeeper residue that is thought to exert allosteric control over catalytic activity. We also found that, even though the canonical motifs interact with the DRS utilizing both charge-charge and hydrophobic interactions, the noncanonical interactions primarily involve the latter. These results demonstrate the feasibility of solution NMR techniques for a comprehensive analysis of docking interactions in a full-length ERK/MAP kinase.

Global chimeric exchanges within the intracellular face of the bradykinin B2 receptor with corresponding angiotensin II type Ia receptor regions: Generation of fully functional hybrids showing characteristic signaling of the AT1a receptor

The intracellular (IC) face of the G‐protein coupled receptors (GPCR), bradykinin (BK) B2 and angiotensin (AT) 1a, is similar in sequence homology and in size. Both receptors are known to link to Gαi and Gαq but differ markedly in a number of physiologic actions, particularly with respect to their hemodynamic action. We made single as well as multiple, global replacements within the IC of BKB2R with the corresponding regions of the AT1aR. When stably transfected into Rat‐1 cells, these hybrid receptors all bound BK with high affinity. Single replacement of the intracellular loop 2 (IC2) or the distal 34 residues of the C‐terminus (dCt) with the corresponding regions of AT1aR resulted in chimera, which turned over phosphotidylinositol (PI) and released arachidonic acid (ARA) as WT BKB2R. In contrast, incorporation of the AT1aR IC3 in a single replacement abolished signal transduction. However, the simultaneous exchange of IC2 and IC3 of BKB2R with AT1aR resulted in a receptor responding to BK with PI turnover and ARA release approximately 4‐fold greater than WT BKB2R. Likewise, the simultaneous replacement of IC2 and dCt resulted in a 2.8‐ and 1.6‐fold increase in PI turnover and ARA release, respectively. In contrast, the dual replacement of IC3 and dCt could not overcome the deleterious effects of the IC3 replacement, resulting in very low PI activation and ARA release. Replacement of all three IC domains (IC2, IC3, and dCt) resulted in PI closer to that of AT1aR than BKB2R. The uptake of the receptor chimeras was similar to that of WT BKB2R with the exception of the IC3/dCt dual mutant, which exhibited very poor internalization (18% at 60′). When transfected into Rat‐1 cells, the AT1aR markedly increased the expression of connective tissue growth factor (CTGF) mRNA, while BK slightly decreased it. The dual IC2/dCt and triple IC2/IC3/dCt hybrids both upregulated CTGF mRNA in response to BK. These results show that the IC face of the BKB2R can be exchanged with that of AT1aR, producing hybrid receptors, which take on the functional characteristics of AT1aR. The characterization of the chimera with stepwise replacement of the IC domains should allow for assignment of specific roles to the individual loops and C‐terminus in the signaling and internalization of the BKB2R and facilitate the generation of a receptor with BKB2R binding and AT1aR function. J. Cell. Biochem. 85: 809–819, 2002.

A Model of a MAPK•Substrate Complex in an Active Conformation: A Computational and Experimental Approach

The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)2-3-X2-6-ΦA-X-ΦB, where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus 7RK-X2-ΦA-X-ΦB 13. This results in a 2-fold increase in k cat/K m for the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves k cat/K m by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 of Ets results in a 14-fold decrease in k cat/K m, with little apparent change in k cat. A peptide that binds to the DRS of ERK2 affects K m, but not k cat. Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction.

Bradykinin B2 receptor signaling: Structural and functional characterization of the C‐terminus

Over the last few years the importance of the intracellular C‐terminus in the signaling of G‐protein coupled receptors (GPCR) has become increasingly evident. In an effort to provide a structural framework for biological function, we have determined the conformation of the C‐terminus of the bradykinin (BK) B2 receptor. Using a uniformly 15N‐ and 13C‐enriched sample of the BKB2 receptor [309–366], NMR results clearly define three α‐helices lying on the zwitterionic surface of the dodecylphosphocholine. The proximal helix consisting of residues 311–326 was previously predicted based on homology modeling with rhodopsin. This corresponds to what is often called helix‐8 of the GPCRs. The two distal helices, residues 333–345 and 348–363, are clearly borne out by the NMR data. The functional importance of these secondary structural elements was probed by determination of the signaling properties (inositol phosphate formation) of mutant BKB2 receptors lacking the domains (deletion mutants) or containing the corresponding region from the related GPCR, angiotensin II AT1a (chimera receptors). We demonstrate that the regions between the helices (residues 327–333 and 346–347) can be exchanged without loss of signaling. In contrast, modification of the three helices, particularly the hydroxyl‐containing residues, has drastic effects on the signaling profile of the BKB2 receptor. By coupling of the structural features with the functional data, the molecular mechanisms of signaling by the BKB2 receptor are beginning to be established.© 2005 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2005

Docking interactions of hematopoietic tyrosine phosphatase with MAP kinases ERK2 and p38α.

Hematopoietic tyrosine phosphatase (HePTP) regulates orthogonal MAP kinase signaling cascades by dephosphorylating both extracellular signal-regulated kinase (ERK) and p38. HePTP recognizes a docking site (D-recruitment site, DRS) on its targets using a conserved N-terminal sequence motif (D-motif). Using solution nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, we compare, for the first time, the docking interactions of HePTP with ERK2 and p38α. Our results demonstrate that ERK2-HePTP interactions primarily involve the D-motif, while a contiguous region called the kinase specificity motif also plays a key role in p38α-HePTP interactions. D-Motif-DRS interactions for the two kinases, while similar overall, do show some specific differences.

Structural characterization of the parathyroid hormone receptor domains determinant for ligand binding

Over the years, the association of peptide ligands to Family B GPCRs (G-protein coupled receptors) has been characterized by a number of experimental and theoretical techniques. For the PTH (parathyroid hormone) ligand-receptor system, important insight has been provided by photoaffinity labelling experiments and the elucidation of direct contact points between ligand and receptor. Our research has focused on the structural elucidation of the receptor domains shown to be involved in the binding of PTH. Employing a combination of carefully designed receptor domains, solution-state NMR carried out in the presence of membrane mimetics and extensive computer simulations, we have obtained a well-resolved model of the ligand-receptor complex for PTH. Here, we review the development of this model and highlight some inherent limitations of the methods employed and their consequences on interpretation of the ligand-receptor model.

Optimized bacterial expression and purification of the c-Src catalytic domain for solution NMR studies

Progression of a host of human cancers is associated with elevated levels of expression and catalytic activity of the Src family of tyrosine kinases (SFKs), making them key therapeutic targets. Even with the availability of multiple crystal structures of active and inactive forms of the SFK catalytic domain (CD), a complete understanding of its catalytic regulation is unavailable. Also unavailable are atomic or near-atomic resolution information about their interactions, often weak or transient, with regulating phosphatases and downstream targets. Solution NMR, the biophysical method best suited to tackle this problem, was previously hindered by difficulties in bacterial expression and purification of sufficient quantities of soluble, properly folded protein for economically viable labeling with NMR-active isotopes. Through a choice of optimal constructs, co-expression with chaperones and optimization of the purification protocol, we have achieved the ability to bacterially produce large quantities of the isotopically-labeled CD of c-Src, the prototypical SFK, and of its activating Tyr-phosphorylated form. All constructs produce excellent spectra allowing solution NMR studies of this family in an efficient manner.

Assignment of backbone resonances in a eukaryotic protein kinase - ERK2 as a representative example.

A first step toward the analysis of the structure, dynamics, and interactions of proteins by NMR is obtaining an acceptable level of resonance assignments. This process is nontrivial in most eukaryotic kinases given their size and suboptimal behavior in solution. Using inactive ERK2 as a representative example, we describe the procedures we utilized to achieve a significant degree of completeness of backbone resonance assignment.