|Jeanine F. Amacher
University of Oregon. 2007
Joined the Madden lab in 2009
CFTR Trafficking. From the table of contents figure of Cushing et. al Angew Chem Int Ed. (2010) 49:9907-9911.
Structural Analysis of PDZ:peptide interactions that regulate CFTR half-life
The PDZ domain is the most common protein interaction module in the human genome, typically recognizing the C-terminal residues of its target proteins. Individual PDZ binding motifs reveal interactions with up to seven residues, denoted by their distance from the terminus, e.g., P0, P-1. In many cases, however, binding motifs reveal only two or three sequence constraints on the target sequence, and PDZ binding is notoriously promiscuous, with one domain binding multiple targets. Nevertheless, individual domains also exhibit significant affinity differences among sequences with shared motifs. The basis for these “cryptic” stereochemical preferences remains unclear.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a target protein recognized by multiple PDZ domains, including NHERF1, NHERF2, NHERF3, and the CFTR-associated ligand (CAL). Of these PDZ partners, CAL is the only one which negatively regulates the abundance of CFTR at the cell surface, making it an attractive therapeutic drug target. Global motif analysis of the CAL PDZ domain shows strong residue preferences only at the P0 and P-2 positions. Using a peptide array engineering approach however, we identified robust affinity determinants reaching back to the P-5 position. The result was the inhibitor peptide iCAL36 (ANSRWPTSII), which increases apical abundance of functional ΔF508-CFTR in CFBE cells. We also previously showed that applying iCAL36 to an epithelial monolayer increases the activity of ΔF508-CFTR by about 25%. However, studies using siRNA to inhibit CAL expression in the same system show a >200% increase in chloride efflux by ΔF508-CFTR. In order to design a better inhibitor of the CFTR:CAL interaction, and more effectively recapitulate the effect of siCAL, we first need to understand the basis, and extent, of selectivity of our inhibitor peptide.
TIP-1:iCAL36 co-complex structure. The tryptophan-selective binding pocket accommodates the P-5 residue in iCAL36.
We know that iCAL36 binds the CAL, but not the NHERF, PDZ domain(s). However, iCAL36 is capable of binding an additional PDZ domain-containing protein, Tax Interacting Protein 1 (TIP-1), which is known to inhibit ß-catenin in Wnt signaling pathways. To understand CAL and TIP-1’s upstream affinity, as well as design better CAL inhibitors, my project involves co-crystallizing the CAL and TIP-1 PDZ domains with iCAL36, and asking a number of questions. First, can the structure reveal why iCAL36 can bind CAL, but not the NHERF proteins? Furthermore, why does iCAL36 bind TIP-1, which is not known to interact with CFTR endogenously? Finally, can we use the structure to design more efficacious CFTR:CAL inhibitors?
A. Surface representation of the contact surface area per peptide position in CAL PDZ:iCAL36 co-complex structure.
B. Superposition of CAL PDZ:peptide structures, where the tryptophan residue at the P-5 position of iCAL36 is substituted. Sequences of the peptides are shown in the table.
Understanding the PDZ:peptide interaction Although PDZ binding motifs only describe preferences for two crucial C-terminal residues, the CAL PDZ:iCAL36 structure reveals interactions up to the P-5 position of the peptide. Do these positions contain selectivity determinants for CAL PDZ? What about the same positions on TIP-1 or other PDZ domains? Our structural and biochemical tools allow us to make single amino acid substitutions in iCAL36, or other PDZ target peptides, and ask what happens to the resulting affinity and stereochemistry of binding for each interaction. We can also directly visualize the interplay between C-terminal and upstream binding determinants. Our results suggest different C-terminal “core” sequences impose different upstream preferences for PDZ domain interactions. Understanding how CAL binds its peptide targets will hopefully allow us to improve inhibition of CFTR:CAL. We also want to gain an insight into not only how CFTR interacts with its other PDZ domain binding partners, but how all PDZ domain-containing proteins recognize their target sequences.
J.F. Amacher, P.R. Cushing, J.A. Weiner, D.R. Madden (2011) Crystallization and preliminary diffraction analysis of the CAL PDZ domain in complex with a selective peptide inhibitor. Acta Crystallogr. F67, 600-603.
|Molecular and Cellular Biology Program Retreat||September, 2009|
|Biochemistry Department Retreat||November, 2010|
|Dartmouth Lung Biology COBRE EAC Meeting||March, 2011|
|Dartmouth Integrative Biology Symposium||April, 2011|
|Biophysical Society Meeting||March, 2011|
|North American Cystic Fibrosis Conference (NACFC)||November, 2011|
|2011-present||Albert J. Ryan Fellow|
|2010-present||NRSA Institutional Research Training Grant Fellow
T32-GM008704 Molecular and Cellular Biology at Dartmouth