Defining the Interacting Regions of the Translational Regulators Polyadenylate Binding Protein (PABP) 1 and the Programmed Cell Death Protein (Pdcd) 4.

Abstract

Programmed Cell Death protein (Pdcd) 4 is a highly conserved nuclear/cytoplasmic shuttling and RNA binding protein that has been shown to suppress tumour progression by regulating transcription and translation and is therefore considered a promising target for anti-cancer therapies. Until recently regulation of translation inhibition was exclusively thought to be eIF4A mediated, and was shown to involve the interaction of the tandem MA3 domains of Pdcd4 with the RNA helicase, eIF4A. Recent evidence has shown that Pdcd4 can also inhibit translation in an eIF4A independent mechanism, via direct interactions with both the Poly Adenylate Binding Protein (PABP) 1 and specific regions of mRNA. The work in this thesis describes the crystallisation of the RRM 2 construct that confirmed previously described structural features. It also describes how models of RRM 3 and RRM 2-3 constructs were designed due to the constraints of obtaining respective crystals. The latter chapters contain NMR chemical shift perturbation experiments that identified the RNA Recognition Motifs (RRM) 2 and 3 of PABP1 as the main contact interface with the Pdcd4 protein. These chapters also showed successful generation of isotopically labeled individual RRM 2 and RRM 3 and tandem RRM 2-3 constructs that enabled sequence-specific backbone assignments to be obtained using triple resonance NMR experiments. This allowed for structural characterisation of the RRM2-3 region and identification of specific residues (primarily the β strands and linker region between RRM 2 and RRM 3) that are believed to form part of the Pdcd4 binding site. Analogous experiments suggest the RNA Binding Region (RBD) and middle MA3 domains of Pdcd4 contain the main PABP1 binding sites. The stoichiometry of the complex suggests a 1:1 transient interaction in solution with a Kd value in the low micromolar region.