Molecular Basis for Signal Transduction in the Bimodular Cell-Cycle Enzyme Pin1

It has become more evident that many proteins experience activity regulation via binding of small molecules or other proteins to sites that are distal from the active site. This phenomenon, commonly referred to as allostery, plays an important role as a mechanism for signal transduction within and between proteins to make up the complex cellular information networks. Proteins of modular design represent a unique class of proteins that construct such information systems whereby a docking module and a loosely connected module of catalytic function operate in concert to achieve optimal activity for the viability of the cell. In this case, inter-domain signal transduction is carried out by the process known as modular allostery. Unlike the classic intra-domain allosteric mechanism, modular allostery is also associated with large inter-domain structural transitions to regulate activity. For instance, ligand binding at one domain may control access of substrate to the active site of another domain by directly blocking the active site or indirectly via a distal allosteric site. Pin1 is a model system of an allosteric protein of modular design which consists of the binding WW domain that is flexibly tethered to a peptidyl-prolyl cis-trans isomerase (PPIase) domain. Pin1 recognizes and catalyses the cis-trans conversion of the peptidyl pSer/pThr-Pro bond of many targets. Variations in the substrate 'input' sequence induces differential processing by Pin1, thereby modulating the resultant 'output' activities for binding and/or catalysis. The isolated domains only hold part of the full activity that is observed when they are linked. Although the WW domain has no catalytic activity, its absence has deleterious consequences on the catalytic activity of Pin1. In addition, ligand interaction results in inter-domain coupling. Therefore, a mode of information transformation exists that qualifies the WW as an allosteric modulator of the activity of Pin1-PPIase. The underlying molecular processes for site to site communication in proteins are thought to involve conformational transitions that are propagated between the active site and the allosteric site. The time scales for such motions can be in the order of slow (Ì_å_s-ms) displacements to fast (ps-ns) fluctuating motions. Nuclear Magnetic Resonance (NMR) is a versatile non-invasive technique that probes such molecular dynamics in solution and at multiple sites. This dissertation reports the research on the discovery of a novel internal dynamic conduit of modular allosteric communication between the catalytic site of Pin1-PPIase and the allosteric WW interaction site located about 12 angstroms away. In particular, deuterium (2D) NMR relaxation experiments were used to probe the internal flexibility of methyl side chains of Pin1-PPIase upon ligand interaction. Ligands of different chemical compositions, configurations and flexibilities induced varying rigidification magnitudes of Pin1's internal fast (ps-ns) fluctuating motions. The greatest flexibility loss was observed for ligands with high binding affinities. Because the locked-cis ground state analog of substrate showed a greater response than the trans counterpart, the dynamic conduit observed with peptide substrate must coordinate the interaction of cis-like substrate conformers. Although the cis-locked isostere does not bind Pin1-WW, it caused a much weaker response in the internal conduit for the isolated PPIase domain and bound weakly than Pin1-PPIase. This suggests that the dynamic conduit links ligand binding activity at the PPIase active site and the interaction of the WW domain at the domain interface on Pin1-PPIase. In addition to probing the dynamics-function relationships of Pin1, natural abundance carbon (13CÌ_å±) NMR relaxation dispersion experiments were used to probe the dynamics of the backbone dihedrals of a Pin1 ligand system comprised of a peptide substrate and its representative ground state cis and trans locked analogues. From this, newer findings on the site-specific kinetics and thermodynamics of the PPIase rotomerization mechanism are hereby reported. In particular, the cis conformer is more conformationally restricted and primed for docking PPIase than the trans counterpart which experiences pre-organization at the terminal ends upon binding PPIase. Substrate isomerization proceeds with anchoring of residues located at the N-terminal of the peptidyl-prolyl bond and rotation at the C-terminal end with activation free energies for cis-trans conversion of Ì_'GÌ¢‰âÂåÊCT 13.3 kcal/mol and Ì_'GÌ¢‰âÂåÊTC 15.4 kcal/mol.