Molecular Simulations of the Dynamics of Disordered Proteins

Abstract

regions in cis to ordered domains. Using atomistic simulations and fluorescence studies we characterize the conformational properties of several archetypes of intrinsically disordered sequences in the presence and absence of cis-acting ordered domains. In particular, we quantify the effects of sequence composition, chain length, and sequence patterning of disordered sequences on the inter-domain coupling. The balance between intra-domain and interdomain interactions can modulate intrinsic conformational propensities of the disordered regions. The circumstances giving rise to convergence toward generic random-coil behavior for disordered regions in cis to ordered domains will be highlighted. 2154-PlatThe C-Terminal V5 Domain of Protein Kinase Ca is a Multi-Functional Intrinsically Disordered Protein Module Yuan Yang, Tatyana I. Igumenova. Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA. Protein Kinase C (PKC) family of isozymes regulate a multitude of signaling pathways that control cell growth, differentiation, and apoptosis. The extreme C-terminal V5 domain has been identified as a key player in maturation, activation and down-regulation of PKCs, but the molecular basis of these events remains poorly understood. We report the first large-scale purification and NMR characterization of the V5 domain from PKCa (V5a) and its phosphorylation-mimicking variant. The combined analysis of NMR chemical shifts and circular dichroism data revealed that both V5a constructs are intrinsically disordered protein domains. Unexpectedly, we found that V5a has a propensity to partition into membrane mimetics acquiring a partial helical structure. Our data suggest that V5a anchors its parent enzyme to membranes during the maturation process. Using NMR techniques, we obtained direct evidence that V5a interacts with another domain of PKCa -the C2 regulatory domain. This interaction is mediated by the phosphorylated hydrophobic motif of V5a and is enhanced by Ca 2þ . Similarly, the affinity of C2 to Ca 2þ is enhanced in the presence of the phosphorylated hydrophobic motif. These findings indicate that V5a may function as an intra-molecular protein interaction module that sensitizes PKCa to Ca 2þ ions. The third aspect of V5a function pertains to its interactions with Pin1, a peptidyl-prolyl isomerase that has been implicated in the down-regulation of conventional PKCs. Pin1 catalyzes proline isomerization of the phosphorylated Ser/Thr-Pro motif. Our preliminary NMR data demonstrate that Pin1 interacts with the hydrophobic motif of V5a, which is a non-canonical site that lacks a proline after the phosphorylated serine. Our data support the hypothesis that V5a serves as a phosphorylation-dependent docking site for Pin1. Increased activity of extracellular signal regulated kinase (ERK)1/2 plays a central role in cancer pathology. More recently, the membrane protein Na þ /H þ exchanger 1 (NHE1) has also been assigned important roles in cancer development and chemotherapy resistance. Using a combination of fluore...