Development of novel methodology to determine the functional role of disorder in the FCP1/RAP74 binding interactio

Modern structural biology currently holds the paradigm that structure determines function. Intrinsically disordered proteins are a unique class of proteins that forces the structure/function paradigm to be expanded. IDPs, unlike globular proteins, have no unique secondary or tertiary structure and yet carry out vital biological functions such as transcription, translation, and cellular signal transduction. The unique structural properties associated with IDPs are due to a sequence bias towards charged, hydrophilic amino acids and away from hydrophobic amino acids. Given of this bias, there are little to no hydrophobic amino acids to be removed from the protein/solvent interface to form a hydrophobic core, which is the primary motivation for protein folding. However, IDPs are known to undergo a cooperative folding upon binding process during protein-protein or protein-nucleic acid interactions. Here we will study FCP1, which is one such IDP that gains helical structure upon its interaction with RAP74, a globular protein.;TFIIF-associating RNA Polymerase II C-terminal domain phosphatase (FCP1) is an IDP that plays a vital role in the RNA Polymerase II (RNAPII) transcription cycle. During the elongation cycle of transcription the C-terminal domain (CTD) of RNAPII becomes increasingly phosphorylated and until it becomes dephosphorylated RNAPII cannot undergo another round of transcription. Interaction with the winged helix domain of RAP74, a subdomain of TFIIF, brings FCP1 into contact with CTD so that it may carry out this dephosphorylation. The residues that compose the binding interface of FCP1 have been shown in the literature to adopt an alpha-helical structure while in complex with RAP74. However, there is little information regarding the structure and dynamics of FCP1 in its disordered unbound state and what role that disorder plays in the overall binding mechanism.;13C-direct detected NMR provides an alternative to traditional 1H-detected NMR for the study of IDP structure and dynamics. Due to the low sequence complexity known for IDPs and the low chemical shift dispersion associated with the 1H nucleus, the NMR spectra of IDPs suffer from severe overlap. This property has prevented many IDPs from being structurally and dynamically characterized by NMR. 13C-direct detected NMR, on the other hand, has been shown by our lab and others to have a large chemical shift dispersion. Using 13C direct-detected NMR methods our lab has been able to acquire full chemical shift assignment FCP1 in the unbound state. Here we will show that even in the apo-state of FCP1, the residues that lie within the RAP74 binding interface show nascent helical structure through CD and recently developed 13C direct detected NMR. We will also show that FCP1 remains disordered while in 30% Dextran and HeLa cell extract, representing a cellular environment. Given the highly flexible nature of FCP1 in its disordered state, it is necessary to be able to characterize the local dynamic information of FCP1. We will show through the development of novel 13C-direct detected experiments CON(T1)-IPAP and CON(T2)-IPAP accurate T1 and T2 relaxation times can be measured for FCP1 in both the apo and holo-state. After analysis of the relaxation data, only the residues of FCP1 in the RAP74 binding region experience a change in dynamics. Additional 15N spin relaxation of RAP74, studied in parallel to FCP1, show that RAP74 experiences only a limited amount of ordering upon association with FCP1. In order to determine what role disorder plays in the binding interaction, the FCP1/RAP74 binding interaction was characterized by ITC.;ITC is a powerful method that allows one to determine the thermodynamic parameters that govern a binding interaction. Here we will show that the FCP1/RAP74 binding interaction is dominated by the removal of hydrophobic surface from solvent, characterized by the large, negative DeltaCP associated with complex formation. Additionally, regardless of the entropic penalty associated with FCP1 acquiring structure, the overall interaction is entropically favorable. We will also show through the use of osmolytes to stabilize secondary structure, that pre-formed helical structure in the binding region of FCP1 has a large effect on the overall binding interaction.;To fully understand the behavior of IDPs in solution one must be able to generate a structure. However, IDPs do not exist as a statically folded globular domain, but, instead, exist as an ensemble of rapidly interconverting conformers. The experimental data reported is only reflective of the ensemble average of FCP1 and not of any of the individual conformers that make up its ensemble structure. So, ensemble modeling then becomes the preferred approach to structurally characterize the FCP1 ensemble. we will show that through a combination of SAXS and the ensemble modeling software EOM, flexible-meccano, and ENSEMBLE that the overall ensemble structure is dominated by the by completely disordered N-terminus and that a random coil model, with a locally fixed structural propensity is sufficient to describe it.