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Intrinsically disordered protein regions (IDRs) partially or completely lack a cooperatively folded structure under native conditions, preventing their equilibrium state from being adequately described by a single structural model. As a direct consequence of their disorder, remarkably few experimental studies have quantified the ensembles IDRs adopt in solution. Here, we conduct unbiased computer simulations of the RAP74 interaction motif from the human phosphatase FCP1 in the unbound state, which provides an ensemble in quantitative agreement with both experimental NMR chemical shift information and small-angle X-ray scattering data. The partially α-helical short linear motif found in the C-terminus of FCP1 has been the subject of extensive biophysical characterization aimed at developing a molecular description for the mechanism of coupled folding and binding and establishing the functional relevance of partial order in the unbound state. The analysis presented here yields a remarkably consistent molecular picture enumerating the diversity of structures present in a "partially formed" helix. Specific interactions, including anticorrelations in backbone dihedral angle fluctuations as well as the transient formation of a helix-stabilizing salt bridge, stabilize the preformed structure in the unbound state. The general consequences of these findings for mechanistic analysis of protein-protein interactions are discussed.

By dephosphorylating the C-terminal domain (CTD) of RNA polymerase II (Pol II), the Transcription Factor IIF (TFIIF)-associating CTD phosphatase (FCP1) performs an essential function in recycling Pol II for subsequent rounds of transcription. The interaction between FCP1 and TFIIF is mediated by the disordered C-terminal tail of FCP1, which folds to form an α-helix upon binding the RAP74 subunit of TFIIF. The present work reports a structure-based simulation study of this interaction between the folded winged-helix domain of RAP74 and the disordered C-terminal tail of FCP1. The comparison of measured and simulated chemical shifts suggests that the FCP1 peptide samples 40-60% of its native helical structure in the unbound disordered ensemble. Free energy calculations suggest that productive binding begins when RAP74 makes hydrophobic contacts with the C-terminal region of the FCP1 peptide. The FCP1 peptide then folds into an amphipathic helix by zipping up the binding interface. The relative plasticity of FCP1 results in a more cooperative binding mechanism, allows for a greater diversity of pathways leading to the bound complex, and may also eliminate the need for "backtracking" from contacts that form out of sequence.

We report atomically detailed molecular dynamics simulations characterizing the interaction of the RAP74 winged helix domain with the intrinsically disordered C-terminal of FCP1. The RAP74-FCP1 complex promotes the essential dephosphorylation of RNA polymerase II prior to initiation of transcription. Although disordered in solution, the C-terminal of FCP1 forms an amphipathic helix when bound to RAP74. Our simulations demonstrate that this interaction also reorganizes and stabilizes RAP74. These simulations illuminate the significance of hydrophobic contacts for stabilizing disordered protein complexes, provide new insight into the mechanism of protein binding by winged helix domains, and also reveal "dynamic fuzziness" in the complex as FCP1 retains significant flexibility after binding. In conjunction with our recent NMR experiments identifying residual structure in unbound FCP1, these simulations suggest that FCP1 loses relatively little conformational entropy upon binding and that the associated coupled folding-binding transition may be less sharp than expected., (© 2011 American Chemical Society)

Intrinsically disordered proteins (IDPs) lack unique 3D structures under native conditions and yet retain critical functions. Recycling of RNA Polymerase II after transcription is promoted by an interaction between the winged helix domain of RAP74, a component of the general transcription factor IIF (TFIIF), and the C-terminus of the TFIIF-associating CTD phosphatase (FCP1). Sixteen residues from the C-terminus of FCP1 form an α-helix in the complex, but the protein is otherwise agreed in the literature to be intrinsically disordered. Here we show through CD and recently developed carbon-detected NMR that, although FCP1 is intrinsically disordered, the above 16 residues composing the RAP74 binding surface form nascent α-helical structure in the unbound state. We further show retention of general FCP1 disorder and the nascent helical content in HeLa extract, establishing cellular relevance. The conformational bias observed leads to a mechanistic proposal for FCP1's transition from a disordered ensemble to an ordered conformation upon binding., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The phosphorylation state of the RNA polymerase II C-terminal repeat domain (CTD) regulates progression through the mRNA biogenesis cycle. Termination of transcription and recycling of RNA polymerase II is promoted by an interaction between the general transcription factor IIF (TFIIF) and the TFIIF-associating CTD phosphatase (FCP1). The acidic C-terminal region of FCP1 is disordered in the free state, but adopts an alpha-helical conformation upon binding to the heavy chain of TFIIF. Here we report (1)H, (13)C, and (15)N resonance assignments for the intrinsically disordered unbound form of human C-terminal FCP1 (residues 879-961). The use of recently developed (13)C direct detected "protonless" NMR experiments allowed the nearly complete assignment of FCP1 reported here and is likely to be a generally effective strategy for the chemical shift assignment of disordered proteins.