The Disordered C-Terminus of the RNA Polymerase II Phosphatase FCP1 is Partially Helical in the Unbound State.
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 form the C-terminus of FCP1 form an alpha helix in the complex, but the protein is otherwise agreed in the literature to be intrinsically disordered. Here we show thorough CD and recently developed carbon-detected NMR that, although FCP1 is intrinsically disordered, the above 16 residues composing the RAP74 binding surface form nascent alpha 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.
The 15N,13C-CON spectra of apo-ctFCP1 (red) and the ctFCP1-RAP74 complex (black) are overlaid and zoomed to highlight the resonances from the RAP74 interaction region (D947-M961; labeled in the bound state). While the movement of peaks in this region is significant, no other changes beyond the linewidth of the peaks are observed.
We have previously shown that carbon-detected NMR is a very powerful method for studying IDPs. Figure 1 clearly shows both the resolving power of the method and the ease with which protein-protein interactions can be detected, even when IDPs are involved. When extended to a third dimension, complete chemical shift assignment becomes tractable, allowing the application of standard chemical shift indexing methods. This analysis shows that, although minimal, there is a native tendency towards helical structure in the core of the RAP74 binding region of apo-ctFCP1 (Figure 2).
Chemical shift index (CSI) analysis of ctFCP1. Analysis of 13C-alpha, 13C-carbonyl, and 13C-beta chemical shifts provides a consensus indication of secondary structure in ctFCP1 that suggests helix nucleation in the RAP74 binding region may occur in the unbound state. Chemical shifts consistent with alpha helix are shown with black bars, whereas those consistent with extended or beta strand structure are shown in gray.
To further validate our findings, we have measured circular dichroism (CD) spectra of ctFCP1 in aqueous solution and in the presence of increasing concentrations of TFE, which has previously been shown to stabilize alpha helical conformations in proteins. These results show both for ctFCP1 and for a shorter synthetic peptide composed only of the RAP74 binding region that there is minimal alpha helical character to the aqueous state and that it is strengthened through additon of TFE.
TFE-dependent conformational transitions in ctFCP1 monitored by circular dichroism CD spectra of (A) FCP1 (944-961) peptide and (B) ctFCP1 (879-961) in 0% (black), 10% (orange), 20% (green), 30% (blue), and 40% (purple) TFE (v/v). Increasingly negative CD signal with a minimum near 222 nm is indicative of increased alpha helical structure as the TFE volume percent increases.
With the knowledge that TFE can be used to control the extent of helicity in apo-ctFCP1 we next attempted to demonstrate that pre-formed helical conformations could be melted out through the addition of a chaeotrope. Figure 4 displays the change in carbonyl chemical shift with addition of either TFE or urea and the anticorrelated shifts induced by these two co-solutes are consistent with reinforcement and melting out of nascent helical structure, respectively (Figure 4A). There is some interest within the IDP field in demonstrating that disordered states are not in vitro artifacts. To this end, we also show the change in chemical shift upon addition of 30% (w/v) dextran and 70 m/mL HeLa cell extract, neither of which induce significant changes in chemical shift. We therefore conclude that crowding does not induce a significant change in state and that our in vitro conditions provide a picture of nascent helicity in FCP1 that is cellularly relevant.
Structural response of ctFCP1 to co-solute addition. (A) Per-residue ctFCP1 13CO chemical shift changes during TFE titration (progressing in a rainbow from 5%-40% in increments of 5%) indicate increasing helicity. The response to urea titration (increasing from 0.5-3.0 M as the gray darkens) indicates decreasing helicity. (B) 30% (w/v) dextran or (C) 70 mg/mL HeLa extract induce no significant chemical shift changes.
For experimental details, in depth analysis, and full resolution images, please see:
Lawrence, C.W., Bonny, A., & Showalter, S.A. (2011) "The Disordered C-Terminus of the RNA Polymerase II Phosphatase FCP1 is Partially Helical in the Unbound State." Biochem. Biophys. Res. Comm., 410, 461-465.