The functional dynamics of FicD's TPR domain are modulated by the interaction with ATP and BiP

Abstract

The human Fic enzyme FicD plays an important role in regulating the Hsp70 homolog BiP in the endoplasmic reticulum: FicD reversibly modulates BiP's activity through attaching an adenosine monophosphate to the substrate binding domain. This reduces BiP's chaperone activity by shifting it into a conformation with reduced substrate affinity. Crystal structures of FicD in the apo, adenosine triphosphate (ATP)-bound, and BiP-bound states suggested significant conformational variability in the tetratricopeptide repeat (TPR) motifs. However, nothing is known about the underlying dynamics. In this study, we investigate the conformational dynamics of FicD's TPR motifs using two-color, single-molecule Förster resonance energy transfer (smFRET) experiments. We demonstrate that the TPR motifs exhibit conformational dynamics between a TPR-out and a TPR-in conformation on timescales ranging from microseconds to milliseconds. In addition, we extend our investigation on multiple labeling positions within FicD, revealing how conformational dynamics vary depending on the location within the TPR motif. We quantify the motions with dynamic photon distribution analysis for the FRET constructs and generate an ensemble of structures for the different states consistent with the smFRET data using molecular dynamic simulations. We propose a conformational landscape model for FicD where the TPR-in/out states exist in equilibrium and the fraction of dynamic population is altered due to the presence of ATP and BiP. These results indicate that not only is FicD dynamic, but the dynamics are linked to the functionality and interactions of FicD with BiP.

Keywords: AMPylation; BiP; FicD; Hsp70; single-molecule FRET.

Fig. 1.

The structure and conformations of FicD. (A) Domain organization of FicD. The construct used in this study is highlighted by the dotted frame (amino acids 102–458). TM: transmembrane-domain, TPR: tetratricopeptide repeat motif, Fic: filamentation induced by cyclic-AMP domain. (B) Superimposed representations of the monomeric FicD^L258D crystal structures in the apo-state [light blue, PDB 6I7J (21)], bound to ATP (yellow, PDB 6I7K (21), stick representation: ATP, green sphere: Mg²⁺ ion), and in a covalently linked complex (clc) with BiP (light green, PDB 6ZMD (24), surface representation in light gray: BiP). The smFRET labeling positions R118 (magenta) and S288 (teal) are shown as spheres on the Cα-atoms of the amino acid residues.

Fig. 2.

The conformational equilibrium of FicD in solution. (A) SmFRET efficiency histograms of FicD^R118C alone (light blue) and in the presence of ATP (yellow). (B and C) 2D plots of donor fluorescence lifetime in the presence of an acceptor (${\tau }_{D(A)}$) vs FRET efficiency (E) for (B) FicD^R118C:apo and (C) FicD^R118C in the presence of ATP. Black and red lines represent the static and dynamic FRET lines, respectively. (D and E) Accessible volume (AV) calculations of (D) FicD in the ATP-bound state [yellow, PDB 6I7K (21)] and (E) in the apo-state [light blue, PDB 6I7J (21)]. The magenta (donor) and teal (acceptor) surfaces represent the possible spatial distribution of the fluorophores whereas the spheres indicate the mean positions of the donor and acceptor dyes. Black lines indicate the distances between both mean positions (R_DA).

Fig. 3.

Binding to BiP alters the conformational equilibrium of FicD^R118C. (A and B) SmFRET efficiency histograms of (A) FicD:apo (light blue) in comparison to FicD in the presence of BiP (blue) and BiP_AMP (black) and (B) FicD:ATP (yellow) in comparison to FicD:ATP in the presence of BiP (orange) and BiP_AMP (brown). (C and D) 2D plots of donor fluorescence lifetime in the presence of an acceptor (${\tau }_{D(A)}$) vs FRET efficiency (E) for (C) FicD:BiP and (D) FicD:ATP:BiP. Black and red lines represent the static and dynamic FRET lines, respectively. (E and F) 2D plots of donor fluorescence lifetime in the presence of an acceptor (${\tau }_{D(A)}$) vs FRET efficiency (E) for (E) FicD:BiP_AMP and (F) FicD:ATP:BiP_AMP. Black and red lines represent the static and dynamic FRET lines, respectively.

Fig. 4.

Both TPR motifs of FicD are involved in conformational switching. (A) Superimposed representations of monomeric FicD^L258D crystal structures in the apo-state [light blue, PDB 6I7J (21)] and bound to ATP (yellow, PDB 6I7K (21), stick representation ATP, green sphere: Mg²⁺ ion). The various smFRET labeling positions within the TPR domain (magenta) and the Fic domain S288 (teal) are indicated by spheres on the Cα-atoms of the amino acid residues. (B) SmFRET efficiency histograms of FicD^L104C:apo (light blue) in comparison to FicD^L104C in the presence of BiP (blue) and BiP_AMP (black). (C) SmFRET efficiency histograms of FicD^L104C:ATP (yellow) in comparison to FicD^L104C:ATP in the presence of BiP (orange) and BiP_AMP (brown). (D) SmFRET efficiency histograms of FicD^K154C:apo (light blue) in comparison to FicD^K154C in the presence of BiP (blue) and BiP_AMP (black). (E) SmFRET efficiency histograms of FicD^K154C:ATP (yellow) in comparison to FicD^K154C:ATP in the presence of BiP (orange) and BiP_AMP (brown). (F and G) 2D plots of donor fluorescence lifetime in the presence of an acceptor (${\tau }_{D(A)}$) vs FRET efficiency (E) for (F) FicD^K154C:apo and (G) FicD^K154C:ATP. Black and red lines represent the static and dynamic FRET lines, respectively.

Fig. 5.

Analysis of the conformational states and dynamics of FicD^R118C using the dynamic photon distribution analysis (PDA) and MD simulations. (A) Schematic of the PDA model used for fitting the data with two static (purple: donor only, green: static TPR-in) and two dynamic (blue: dynamic TPR-in, orange: dynamic TPR-out) conformational states that were incorporated into a global PDA fit model for FicD^R118C. The contribution of the dynamically interconverting population to the smFRET histogram is shown in yellow. The purple donor-only population was necessary to describe the bins where the acceptor is blinking or photobleached. (B and C) For dynamic PDA, uncorrected FRET efficiency (proximity ratio) histograms with time binnings of 0.5 ms, 0.75 ms, and 1.0 ms were used to calculate the distance distribution for different FRET populations and dynamics between them. The proximity ratio histograms for the time window size of 0.75 ms is shown for FicD^R118C (B) in the apo-state and (C) in the presence of ATP. The proximity ratio histograms were fitted using the 2-state dynamic PDA model that includes the TPR-in (blue) and TPR-out (orange) states and the dynamically interconverting population (yellow) between them. The fits also included the static TPR-in state (green) and additional low-FRET population (purple) to account for impurities. Results from the PDA fits are given in SI Appendix, Table S21. (D) Comparison of the ratio k_out/k_in obtained from PDA of monomeric (L258D) or dimeric FicD labeled at R118C and S288C. (E) RMSD plot of the peptide backbones of the TPR domain and interdomain helix linker for the 4 µs of each MD trajectory (4 replicas of 1 µs each): FicD:apo (light blue), FicD:ATP (yellow), FicD:BiP (light green) and FicD:BiP simulated without BiP (asterisk, dark green). Each simulation is aligned on the Fic domain backbone to highlight TPR fluctuations with respect to the first frame of the respective MD trajectory as a reference structure.

Fig. 6.

The conformational equilibrium of dFicD^R118C. (A) SmFRET efficiency histograms of dFicD^R118C:apo (light blue) in comparison to dFicD^R118C in the presence of BiP (blue) and BiP_AMP (black). (B and C) 2D plots of donor fluorescence lifetime in the presence of an acceptor (${\tau }_{D(A)}$) vs FRET efficiency (E) for (B) dFicD^R118C:BiP_AMP and (C) inactive dFicD^{R118C H363A}:BiP_AMP. Black and red lines represent the static and dynamic FRET lines, respectively. (D) SmFRET efficiency histograms of dFicD^R118C:ATP (yellow) in comparison to dFicD^R118C:ATP in the presence of BiP (orange) and BiP_AMP (brown). (E and F) 2D plots of donor fluorescence lifetime in the presence of an acceptor (${\tau }_{D(A)}$) vs FRET efficiency (E) for (E) dFicD^R118C:ATP:BiP_AMPand (F) inactive dFicD^{R118C H363A}:ATP:BiP_AMP. Black and red lines represent the static and dynamic FRET lines, respectively.

Fig. 7.

Dynamics of the TPR domain of FicD. A schematic representation of the proposed conformational landscape of FicD according to our smFRET data. (A) The static TPR-in conformation (green) as well as the dynamically interconverting TPR-in (light blue) and TPR-out (yellow) states of FicD exist in equilibrium for monomeric and dimeric FicD. The full structure of dimeric FicD is shown in the inset (one protein in green, the other in wheat). While the presence of BiP promotes the static TPR-in state, binding to ATP favors the dynamically interconverting states and furthermore enhances the transition to the TPR-out state (k_out). Furthermore, dimeric FicD showed larger fractions of dynamic molecules compared to monomeric FicD. No transitions between the dynamic and static TPR-in conformation have been observed in the smFRET burst experiments, indicating a timescale much larger than the burst duration (transition rates << 0.1 ms⁻¹). The interconversions between the dynamic states (k_out and k_in) have been observed in a range of 0.75 to 2.84 ms⁻¹ for FicD^R118C. (B and C) The dynamics of FicD are linked to catalysis. While noncatalytic enzyme–target complexes (e.g. monomeric FicD or inactive FicD^H363A interacting with BiP_AMP) are mainly static and k_in is predominant (B), catalytic complexes (e.g. during deAMPylation of BiP_AMP by dimeric FicD) exist in all three conformational states of FicD with similar likelihood (C). Note that binding of ATP to FicD enhances dynamics irrespective of catalysis. Transparency of the TPR domain in B and C indicates the relative population of the conformational states (lighter colors are less populated). FicD is shown as colored cartoon representation [PDB 6I7J, 6I7K (21), BiP as a gray surface (PDB 6ZMD (24)].