FBP21's C-Terminal Domain Remains Dynamic When Wrapped around the c-Sec63 Unit of Brr2 Helicase

Abstract

Based on our recent finding that FBP21 regulates human Brr2 helicase activity involved in the activation of the spliceosomal B-complex, we investigated the structural and dynamic contribution of FBP21 to the interaction. By using NMR spectroscopy, we could show that the 50 C-terminal residues of FBP21 (FBP21^326-376), which are sufficient to fully form the interaction with the C-terminal Sec63 unit of Brr2 (Brr2^C-Sec63), adopt a random-coil conformation in their unbound state. Upon interaction with Brr2^C-Sec63, 42 residues of FBP21^326-376 cover the large binding site on Brr2^C-Sec63 in an extended conformation. Short charged motifs are steering complex formation, still allowing the bound state to retain dynamics. Based on fragment docking in combination with experimental restraints, we present models of the complex structure. The FBP21^326-376/Brr2^C-Sec63 interaction thus presents an example of an intrinsically disordered protein/ordered-protein interaction in which a large binding site provides high specificity and, in combination with conformational disorder, displays a relatively high affinity.

Figure 1

Backbone assignment of free and Brr2^C-Sec63-bound FBP21^326–376. (A) Shown is the assigned ¹H-¹⁵N-TROSY-HSQC of 140 μM ²H-¹⁵N-¹³C-FBP21^326–376 with low signal dispersion, indicating intrinsic disorder. (B) An overlay of ¹H-¹⁵N-TROSY-HSQC spectrum of 140 μM FBP21^326–376 alone and in complex with a threefold molar excess of Brr2^C-Sec63. Assigned peaks deriving from FBP21^326–376 in the Brr2^C-Sec63-bound state are labeled in gray. To see this figure in color, go online.

Figure 2

Secondary structure analysis of free and Brr2^C-Sec63-bound FBP21^326–376. (A) Cα secondary chemical shifts are shown for free and Brr2^C-Sec63-bound FBP21^326–376. Fluctuation around zero indicates no defined secondary structure in free FBP21^326–376. In the Brr2^C-Sec63-bound state, increased negative values indicate regions adopting an extended conformation. (B) The secondary structure was predicted for free and Brr2^C-Sec63-bound FBP21^326–376 by using the web tool CSI3.0 (14). Free FBP21^326–376 is predicted to be disordered (CSI3.0 free), whereas the Brr2^C-Sec63-bound state appears to form regions with extended conformation (CSI3.0 bound, light B: interior β-strand, dark B: edge β-strand). Additionally shown are peptide fragments of FBP21^326–376 investigated here and/or previously (9). Peptides only used for docking analyses are colored as in Fig. S5, with lysine residues in black indicating the positions of restraints derived from cross-linking and residues in bold indicating the position of restraints derived from NOEs. (C) The secondary structure propensity score (ncSPC score: value 1 corresponds to 100% α-helix, value −1 corresponds to 100% β-strand) was calculated for free and Brr2^C-Sec63-bound FBP21^326–376 by using the web tool ncSPC (20). To see this figure in color, go online.

Figure 3

Differences in fold and dynamics between free and Brr2^C-Sec63-bound FBP21^326–376. (A) Plotted are per-residue combined ¹H,¹⁵N chemical shift differences between the two spectra shown in Fig. 1B. The black line indicates the average chemical shift difference (0.27 ppm). (B) Strips of a ¹H-¹⁵N-NOESY-HSQC spectrum of 140 μM ²H-¹⁵N-¹³C-FBP21^326–376 in the presence of 420 μM Brr2^C-Sec63 show H^N-H^N-NOEs. (C) Heteronuclear ¹H-¹⁵N-NOE values and T₁/T₂ relaxation time ratios obtained for 125 μM ¹⁵N-FBP21^326–376 without or with a threefold molar excess of unlabeled Brr2^C-Sec63. Heteronuclear ¹H-¹⁵N-NOE values represent the average of three independent measurements, error bars reflect the standard deviation. Uncertainties of T₁ and T₂ times were obtained from exponential fits. To see this figure in color, go online.

Figure 4

Energy-minimized models generated from docking results (Fig. S6C). (A) Models of FBP21^326–376 are shown with Brr2^C-Sec63 in surface representation. Residues showing chemical shift differences upon addition of FBP21^326–376 (9) are shown in strong (if bigger than mean + 1 SD) or light (if between mean and mean + 1 SD) colors. Chemical shift differences are induced by N-terminal residues (that is upon interaction with FBP21^326–376 but not with N-terminally truncated fragments) by core residues (that is upon interaction with FBP21^326–376, FBP21^346–365, and FBP21^351–370) or by C-terminal residues (that is upon interaction with FBP21^326–376 or FBP21^351–370). (B–E) PRE experiments confirm the models. (B) and (C) show regions of the overlay of ¹H-¹⁵N-TROSY-HSQC spectra of 100 μM ¹⁵N-Brr2^C-Sec63 in complex with equimolar amounts of (B) FBP21^S344C-MTSL and (C) FBP21^S366C-MTSL in its paramagnetic and its diamagnetic states. (D) and (E) illustrate the position of the label in the models (arrows) of FBP21^326–376 and regions experiencing relaxation enhancement in Brr2^C-Sec63 in surface representation. Indicated is a loss in peak intensity beyond the average minus SD (67%) or beyond 25% in light or dark colors respectively. (F–I) Mutations introduced in FBP21 confirm the models. (F) and (G) show regions of the overlay of ¹H-¹⁵N-TROSY-HSQC spectra of 100 μM ¹⁵N-Brr2^C-Sec63 free in complex with a twofold molar excess of FBP21^326–376 and a twofold molar excess of (F) FBP21^GGG or (G) FBP21^ANLA. (H) and (I) show Brr2^C-Sec63 for which chemical shift differences upon complex formation with FBP21^GGG (H) or FBP21^ANLA (I) drops beyond the significance cutoff in the presence of the mutations. The position of the mutations in FBP21^326–376 are indicated with an arrow. PDB: 4f91 (23) is used to present Brr2^C-Sec63 with models of FBP21^326–376. To see this figure in color, go online.