A large intrinsically disordered region in SKIP and its disorder-order transition induced by PPIL1 binding revealed by NMR

Abstract

Intrinsically disordered proteins or protein regions play an important role in fundamental biological processes. During spliceosome activation, a large structural rearrangement occurs. The Prp19 complex and related factors are involved in the catalytic activation of the spliceosome. Recent mass spectrometric analyses have shown that Ski interaction protein (SKIP) and peptidylprolyl isomerase-like protein 1 (PPIL1) are Prp19-related factors that constitute the spliceosome B, B*, and C complexes. Here, we report that a highly flexible region of SKIP (SKIPN, residues 59-129) is intrinsically disordered. Upon binding to PPIL1, SKIPN undergoes a disorder-order transition. A highly conserved fragment of SKIP (residues 59-79) called the PPIL1-binding fragment (PBF) was sufficient to bind PPIL1. The structure of PBF.PPIL1 complex, solved by NMR, shows that PBF exhibits an ordered structure and interacts with PPIL1 through electrostatic and hydrophobic interactions. Three subfragments in the PBF (residues 59-67, 68-73, and 74-79) show hook-like backbone structure, and interactions between these subfragments are necessary for PBF.PPIL1 complex formation. PPIL1 is a cyclophilin family protein. It is recruited by SKIP into the spliceosome by a region other than the peptidylprolyl isomerase active site. This enables the active site of PPIL1 to remain open in the complex and still function as a peptidylprolyl cis/trans-isomerase or molecular chaperon to facilitate the folding of other proteins in the spliceosomes. The large disordered region in SKIP provides an interaction platform. Its disorder-order transition, induced by PPIL1 binding, may adapt the requirement for a large structural rearrangement occurred in the activation of spliceosome.

FIGURE 1.

Tricine-SDS-PAGE of proteins. A, lanes 1–9, proteins are PPIL1-(GGGS)₄-PBF, R131A, R131E, R131K, K91D, K91A, K30D, K30A, and wild-type PPIL1. B, lanes 1–6, proteins are protein molecular mass markers (116.0, 66.2, 45.0, 35.0, 25.0, 18.4, and 14.4 kDa, respectively, from top to bottom), L116A, L116D, I136A/V139A, SKIPN, and SKIP172.

FIGURE 2.

A, ¹H-¹⁵N HSQC spectrum of ∼1. 0 mm ¹⁵N/¹³C/²H-labeled SKIPN alone in Buffer A at 25 °C. B, the two-dimensional ¹⁵N,¹H-TROSY spectrum of 0.6 mm ¹⁵N/¹³C/²H-labeled SKIPN in complex with 0.9 mm unlabeled PPIL1 in Buffer A at 25 °C. C, the backbone assignments of free SKIPN (expressed in ∼100% D₂O media).

FIGURE 3.

Characteristics of protein SKIPN in the free state. A, the deviations of the ¹³CA, ¹³CB, ¹³CO, and ¹H^N chemical shifts from random coil values (Δδ) for protein SKIPN. B, far-UV CD spectra of SKIPN from 5 to 95 °C at 5 °C intervals. C, changes in ellipticity at 200, 208, 220, and 275 nm as function of temperature (from 5 to 95 °C at 5 °C intervals) of SKIPN in 50 mm NaCl, 20 mm phosphate buffer, pH 6.5. For far-UV CD spectra, 0.2 mg/ml SKIPN was used. For near-UV CD spectra, 2 mg/ml SKIPN was used. D, the heat capacity of 1 mg/ml SKIPN (gray line) was recorded by nano-DSC at a rate of 1 °C/min. Lysozyme (black line) at 3 mg/ml was used as control.

FIGURE 4.

Backbone dynamics of 0.3 mm¹⁵N-labeled SKIPN alone in Buffer A at 25 °C. T₁ (A), T₂ (B), and ¹⁵N-¹H NOE (C) values of the backbone amide resonances of protein SKIPN are plotted by residue number. The dashed lines represent the average T₁ and T₂ of SKIPN. Residues with overlapping, vanishing cross-peaks, without assignment, or residue Pro (residues 65, 73, and 121) were excluded.

FIGURE 5.

A, ¹H-¹⁵N HSQC spectrum of ∼0. 7 mm ¹⁵N/¹³C-labeled Ala-Met-PBF of SKIP in complex with 0.9 mm unlabeled PPIL1 in Buffer A at 25 °C. B, T₂ values of 0.6 mm ¹⁵N/¹³C/²H-labeled SKIPN in complex with 0.9 mm unlabeled PPIL1 backbone amides in Buffer A at 25 °C. The lower and higher dashed lines represent the average T₂ of PBF (residues 59–79) and another fragment of SKIPN (residues 80–129). Residues with overlapping, vanishing cross-peaks, without assignment, or residue Pro (residues 65, 73, and 121) were excluded.

FIGURE 6.

Structures of PPIL1·PBF. A, best-fit superposition of the backbone atoms of the final 20 NMR structures of PPIL1·PBF. PBF is shown in green, and PPIL1 is shown in gray. Residues 167–182, the linker (GGGS)₄, have been omitted. B, ribbon representation of the energy-minimized average structure of PPIL1·PBF. C, two-dimensional ¹⁵N,¹³C double half-filter NOESY of ¹⁵N/¹³C/²H-labeled PPIL1 (expressed in ∼75% D₂O media) in complex with unlabeled PBF. D, interface of PPIL1·PBF structure. PBF is shown in brown, and the interface in PPIL1 is shown in gray. Interactions involving Met⁷⁶ (E) and Glu⁶⁶ (F) of SKIPN. For E and F, the backbone of PBF is shown in brown, and the backbone of PPIL1 is shown in steel blue. Proton and carbon atoms of the side chain, nitrogen atoms of the Arg and Lys side chains, oxygen atoms of the Asp side chain, and the methyl group of Met⁷⁶ in PBF are shown in gray, blue, red, and yellow, respectively.

FIGURE 7.

Kinetic study the interaction between SKIPN to PPIL1 or its mutants by a BIAcore 3000 system at 25 °C. Protein SKIPN was immobilized on a CM5 sensor chip by amine coupling. Wild-type PPIL1 or its mutants were injected over sensor chip at concentrations of 5.18 μm in phosphate-buffered saline (A). Wild-type PPIL1 (B) and its mutants K30A (C), K30D (D), and K91A (E) at concentrations from 0 to 5.18 μm: 0.08, 0.16, 0.32, 0.65, 1.30, 2.59, and 5.18 μm in phosphate-buffered saline (pH 7.4). Raw binding data were analyzed by BIAevaluation 4.0 and fit to a 1:1 Langmuir binding model. The equilibrium dissociation constants (K_D) are summarized in Table 2.

FIGURE 8.

Sequence alignments of SKIP172 of SKIP. Alignments of SKIP172 sequences from various species, including SKIP/SNW1/NCoA-62 in H. sapiens (NP_036377), Bx42 in D. melanogaster (NP_511093), Prp45p in S. pombe (CAB41231) and S. cerevisiae (P28004), Cwf13/SkiP in A. fumigatus (XP_748038) and N. fischeri (XP_001266227), SNW1 in C. familiaris (XP_531889), D. rerio (AAI33123), E. caballus (XP_001492173), M. musculus (EDL02956), and S. mansoni (ABQ15152), and SKIP in A. aegypti (XP_001648953), B. taurus (NP_001071302), C. elegans (NP_505950), E. multilocularis (CAI59265), M. mulatta (XP_001096395), M. domestica (XP_001367089), O. anatinus (XP_001505367), P. troglodytes (XP_001165674), S. purpuratus (XP_001188665), and X. tropicalis (NP_001017145). The alignments were generated by ClustalW (53) and decorated using ESPript (10). Conserved residues are colored red in blue frames. Lowly conserved residues are in black columns. The black numbers represent the residue number of Cwf13 of A. fumigatus, and the blue numbers represent the residue number of SKIP of H. sapiens. The thick black line represents the PBF of SKIP.

FIGURE 9.

Characteristics of protein SKIP172 in the free state. A, one-dimensional NMR spectrum of SKIP172 (2 mg/ml) in H₂O containing 10% D₂O at 25 °C in Buffer A. B, far-UV CD spectra of SKIP172 (0.2 mg/ml) in 50 mm NaCl, 20 mm phosphate buffer, pH 6.5, from 25 to 95 °C.