Coupled intra- and interdomain dynamics support domain cross-talk in Pin1

Abstract

The functional mechanisms of multidomain proteins often exploit interdomain interactions, or "cross-talk." An example is human Pin1, an essential mitotic regulator consisting of a Trp-Trp (WW) domain flexibly tethered to a peptidyl-prolyl isomerase (PPIase) domain, resulting in interdomain interactions important for Pin1 function. Substrate binding to the WW domain alters its transient contacts with the PPIase domain via means that are only partially understood. Accordingly, we have investigated Pin1 interdomain interactions using NMR paramagnetic relaxation enhancement (PRE) and molecular dynamics (MD) simulations. The PREs show that apo-Pin1 samples interdomain contacts beyond the range suggested by previous structural studies. They further show that substrate binding to the WW domain simultaneously alters interdomain separation and the internal conformation of the WW domain. A 4.5-μs all-atom MD simulation of apo-Pin1 suggests that the fluctuations of interdomain distances are correlated with fluctuations of WW domain interresidue contacts involved in substrate binding. Thus, the interdomain/WW domain conformations sampled by apo-Pin1 may already include a range of conformations appropriate for binding Pin1's numerous substrates. The proposed coupling between intra-/interdomain conformational fluctuations is a consequence of the dynamic modular architecture of Pin1. Such modular architecture is common among cell-cycle proteins; thus, the WW-PPIase domain cross-talk mechanisms of Pin1 may be relevant for their mechanisms as well.

Keywords: MD; Pin1; allosteric regulation; multidomain protein; nuclear magnetic resonance (NMR); paramagnetic relaxation enhancement; post-translational modification; post-translational modification (PTM); protein dynamic; protein-protein interaction.

Figure 1

Structural features of human Pin1 (PDB entry1PIN). The N-terminal WW (green) and C-terminal PPIase (gray) domains are shown with secondary structure elements labeled. Orange shading denotes the WW domain Loop 2 (residues 27–29) at the interdomain interface. Residue 27 (orange sphere) is the MTSL (nitroxide spin label) attachment site. Red shading highlights the PPIase domain catalytic loop (residues 64–80) and the WW domain substrate-binding site, Loop 1, and Trp³⁴. Ser¹⁶ (red sphere) is a post-translational phosphorylation site.

Figure 2

Paramagnetic MTSL line broadening in 3m-Pin1. Overlays of ¹H-¹⁵N HSQC spectra with sample conditions as follows. A, apo-WT-Pin1 (black) and apo-3m-Pin1 (green); B, apo-3m-Pin1 (green), apo-DIA 3m-Pin (blue), and apo-PARA 3m-Pin1 (red). Residue cross-peaks disappearing in the PARA sample are annotated with dashed or solid ovals indicating substantial or insubstantial CSPs, respectively, in the DIA sample. C, pCdc25C-bound PARA 3m-Pin1 (dark green) and apo-PARA 3m-Pin1 (red). The cross-peaks for Phe²⁵, Ala³¹, and Ser⁹⁸ reappear in the PARA sample spectrum upon pCdc25C binding. D, pCdc25C-bound DIA 3m-Pin1 (magenta) and apo-DIA 3m-Pin1 (blue).

Figure 3

3m-Pin1 preserves WT dynamic response to pCdc25C binding. Linear correlation of backbone ¹⁵N relaxation rate constants, R₂ − R₁/2, for the apo-state (horizontal) versus the pCdc25C-complex state (vertical) for WT and 3m-Pin1. Turquoise circles, WW domain residues; brown circles, PPIase domain residues. A, WT-Pin1, linear regression: WW domain slope = 0.80, correlation coefficient = 0.99; PPIase domain slope = 0.98, correlation coefficient = 0.99. B, 3m-Pin1, linear regression: WW domain slope = 0.81, correlation coefficient = 0.98; PPIase domain slope = 0.94, correlation coefficient = 0.99. In both 3m-Pin1 and WT-Pin1, pCdc25C binding causes differential changes in domain rotational mobility, indicative of reduced interdomain contact. C, residues with R₂ − R₁/2 deviating significantly from the linear fit localize to the interdomain interface in the 1PIN crystal structure.

Figure 4

PREs of the apo-3m-Pin1.Left, bar graph of the PRE rates of apo-3m-Pin1, Γ₂ (¹H^N) = R_{2, apo-PARA}(¹H^N) − R_2, apo-DIA(¹H^N). Secondary structure motifs are indicated at the top of the bar graph. The threshold value (dashed line) indicates the sum of the trimmed mean and 2 times the S.D. of the filtered Γ₂ (¹H^N) (14.5 rad/s) (see “Experimental procedures”). PPIase domain residues with significant Γ₂ (¹H^N) values: α1 (Glu⁸³, Gln⁹⁴, Ser⁹⁸), α1/α2 turn (Gly⁹⁹, Asp¹⁰²), α2 (Phe¹⁰³), α4 (Gln¹³¹-Lys¹³², Phe¹³⁴-Ala¹³⁷, Ala¹⁴⁰), α4/β6 turn (Leu¹⁴¹, Arg¹⁴²), β6 (Thr¹⁴³–Glu¹⁴⁵, Ser¹⁴⁷-Gly¹⁴⁸, Val¹⁵⁰–Thr¹⁵²), β6/β7 turn (Asp¹⁵³-Ser¹⁵⁴), and β7 (His¹⁵⁷-Ile¹⁵⁹). Right, the red gradient denotes the amplitude of Γ₂ (¹H^N) (PDB entry 1PIN) (15). Black shading, residues lacking Γ₂ (¹H^N) values due to peak overlap or poor signal/noise ratio. The orange sphere is residue 27 (His²⁷ in WT-Pin1), the attachment site for the nitroxide spin label MTSL and its diamagnetic counterpart (acetyl-MTSL). The red, numbered spheres are PPIase domain residues disappearing in the presence of paramagnetic MTSL (apo-PARA sample).

Figure 5

pCdc25C binding increases interdomain separation.A, top left, PRE values, Γ₂(¹H^N) = R_{2, CDC-PARA}(¹H^N) − R_2, CDC-DIA(¹H^N) versus sequence for pCdc25C-complexed 3m-Pin1 with secondary structure elements across the top. The dashed green line indicates the significance threshold of 2 S.D. values above the trimmed mean (13.1 rad/s). Top right, 1PIN structure with red gradient shading indicates the location and relative magnitudes of Γ₂(¹H^N) (15); red spheres denote PPIase domain residues that disappear in apo-PARA Pin1. B, bottom left, changes in Γ₂(¹H^N) caused by pCdc25C binding, ΔΓ₂(¹H^N) = Γ_2,apo(¹H^N) − Γ_2,CDC(¹H^N). The red dashed line indicates the significance threshold of 2 S.D. values beyond the trimmed mean (+5.3 and −6.3 rad/s). Bottom right, 1PIN structure with blue-to-red gradient shading for ΔΓ₂ (¹H^N); blue/red, decreased/increased Γ₂(¹H^N), respectively, in the pCdc25C complexed state. Blue spheres, PPIase domain residues showing the largest reduction of ΔΓ₂(¹H^N) upon pCdc25C binding. Black shading, residues lacking Γ₂(¹H^N) values due to peak overlap or poor signal/noise ratio. Orange sphere, MSTL attachment site at position 27 in the WW domain (His²⁷ in WT-Pin1).

Figure 6

MD simulations suggest multiple interdomain contacts.A–C, fluctuations of diagnostic interdomain distances throughout the 4.5-μs MD trajectory, where WW and PPIase denote the centers of mass of the respective domains. The dashed rectangle in B is enlarged in Fig. 7A (bottom). D, MD snapshots aligned by their PPIase domain (dark gray). The snapshots are configurations with H27Cα, the spin label position, at its closest distance to the Cα of other PPIase domain residues that either vanished in the apo-PARA 3m-Pin1 sample (Ser⁹⁸, Phe¹⁰³, Gly¹⁴⁸, and Phe¹⁵¹) or had the largest measurable Γ₂(¹H^N) (Asp¹³⁶). The configurations are distinguished by WW domains colored as follows: Gly¹⁴⁸ Cα and Phe¹⁵¹ Cα (wheat), Ser⁹⁸ Cα and Phe¹⁰³ Cα (hot pink); Asp¹³⁶ Cα (sand). Also shown (in blue white) is the configuration at 610.6 ns with Phe¹⁰³ closer to His²⁷ Cα than Ser⁹⁸ Cα. Spheres indicate the PPIase residues that showed the most prominent PREs in the apo-PARA 3m-Pin1 sample: Ser⁹⁸ (blue), Phe¹⁰³ (red), Asp¹³⁶ (turquoise), Gly¹⁴⁸ (dark green), and Phe¹⁵¹ (orange). In the WW domain, the yellow spheres indicate Ile²⁸ in Loop 2, whereas marine sticks indicate Arg¹⁷ in Loop 1, the substrate-binding site in the WW domain.

Figure 7

MD of apo-WT-Pin1 captures interdomain conformations supporting PRE changes induced by pCdc25C binding.A, top, time series of distance differences (D_{H27Cα-F103Cα} – D_{H27Cα-S98Cα}). A (bottom), zoom-in view of Fig. 6B showing a trajectory segment where interdomain distance D_{H27Cα-F103Cα} < D_{H27Cα-S98Cα}, which could explain the larger PRE observed for Phe¹⁰³ than Ser⁹⁸ in the pCdc25C-bound Pin1. This suggests that the pCdc25C-bound conformation could preexist as a sparse population in the apo-ensemble. B, an MD snapshot at 610.6 ns (also indicated in A (bottom)) with D_{H27Cα-F103Cα} < D_{H27Cα-S98Cα}.

Figure 8

Correlations between inter- and intradomain distance fluctuations.Left, scatter plot correlating interdomain separation (ρ) with the radius of gyration for the WW domain. Each dot is a snapshot from the apo-Pin1 MD simulation. The horizontal histogram refers to ρ, the distance between the domain centers of mass, schematized by the red arrow on the right. The vertical axis histogram refers to the WW domain radius of gyration and gives a measure of its compactness.

Figure 9

Pairwise intradomain residue contacts that correlate with different interdomain distances.A, blue shading denotes residues engaged in pairwise contacts showing the largest-magnitude correlation coefficients (the top 5%) with the PRE-identified interdomain distances (D_H27Ca-S98Ca, D_H27Ca-D136Ca, D_H27Ca-R142Ca, and D_H27Ca-H157Ca). The top dashed oval denotes PPIase residues important for isomerase activity; the bottom dashed ovals highlight WW domain residues Ser¹⁶ and Trp³⁴ that mediate substrate binding. B, color-coded depiction of contact/distance correlation coefficients. Coefficient magnitudes within the top 5, 10, 20, 30, and 40% are red, pink, orange, yellow, and green, respectively. Thus, red denotes the largest-magnitude correlation, whereas green indicates the lowest. The red shading reveals apparent “passageways” linking the WW domain substrate-binding site and the distal PPIase active site, for each of the four interdomain distances.

Figure 10

Markers of WW domain conformation correlating with interdomain distance. Increased interdomain distances are accompanied by weaker contact of C_R14–Y23 and stronger contacts of C_Y23–Q33, C_Y23–S32, C_S16–Y23, and C_S16–W34.

Figure 11

Response of H-bonds to local conformational changes in the WW domain. The average occupancy H-bonds involving Tyr²³, Glu³⁵, Ser¹⁶, and Trp³⁴ in the two clusters corresponding to the compact (black) and extended (red) form of Pin1.

Figure 12

Backbone NH CSPs from different perturbations to the WW domain.Top, pCdc25C binding to the WW domain; the CSPs reflect pCdc25C-complexed WT-Pin1 versus apo-Pin1. Bottom, S16E substitution to mimic phosphorylated Ser¹⁶; the CSPs reflect apo-S16E-Pin1 versus apo-WT Pin1. NH CSP surges in PPIase regions for interdomain contact are prominent in both cases (dotted rectangles).