PMFs of iASPP-p53 dissociation starting from the crystallography-determined binding mode (PDB 6RZ3), and from the PDB 1YCS binding mode (assuming iASPP and p53 can bind in this mode).

PMFs of iASPP-p53 dissociation starting from the crystallography-determined binding mode (PDB 6RZ3), and from the PDB 1YCS binding mode (assuming iASPP and p53 can bind in this mode).</p

Structural background of p53-ASPP PPI.

(A) Domain organizations of p53 and ASPP proteins. p53’s DBD and ASPP’s ANK-SH3 are folded domains whereas the remaining sequences are intrinsically disordered. (B) Crystal structures of ASPP2-p53 complex (PDB 1YCS [5]) and iASPP-p53 complex (PDB 6RZ3 [6]). Solution NMR studies [4] also suggest a iASPP-p53 binding mode that is different from PDB 6RZ3. (C) In this study, we combined all-atom and coarse-grained (CG) MDs plus free energy calculations to explore the binding mechanisms between three p53 constructs (p53DBD p53P-DBD and p53P-DBD-L) and the ANK-SH3 domains of ASPP2/iASPP.</p

Energetic characterizations of Martini CGMD simulated binding complexes.

(A) PMF curves of the dissociation process between p53DBD and ASPPs, starting from the crystal-structure binding modes. The PMF errors were estimated by the Monte Carlo bootstrapping procedure of the WHAM program. PMF-derived binding free energy (ΔG) was calculated by defining the bound state at RC = 1.5 Å and the unbound state at RC = 14.5 Å. The experimentally measured ΔGs were shown as red dashed lines. (B) Martini CGMD simulated binding between p53DBD and ASPPs. The trajectories are projected onto the distance (COM of p53DBD to COM of ANK-SH3 domain), and the RMSD (with respect to crystal structures) axes. The color scale means frequency of binding. The red shaded areas highlight the binding poses that are close to crystal structures, and those resemble exactly to crystal structures were superimposed onto crystal structures. (C) Overall atom density distribution of p53DBD (cyan) around ASPP (green) in the 3D space. (D) Comparison of experimentally measured ΔGs, calculated ΔGs based on crystal structures, and ΔGs calculated from multiple representative Martini complexes (detailed PMF curves are shown in S7 Fig). t-tests were performed to assess the statistical significance.</p

Effects of p53’s IDRs on p53-ASPP binding.

(A) First contact formation times of ASPPs binding to p53DBD and p53P-DBD-L. For p53P-DBD-L, besides the normal definition of first contact as “any ASPP residue to any p53 residue”, we also counted the first contact defined as “any ASPP residue to residues only from p53 DBD domain” (hatched bars). For ASPP2-p53P-DBD-L binding, an additional set of 50 × 4 μs Martini CGMD was performed in which p53’s IDRs were rigidified by constraints (green bar, using the normal definition of first contact). (B) Average time-dependent number of contacts between ASPP and different p53 domains of p53P-DBD-L calculated from 50 × 4 μs Martini CGMD simulations. The contacts based on p53DBD-ASPP simulations were also shown as gray bars. (C) Projections of Martini CGMD sampled ASPP-p53 complexes onto 2D plane using the sketch-map dimentionality reduction method. We defined “prime” and “secondary” binding modes to refer to the most highly-sampled states, and the less sampled but still have considerable population states (if present), respectively. Representative complexes were shown right to the sketch-map projection.</p

Examining the interactions between p53’s C-terminal linker and iASPP’s RT loop.

(A) Samplings of p53’s C-terminal linker (yellow) and iASPP’s RT loop (red) from 3 ×1 μs all-atom MDs starting from the PDB 6RZ3 binding mode. (B) Starting from PDB 1YCS binding mode. For each case, MD trajectories were concatenated and were aligned on p53’s DBD against the shown respective reference structure. The X and Y Cartesian coordinates of the protein backbone atoms were used for the projection. ASPP and p53 are colored green and cyan, respectively. The gray lines depict the average structures of protein backbone.</p

Assessing the refolding of Martini CGMD sampled ASPP-p53 complexes.

(A) Refolding is gauged by a RMSD metric, namely, starting from the moment when ASPP and p53 first contact, the subsequent complexes’ RMSDs with respect to the first contact complex were calculated. (B-C) Five Martini CGMD trajectories were randomly selected to assess the refolding for ASPP2 and iASPP in the binding of p53DBD, respectively.</p

Effects of Zn²⁺ on simulation results.

(A) Superimposing MD-sampled p53DBD conformations, w/ Zn2+ (blue) and w/o Zn2+ (red), on crystal structure (green). (B) RMSD and RMSF of p53DBD from MD simulations w/ and w/o Zn2+. (C-D) PMF of p53DBD disassociation from ASPP2 and iASPP w/ and w/o Zn2+. Errors bars were drawn in black, and were estimated by the built-in bootstrap error analysis of the WHAM program (num_MC_trials = 100). The relative small errors (∼0.1 kcal/mol) reflect that our 10 ns samplings per window in the umbrella sampling is sufficient. (TIF)</p

20 representative p53_DBD-ASPP2 complexes sampled by Martini CGMD.

The pair-wise RMSDs of ASPP2 (after aligning on p53) were shown on the right to illustrate the structural difference of ASPP2. Procedure for selecting the complexes is: A center of mass (COM) distance criteria (DBD and were subject to following processes: ASPP protein densities around p53DBD were calculated using the grid command from the CPPTRAJ program. Regions have high relative density (> 0.6) were identified as the most probable binding patterns. The COM of ASPP was drawn around p53 for each frame. For those frames their COMs are located within the high density regions, they are identified as candidates. 20 frames were randomly picked out from the candidate pool. (TIF)</p

PMF curves of representative Martini CGMD sampled complexes.

(A) Umbrella sampling using COM-COM distance as CV can lead to protein gliding on another protein’s surface instead of driving complex directly disassociate. We show that during the umbrella sampling, a successful disassociation has inter-protein contacts smoothly disappear as window number increases, indicating a clean one-way disassociation, while for a “gliding” event, newly formed inter-protein contacts are keep emerging as window number increases. (B-C) For the 20 representative Martini CGMD-sampled complexes for each ASPP protein, the PMF curves for successful dissociations (n = 13/20 for ASPP2, and n = 11/20 for iASPP), and for “glidings” are given. (TIF)</p

The intrinsically disordered domains of p53 as predicted by the online-website PONDR [http://pondr.com].

The Pro-domain and linker-domain flanking p53DBD are disordered. (TIF)</p