# Reconciling ASPP-p53 binding mode discrepancies through an ensemble binding framework that bridges crystallography and NMR data

## TL;DR

This paper resolves conflicting data on how ASPP proteins bind to p53 by using simulations to show that multiple binding modes exist dynamically.

## Contribution

The study introduces an ensemble binding framework that reconciles NMR and crystallography data for p53-ASPP interactions.

## Key findings

- Ensemble simulations reveal that iASPP-p53 interactions include a verified RT loop engagement not seen in single crystal structures.
- NMR and crystallography binding modes are both valid and represent different states in a dynamic ensemble.
- p53's disordered regions influence binding and are essential for biological function.

## Abstract

ASPP2 and iASPP bind to p53 through their conserved ANK-SH3 domains to respectively promote and inhibit p53-dependent cell apoptosis. While crystallography has indicated that these two proteins employ distinct surfaces of their ANK-SH3 domains to bind to p53, solution NMR data has suggested similar surfaces. In this study, we employed multi-scale molecular dynamics (MD) simulations combined with free energy calculations to reconcile the discrepancy in the binding modes. We demonstrated that the binding mode based solely on a single crystal structure does not enable iASPP’s RT loop to engage with p53’s C-terminal linker—a verified interaction. Instead, an ensemble of simulated iASPP-p53 complexes facilitates this interaction. We showed that the ensemble-average inter-protein contacting residues and NMR-detected interfacial residues qualitatively overlap on ASPP proteins, and the ensemble-average binding free energies better match experimental KD values compared to single crystallgarphy-determined binding mode. For iASPP, the sampled ensemble complexes can be grouped into two classes, resembling the binding modes determined by crystallography and solution NMR. We thus propose that crystal packing shifts the equilibrium of binding modes towards the crystallography-determined one. Lastly, we showed that the ensemble binding complexes are sensitive to p53’s intrinsically disordered regions (IDRs), attesting to experimental observations that these IDRs contribute to biological functions. Our results provide a dynamic and ensemble perspective for scrutinizing these important cancer-related protein-protein interactions (PPIs).

Solution NMR and crystallography often yield disparate results when determining the biologically relevant binding modes of protein-protein interactions. Through computational modeling, we successfully reconciled the discrepancies in binding modes reported by NMR and crystallography for the tumor suppressor protein p53 and its regulators, ASPP2 and iASPP. We demonstrated that the controversial binding modes determined by NMR and crystallography for iASPP-p53 are, in fact, the two major binding modes captured in free protein-protein binding simulations. Therefore, both reported binding modes are plausible, and crystal packing influences the preference for the mode captured in crystallography. Our results underscore the concept that proteins function through dynamic ensembles, and under physiological conditions, protein-protein interactions can have more than one binding mode. Using this ensemble binding framework, we explore the roles of disordered regions of p53 in complex formation, demonstrating how the overlooked IDRs can fine-tune protein-protein interactions.