Pushing the size limit of de novo structure ensemble prediction guided by sparse SDSL-EPR restraints to 200 residues: The monomeric and homodimeric forms of BAX

Abstract

Structure determination remains a challenge for many biologically important proteins. In particular, proteins that adopt multiple conformations often evade crystallization in all biologically relevant states. Although computational de novo protein folding approaches often sample biologically relevant conformations, the selection of the most accurate model for different functional states remains a formidable challenge, in particular, for proteins with more than about 150 residues. Electron paramagnetic resonance (EPR) spectroscopy can obtain limited structural information for proteins in well-defined biological states and thereby assist in selecting biologically relevant conformations. The present study demonstrates that de novo folding methods are able to accurately sample the folds of 192-residue long soluble monomeric Bcl-2-associated X protein (BAX). The tertiary structures of the monomeric and homodimeric forms of BAX were predicted using the primary structure as well as 25 and 11 EPR distance restraints, respectively. The predicted models were subsequently compared to respective NMR/X-ray structures of BAX. EPR restraints improve the protein-size normalized root-mean-square-deviation (RMSD100) of the most accurate models with respect to the NMR/crystal structure from 5.9 Å to 3.9 Å and from 5.7 Å to 3.3 Å, respectively. Additionally, the model discrimination is improved, which is demonstrated by an improvement of the enrichment from 5% to 15% and from 13% to 21%, respectively.

Structure and Dynamics of Type III Secretion Effector Protein ExoU As determined by SDSL-EPR Spectroscopy in Conjunction with De Novo Protein Folding

“…This approach has been successfully used for de novo prediction of membrane proteins 18 and soluble proteins that exist in multiple relevant states. 17 …”

Conformational Heterogeneity in the Activation Mechanism of Bax

“…Similar to their work, model 8 of the NMR structural ensemble of Bax provided a good fit of the data using MMM2013.2 (Polyhach et al, 2011). In addition, the 25 distances acquired in their study were used as restraints for in silico folding of Bax to the NMR structure (Fischer et al, 2016). …”

The contribution of modern EPR to structural biology

“…Favourable labelling sites are selected by maximizing sequence separation under the constraint that predicted secondary structure elements are pairwise connected [50]. With a moderately sized set of 25 EPR distance distribution restraints, such de novo modelling provided useful results for the soluble form of Bax with 192 residues [51].…”