MutateX: an automated pipeline for in silico saturation mutagenesis of protein structures and structural ensembles

Abstract

Mutations, which result in amino acid substitutions, influence the stability of proteins and their binding to biomolecules. A molecular understanding of the effects of protein mutations is both of biotechnological and medical relevance. Empirical free energy functions that quickly estimate the free energy change upon mutation (ΔΔG) can be exploited for systematic screenings of proteins and protein complexes. In silico saturation mutagenesis can guide the design of new experiments or rationalize the consequences of known mutations. Often software such as FoldX, while fast and reliable, lack the necessary automation features to apply them in a high-throughput manner. We introduce MutateX, a software to automate the prediction of ΔΔGs associated with the systematic mutation of each residue within a protein, or protein complex to all other possible residue types, using the FoldX energy function. MutateX also supports ΔΔG calculations over protein ensembles, upon post-translational modifications and in multimeric assemblies. At the heart of MutateX lies an automated pipeline engine that handles input preparation, parallelization and outputs publication-ready figures. We illustrate the MutateX protocol applied to different case studies. The results of the high-throughput scan provided by our tools can help in different applications, such as the analysis of disease-associated mutations, to complement experimental deep mutational scans, or assist the design of variants for industrial applications. MutateX is a collection of Python tools that relies on open-source libraries. It is available free of charge under the GNU General Public License from https://github.com/ELELAB/mutatex.

The Cancermuts software package for the prioritization of missense cancer variants: a case study of AMBRA1 in melanoma

“…We have used an in silico mutational scan based on MutateX [ 46 ] and the FoldX energy function [ 47 ] to investigate whether AMBRA1 mutations validated for this study were likely to affect the stability of the β-propeller domain (Fig. 6 ).…”

Directed Evolution of (R)-2-Hydroxyglutarate Dehydrogenase Improves 2-Oxoadipate Reduction by 2 Orders of Magnitude

“…In addition, we included residues proposed to be involved in the binding of 2-oxoglutarate. 19 We complemented the FuncLib scans with in silico saturation mutagenesis using MutateX, an automated pipeline 22 based on the FoldX energy function. 21 It has been shown that FoldX is effective in predicting destabilizing mutations in proteins.…”

“…We employed the FoldX energy function 21 to perform in silico saturation mutagenesis using MutateX, an automated pipeline that we recently developed. 22 Thus, we employed the same protocol we had applied to other proteins 29 , 30 to estimate the changes caused by the selected mutations on the structural stability of Hgdh. To this end, we performed the calculations on a monomer derived from the X-ray structure of Hgdh (PDB ID 1XDW , monomer in chain A).…”

“…Protein engineering strategies and workflow employed in the present study. A computational analysis combining the evolution-guided approach of FuncLib and in silico high-throughput saturation mutagenesis , was carried out to design mutant variants that could improve 2-oxoadipate activity. Seven residues were targeted for saturation mutagenesis.…”

Epistasis between promoter activity and coding mutations shapes gene evolvability

“…-Biophysical effects of mutations: FoldX version 5.0 (56, 57) was used with the MutateX wrapper (58) to simulate all possible mutations on the biological assembly of DfrB1 (PDB: 2RK1).…”