Disentangling the mutational effects on protein stability and interaction of human MLH1
Sven Larsen-Ledet, Aleksandra Panfilova, Amelie Stein
Abstract
Missense mutations can have diverse effects on proteins, depending on their location within the protein and the specific amino acid substitution. Mutations in the DNA mismatch repair gene MLH1 are associated with Lynch syndrome, yet the underlying mechanism of most disease-causing mutations remains elusive. To address this gap, we aim to disentangle the mutational effects on two essential properties for MLH1 function: protein stability and protein-protein interaction. We systematically examine the cellular abundance and interaction with PMS2 of 4839 (94%) MLH1 variants in the C-terminal domain.
Introduction
Proteins constitute the functional units of the cell and perform essential cellular processes to sustain cell function and health. To obtain biological function, most proteins must fold into a marginally stable native structure, which is largely determined by the amino acid sequence [1]. This makes proteins extremely sensitive to missense mutations that disrupt the structural stability, leading to protein degradation and reduced cellular abundance. Thus, protein stability is fundamental for protein function and, indeed, the most frequent mechanism of disease-causing missense mutations is through destabilization and degradation [2–9].
Materials and method
Plasmids and cloning
The DNA sequence of human wild-type MLH1 (UniProt ID: P40692) and human wild-type PMS2 (UniProt ID: P54278) were codon-optimized for yeast expression and cloned into pDONR221 (Genscript). Missense variants were created by Genscript. For the Y2H assay, entry clones were cloned into pDEST22 (prey) or pDEST32 (bait) from the ProQuest Two-Hybrid System (Invitrogen) using Gateway cloning (Invitrogen). For the DHFR-PCA, entry clones were cloned into pDEST-DHFR-PCA using Gateway cloning (Invitrogen).
Results
Yeast-based assays to measure MLH1 abundance and interaction with PMS2
To disentangle the mutational effects on protein abundance and interaction, we selected the interaction between the human proteins MLH1 and PMS2. MLH1 and PMS2 form the MutLα complex through their C-terminal domains, an essential component of the DNA mismatch repair pathway (Fig 1A).
Discussion
Most proteins have several properties that contribute to their function. Here, we have combined two multiplexed assays of variant effects (MAVEs), one probing stability and another probing interaction, to disentangle which variants affect either or both properties in the C-terminal domain of human MLH1.
Acknowledgments
We acknowledge the use of the sequencing and computing core facilities at the Department of Biology, University of Copenhagen. We thank Vasileios Voutsinos for assistance with Illumina sequencing, Matteo Cagiada for running Rosetta and GEMME predictions, and members of the Linderstrøm-Lang Centre for insightful discussions. pDEST-DHFR-PCA was a gift from Professor Rasmus Hartmann-Petersen (University of Copenhagen).
Citation: Larsen-Ledet S, Panfilova A, Stein A (2025) Disentangling the mutational effects on protein stability and interaction of human MLH1. PLoS Genet 21(4): e1011681. https://doi.org/10.1371/journal.pgen.1011681
Editor: John Prensner, University of Michigan, UNITED STATES OF AMERICA
Received: October 30, 2024; Accepted: April 8, 2025; Published: April 28, 2025
Copyright: © 2025 Larsen-Ledet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data generated are included in the figures and supplemental files. Sequencing reads are available at the NCBI Gene Expression Omnibus (GEO) repository (accession number: GSE273652). Processed cellular abundance and PMS2 interaction scores are available in MaveDB (accession number: urn:mavedb:00001218-a). Data and code are available at GitHub (https://github.com/KULL-Centre/_2024_Larsen-Ledet_MLH1).
Funding: The present work was supported by grants from the Lundbeck Foundation (R272-2017–452, to A.S.) and the Novo Nordisk Foundation challenge program PRISM (NNF18OC0033950, to A.S.). S.L-L. and A.P. received salary from R272-2017–452. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
