Disentangling the mutational effects on protein stability and interaction of human MLH1

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. Our combined data shows that most MLH1 variants lose interaction with PMS2 due to reduced cellular abundance. However, substitutions to charged residues in the canonical interface lead to reduced interaction with PMS2. Unexpectedly, we also identify a distal region in the C-terminal domain of MLH1 where substitutions cause both decreased and increased binding with PMS2, and propose a region in PMS2 as the binding site. Our data successfully distinguish benign from pathogenic MLH1 variants and correlate with thermodynamic stability predictions and evolutionary conservation. This work provides mechanistic insights into variant consequences and may help interpret MLH1 variants. Author SummaryMutations in proteins, including single amino acid substitutions, can disrupt normal cellular function and lead to diseases. Hence, there is great interest in understanding their consequences from both basic science and clinical genetics perspectives. Loss-of-function variants in the human mismatch repair protein MLH1 are associated with Lynch syndrome, a hereditary condition that significantly increases the risk of certain cancers. Detailed insights into the molecular consequences of MLH1 variants are essential for understanding the mechanisms underlying Lynch syndrome. In this study, we use high-throughput experimental approaches to test how missense mutations affect the cellular abundance of MLH1 and the interaction with its partner, PMS2. We find that many variants disrupting the interaction with PMS2 also lead to reduced cellular abundance levels, suggesting a strong relationship between protein stability and interaction in MLH1. Additionally, we identify a potential novel interaction site between MLH1 and PMS2, where mutations in MLH1 either increase or decrease interaction without affecting abundance. Our datasets are in line with the classification of clinically annotated MLH1 variants and may aid in interpreting variants of uncertain significance, while also providing new insights into the molecular details of the interaction with PMS2.