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

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 remain 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. 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. Overall design: The C-terminal domain of human MLH1 was divided into seven tiles. In each tile, we generated site saturation mutagenesis libraries comprising all possible missense variants and cloned them into pDONR221. The libraries were subsequently cloned into pDEST-DHFR-PCA for the abundance assay and pDEST22 for the interaction assay and transformed into the wild-type BY4741 yeast strain and the MaV203 yeast strain, respectively. For the abundance assay, cells were plated on medium without uracil and with (selection) or without (control) methotrexate. For the interaction assay, cells were plated on medium with (control) or without (selection) histidine. After selection, cells were harvested and plasmids were isolated to prepare amplicons that was sequenced using the Illumina NextSeq 550 System. Based on the relative change in frequency of variants between the control and selection conditions compared to synonymous wild-type variants, we calcuated an abundance and interaction score for each variant.