Deep indel mutagenesis reveals the impact of amino acid insertions and deletions on protein stability and function

Abstract

Amino acid insertions and deletions (indels) are an abundant class of genetic variants. However, compared to substitutions, the effects of indels are not well understood and poorly predicted. Here we address this shortcoming by performing deep indel mutagenesis (DIM) of structurally diverse proteins. Indel tolerance is strikingly different to substitution tolerance and varies extensively both between different proteins and within different regions of the same protein. Although state of the art variant effect predictors perform poorly on indels, we show that both experimentally-measured and computationally-predicted substitution scores can be repurposed as good indel variant effect predictors by incorporating information on protein secondary structures. Quantifying the effects of indels on protein-protein interactions reveals that insertions can be an important class of gain-of-function variants. Our results provide an overview of the impact of indels on proteins and a method to predict their effects genome-wide.

KBTBD4 Cancer Hotspot Mutations Drive Neomorphic Degradation of HDAC1/2 Corepressor Complexes

“…Moreover, these findings underscore how insertions at a protein surface, in comparison to point substitutions, can be particularly effective at promoting neo-PPIs. 28 Leveraging these data with cryo-EM, we reveal the mechanistic basis by which MB mutations reconfigure the 2b-2c loop of KBTBD4 to extend into the HDAC1 active site in a shape- complementary fashion. Remarkably, these additional contacts made by the cancer mutations precisely mimic the effects of UM171 in gluing the suboptimal KBTBD4-HDAC1 interface, showcasing how chemical and human genetic perturbations can act as molecular facsimiles.…”

“…2g , Pearson’s r = –0.04, n.s.). These data suggest that (1) single and double insertions remodel the 2b-2c loop by distinct mechanisms, in contrast to single and double substitutions, and that (2) insertions are particularly effective in promoting gain- of-function protein-protein interactions 28 . Altogether, our deep mutational scanning supports the notion that double insertions are enriched in KBTDB4-mutant MBs due to their propensity to promote neomorphic activity.…”

Site saturation mutagenesis of 500 human protein domains reveals the contribution of protein destabilization to genetic disease

“…However the same libraries can be re-used in future work to quantify variant effects on additional molecular traits, for example abundance in different cellular contexts 8,44,45 , in vitro fold stability 23 , protein-protein 46,47 and protein-nucleic acid 48 interactions, protein localisation 49 , aggregation 50 , and allostery 25,36 . It will also be important to include more complex genetic variants such as insertions and deletions 51 and to extend to proteome-wide coverage, including extracellular and transmembrane proteins 52 . Domainome 1.0 is a first important step in the comprehensive experimental analysis of human protein variants.…”

Indels allow antiviral proteins to evolve functional novelty inaccessible by missense mutations

“…For example, indels introduced during genetic recombination and later affinity maturation enable B cell receptor genes to encode antibodies with improved antigen affinity ( 32 ). Moreover, in some instances, indels are more likely than missense mutations to improve enzymatic activity ( 19 ) or ligand binding affinity ( 17 ), although they also more frequently compromise these functions.…”