Using structural analysis In Silico to assess the impact of missense variants in MEN1

Despite the rapid expansion in recent years of databases reporting either benign or pathogenic genetic variations, the interpretation of novel missense variants remains challenging, particularly for clinical or genetic testing laboratories where functional analysis is often unfeasible. Previous studies have shown that thermodynamic analysis of protein structure in silico can discriminate between groups of benign and pathogenic missense variants. However, although structures exist for many human disease‒associated proteins, such analysis remains largely unexploited in clinical laboratories. Here, we analyzed the predicted effect of 338 known missense variants on the structure of menin, the MEN1 gene product. Results provided strong discrimination between pathogenic and benign variants, with a threshold of >4 kcal/mol for the predicted change in stability, providing a strong indicator of pathogenicity. Subsequent analysis of seven novel missense variants identified during clinical testing of patients with MEN1 showed that all seven were predicted to destabilize menin by >4 kcal/mol. We conclude that structural analysis provides a useful tool in understanding the effect of missense variants in MEN1 and that integration of proteomic with genomic data could potentially contribute to the classification of novel variants in this disease.This article is freely available via Open Access. Click on the Publisher URL to access the full-text.Open Acces

Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity

The retargeting of protein-DNA specificity, outside of extremely modular DNA binding proteins such as TAL effectors, has generally proved to be quite challenging. Here, we describe structural analyses of five different extensively retargeted variants of a single homing endonuclease, that have been shown to function efficiently in ex vivo and in vivo applications. The redesigned proteins harbor mutations at up to 53 residues (18%) of their amino acid sequence, primarily distributed across the DNA binding surface, making them among the most significantly reengineered ligand-binding proteins to date. Specificity is derived from the combined contributions of DNA-contacting residues and of neighboring residues that influence local structural organization. Changes in specificity are facilitated by the ability of all those residues to readily exchange both form and function. The fidelity of recognition is not precisely correlated with the fraction or total number of residues in the protein-DNA interface that are actually involved in DNA contacts, including directional hydrogen bonds. The plasticity of the DNA-recognition surface of this protein, which allows substantial retargeting of recognition specificity without requiring significant alteration of the surrounding protein architecture, reflects the ability of the corresponding genetic elements to maintain mobility and persistence in the face of genetic drift within potential host target sites