Abstract
Purpose :
Calpain-5 (CAPN5) is a calcium-dependent cysteine protease in the retina with pathogenic genetic variants that cause an inherited eye disease, Neovascular Inflammatory Vitreoretinopathy (NIV, OMIM# 193235). How CAPN5 missense mutations cause enzymatic hyperactivity and result in inflammation, neuronal cell death, and neovascularization is not known. We integrated protein structure- and synthetic-biology approaches to determine the structure-pathology relationship behind the CAPN5 NIV-mutants, which are scattered throughout CAPN5, suggesting that each mutant may have a different structure-pathology relationship for hyperactivity.
Methods :
Protein structures of the CAPN5 NIV-mutant were determined by X-ray crystallography. The structure dynamics of each CAPN5 variant was calculated by Normal mode analysis (NMA) and Perturbation Response Scanning (PRS) in silico. By combining the empirical and the computational information, de novo CAPN5 mutants, which were expected to share a similar structure-pathology relationship to hyperactive CAPN5 NIV-mutants, were designed. The enzymatic activity levels of these synthetic CAPN5 mutants were validated in cells by a CAPN5 activity assay.
Results :
The X-ray crystallographic structures of two CAPN5 NIV-mutants (p.G267S and p.R289W) showed nearly identical conformations as the wild-type structure. Since X-ray structures provide only a snapshot of many possible conformations, we performed in silico structure dynamics calculations to better sample the impact of the NIV-mutants. NMA calculations found the NIV-mutants localized to structural hotspots on flexible loops in the CAPN5 protein. PRS calculation profiling suggested each NIV-mutation had a different structural effect leading to a hyperactivity. We generated and tested a series of de novo CAPN5 mutants predicted to either increase or decrease enzymatic activity based on their structural perturbations. Most synthetic mutants showed enzymatic activity that met their design expectations.
Conclusions :
Each NIV mutation has a unique biophysical effect on the CAPN5 structure dynamics leading to hyperactivity. Mutations within a protein’s flexible loops may represent an evolutionary mechanism to tune enzymatic activity without altering the native protein folding and structure.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.