Abstract
Purpose :
Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the TYRP1 gene. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of dihydroxyindole carboxylic acid oxidase (DHICA) to indolequinone carboxylic acid (IQCA). Here, we used computational methods to assess molecular docking of DHICA and IQCA in Tyrp1 and associated OCA3 mutants.
Methods :
The mutations for analysis, C30R, H215Y, D308N, and R326H, were selected from ClinVar and The Human Gene Mutation Database. The crystal structure of human Tyrp1 (5M8L) was minimized using molecular dynamics in water and subjected to global mutagenesis to ascertain the ΔΔG values of folding. In total, four known mutations of Tyrp1 and the wild type (chain A of 5M8L) were simulated for 100 ns in pH 5.5. Global mutagenesis was conducted to identify residues critical to protein folding, a Weighted Histogram Analysis Method (WHAM) was applied to compare free energy landscapes of the active site between the wild type and mutants, and Maestro software was used to dock DHICA and IQCA.
Results :
C30R and H215Y exhibit greater instability according to experimental results, which is consistent with their unfolding parameters of 1, indicating protein misfolding. Global mutagenesis suggests that of the four mutants, D308N and R326H were the only ones that were not completely unfolded or misfolded, with unfolding parameters of 0.56 and 0.98, respectively. Free energy landscapes generated by WHAM indicate that the binding cleft (residues Y362, N378, and T391) of both of these mutants are larger and less well defined. The docking of DHICA in Tyrp1 highlights four interactions that remain constant between DHICA and the protein: three hydrogen bonds and one salt bridge. D212 and E216 are the most common recipients of these interactions.
Conclusions :
Our observations are consistent with the results of biochemical analysis of recombinant Tyrp1 and four mutant variants from this study. The docking of DHICA, in the most stable protein conformations as determined by WHAM, indicate that the hydrogen bond and salt bridge interactions that stabilize DHICA remain similar among wild type Tyrp1, D308N, and R326H. However, the strength of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined nature of the binding cleft in mutants.
This is a 2021 ARVO Annual Meeting abstract.