Proteins From Hereditary Eye Diseases: In silico and Experimental Studies

National Eye Institute

Investigators

Linked publications & trials

Abstract

To understand how a pathogenic mutation causes inherited eye disease, it is necessary to recognize how pathogenic mutations could affect protein structure-function, metabolic pathways, and how these perturbations could be associated with clinical parameters describing the disease phenotype. This year we purified the truncated versions of human recombinant tyrosinase (Tyr), mutant variants P406L and R422Q, Tyrp1, and 5 mutant variants. Also, we developed a new method for the isolation of dopachrome and melanin particles from the Tyr diphenol oxidase reaction with L-DOPA. The method is using tyrosinase immobilized to magnetic beads (Tyr-MB). We also characterize thermodynamic driving forces involved in tyrosinase-related reactions of the mutant variants in vitro. From molecular modeling, we suggested a mechanism for establishing the link between genetic alterations in tyrosinase structure and the effects leading to either form of OCA1. In the melanogenesis pathway, Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. The characterization of dopachrome-related products is difficult. In our work, we found a simple way to partition dopachrome from protein fractions. For this purpose, we immobilize catalytically pure recombinant human Tyr domain (residues 19-469) containing 6xHis tag to Ni-loaded magnetic beads and performed the diphenol oxidase reaction with L-DOPA (Varghese et all, 2021). Using Hill kinetics, we showed that Tyr-MB has a catalytic activity similar to that of intact Tyr. Surprisingly, the immobilization of Tyr to magnetic beads makes the protein more stable. At 50 C, intact Tyr does not show catalytic activity. In the same condition, Tyr-MB shows residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. Also, we characterized the sizes of Tyr-MB and isolated melanin particles. Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 nm (24.4 nm) while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 nm (18.1 nm). Thus, this method could be used to partition the products of biochemical reactions. Currently, we started to use this approach for the analysis activity of tyrosinases from the melanogenic pathway, including Tyr and tyrosinase-related proteins 1 and 2 (Tyrp1 and Tyrp2). Tyr is involved in pigment biosynthesis, where mutations in its corresponding gene TYR have been linked to oculocutaneous albinism 1, an autosomal recessive disorder. Although the enzymatic capabilities of Tyr have been well-characterized, the thermodynamic driving forces underlying melanogenesis remain unknown. Protein binding was analyzed using the diphenol oxidase behavior of Tyr and Vant Hoff temperature-dependent analysis was performed (Young et al, 2020). In the present year, we characterized the temperature-dependent kinetics and thermodynamic signatures of Tyr and two OCA1B mutants, R422Q, and P406L using diphenol oxidase activities at 28, 31, 37, and 43 oC (Wachamo et al., 2021). To obtain the kinetic and thermodynamic parameters, we used Michaelis-Menten and Vant Hoff analyses. We also assessed the role of temperature in the production of dopachrome through the diphenol oxidase reaction. Our results revealed, for the first time, that the association of L-DOPA with R422Q and P406L is a complex reaction supported by enthalpy and entropy forces. We further showed that the Tyr had a higher turnover number as compared with both R422Q and P406L. Moreover, the production of dopachrome increased with increasing temperature for WT, R422Q, and P406L, but it was significantly higher for the Tyr at all temperatures examined. Elucidating the kinetics and thermodynamics of mutant variants of Tyr in OCA1B helps to understand the mechanisms by which they lower Tyr catalytic activity. Under the same conditions, we performed computational simulations of the association of L-DOPA and Tyr (Patel M., in Wachamoet al., 2021). Computer simulations of Vant Hoff relationships were conducted to assess the temperature-dependent association between Tyr (R422Q, P406L) and L-DOPA and to explain the experimental observations. Both methods, in silico docking and Michaelis-Menten kinetics, show results with negative apparent enthalpy of 22.48-26.12 kJ/mol (experimental) and 30.16-43.11 kJ/mol (calculated). Negative enthalpy suggests the loss of ionic interactions and hydrogen bonds. The apparent entropy were negative: 0.13-0.13 kJ/(K mol) (experimental) and 0.13-0.16 kJ/(K mol) (calculated), respectively. Negative entropy indicates the decrease of disorder in the system. This decrease could be related to the formation of stable associates or particles (melanin), loss of rotational and translational freedom. Indeed, isolated particles were observed in our experiments of dopachrome isolation using magnetic beads. The Vant Hoff analysis provides a thermodynamic explanation of how melanin associates formed in vitro indicating that the association of L-DOPA and mutant tyrosinases are supported by enthalpy and entropy forces. Our computational work contributes to the understanding of inherited disease from the atomic level of protein structure and analysis of the impact of genetic mutations on disease phenotype. Recently, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally (Patel, 2021). Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Consistent with the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. SDS-PAGE and Western blot analysis of the purified recombinant proteins (Dolinska, 2021) confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. This study suggests that predictions of the effect of the genetic mutation at the protein level are supported by our experiments in vitro. Another example of this analysis is related to Myosin VIIA. Usher syndrome type 1B (USH1B) is a genetic disorder caused by mutations in the unconventional Myosin VIIa (MYO7A) protein. USH1B is characterized by hearing loss due to abnormalities in the inner ear and vision loss due to retinitis pigmentosa. We generated the homology model of the human MYO7A Isoform 1 (residues 1-2,215), built the homodimer using this model, and refined the dimer using 5 ns molecular dynamics in water. Global computational mutagenesis (McCafferty&Sergeev, 2016, 2017; Ortiz&Sergeev, 2019) was applied to evaluate the effect of missense mutations that are critical for maintaining protein structure and stability of MYO7A in inherited eye disease. We found that 43.26% (77 out of 178 in HGMD) and 41.9% (221 out of 528 in ClinVar) of the disease-related missense mutations were associated with higher protein structure destabilizing effects. Overall, most mutations destabilizing the MYO7A protein were found to associate with USH1 and USH1B. Particularly, motor domain and MyTH4 domains were found to be most susceptible to mutations causing the USH1B phenotype. Results of our computational study were incorporated in the ocular proteome website at the NEI Commons (https://neicommons.nei.nih.gov/#/proteomeData). The latest version of the ocular proteome website contains in-silico predictions for 1,411,100 missense mutations associated with 112 protein structures from 164 inherited eye diseases.