Proteins From Hereditary Eye Diseases: In silico and Experimental Studies

National Eye Institute

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Abstract

To understand how a pathogenic mutation causes inherited eye disease, it is necessary to recognize how pathogenic mutations could affect protein structure-function and metabolic pathways, and how these perturbations could be associated with clinical parameters describing the disease phenotype. Our major focus was on the intra-melanosomal domains (IMD) of tyrosinases from the melanogenic pathway. Last year, we purified and characterized the IMD of human recombinant tyrosinase-related protein 2 (Tyrp2) (residues 1-474) and missense variants C40S and C61W mimicking the alterations found in genetic studies in patients with oculocutaneous albinism type 8 (Dolinska et al., 2022). The catalytic activity of Tyrp2 catalyzing the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) was studied in more detail this year (Osuna et al., 2023). Human recombinant IMDs of key enzymes from the melanogenic pathway were produced in T. ni larvae and then purified using a combination of chromatography techniques in catalytically active form. Using MichaelisMenten kinetics, the diphenol oxidase activity of tyrosinase achieved the maximum production of native dopachrome at 10 min of incubation at 37 oC for TYR immobilized to magnetic beads (TYR-MB). The presence of dopachrome was confirmed spectrophotometrically at 475 nm through HPLC analysis and in the TYRP2-catalyzed reaction, yielding DHICA. In the TYRP1-driven oxidation of DHICA, the formation of 5,6-indolequinone-2-carboxylic acid (IQCA) was confirmed at 560 nm. In this work, we were able to show the activity of TYRP1 with the formation of IQCA without the use of MBTH for the first time. We demonstrated the success of dopachrome as a substrate through the characterization of TYR-MB particle activity, confirmed the enzymatic activity of both TYRP2 and TYRP1 and identified the substrates in the melanogenesis pathway through HPLC analysis. This is the first in vitro reconstitution of the reactions from the melanogenic pathway based on IMD. This approach could be used for quantitative in vitro analysis of proteins in the melanin pathway, biochemical effects associated with inherited disease-related mutations, and drug screens. Also, we characterize in vitro the IMD of human recombinant tyrosinase-related protein 1 (TYRP1) and its oculocutaneous albinism type 3 (OCA3) related mutant variants (Dolinska et al., 2023). TYRP1 is the most abundant melanosomal protein of the melanocyte, which plays an important role in the synthesis of eumelanin, catalyzing the oxidation of DHICA to IQCA. Mutations to the TYRP1 gene can result in OCA3, a rare disease characterized by reduced synthesis of melanin in skin, hair, and eyes. To investigate the effect of genetic mutations on the TYRP1 structure, function, and stability, we engineered the TYRP1 IMD and its mutant variants mimicking either OCA3-related changes, C30R, H215Y, D308N, and R326H or R87G mutant variant, analogous to OCA1-related pathogenic effect in tyrosinase. Proteins were produced in T. ni larvae, then purified, and analyzed by biochemical methods. Data shows that D308N and R326H mutants keep the native conformations and demonstrate no change in their stability and enzymatic activity. In contrast, mutations C30R and R87G localized in the Cys-rich subdomain show the variants' misfolding during the purification process. The H215Y variant disrupts the binding of Zn2+ in the active site and thus reduces the strength of the enzyme/substrate interactions. Our results, consistent with the clinical and in silico studies, show that mutations at the protein surface are expected to have a negligible phenotype change compared to that of TYRP1. Our results, consistent with the clinical (ClinVar) and in silico studies (Patel et al., 2022), can help to understand the mechanism of protein malfunction due to inherited mutations, and their link to the phenotype of the disease. therefore, may aid in finding a potential treatment for OCA3 patients. In the Tyr IMD, the cysteine-rich and tyrosinase catalytic subdomains are essential for enzymatic activity. Here, we performed 6 molecular dynamics simulations at room temperature for Tyr and OCA1-related mutant variants P406L and R402Q intra-melanosomal domains to understand structural changes in protein under chemical denaturation (Woods & Sergeev, 2023). The proteins were simulated for 1 ms in water and urea to induce unfolding. In urea, we observed increases in surface area, decreases in intramolecular hydrogen bonding, and decreases in hydrophobic interactions suggesting a molten globule state for each protein. Between all conditions, the cysteine-rich subdomain remains stable, whereas the catalytic subdomain shows increased flexibility. This flexibility is intensified by the P406L mutation, while R402Q increases the catalytic domains rigidity. The cysteine-rich subdomain is rigid, preventing the protein from unfolding, whereas the flexibility of the catalytic subdomain accommodates mutational changes which could inhibit activity. These findings match the conclusions from our experimental work suggesting the function alteration by the P406L mutation, and the potential role of R402Q as a polymorphism. The finding of the molten globula state in tyrosinase raises a question about the potential existence of a similar state in the native environment of tyrosinase mutant variants. In summary, we showed that using in vitro and computational methods, we proved that the localized in the Cys-rich domain of Tyr mutations strongly impacted protein folding and stability. This result suggests the importance of the Cys-rich domain for the tyrosinase family of proteins and agrees with our previous results on the Tyrp1 protein (Patel et al., 2001). In collaboration with a team from NHGRI, we computationally analyzed the impact of tyrosinase mutations on protein structure (Loftus et al., 2023). Initially, molecular dynamics simulations were performed using the Tyr Alphafold model. Alphafold structures for molecules with each of the two variants, S192Y and R402Q, alone and in combination S192Y; R402Q were simulated in comparison to the Tyr reference predicted structure. The Alphafold model predicted a roughly additive effect for the two-variant model. However, the AlfaFold model is inaccurate because does not contain the copper ions in an active site. Therefore, our IMD Tyr model which includes copper ions and two oxygen atoms was used next. Using the model, we performed a 10 ns molecular dynamics simulation with the same three modifications. Changes in interatomic distances across multiple critical histidines are predicted, with the strongest impact predicted to occur at H363 in both models for the S192Y; R402Q double variant. The Alphafold structure predictions are consistent with a hypothesis of the additive effect of two-component cis-YQ mutations on the positions of critical copper-coordinating histidine residues. The IMD model showed smaller absolute changes in histidine positions but supported an overall effect on copper positions. Our experimental and 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. Our computational achievements in modeling of effects of genetic mutations were recently reviewed (Sergeev, 2023). The results of our computational study were incorporated into the ocular proteome website at the NEI Commons. The latest version of the ocular proteome website contains in-silico predictions for 1,411,700 missense mutations associated with 112 protein structures from 164 inherited eye diseases.