Mature Tyrosinase (TYR, monophenol monoxygenase EC 1.14.18.1, UniProt #14679) is a melanosomal membrane-bound glycoenzyme with a type-3 copper active site. TYR undergoes extensive processing inside endoplasmic reticulum (ER) aided by the chaperones Bip, Calnexin and Calreticulin, from where, it reaches the Golgi and next the melanosomes, the site of its function and subsequent pigment production. Though mammalian TYR in its pure and complete form has not been crystallized yet, the domains of the protein are well characterized. Mature TYR harbours seven N-glycosylation sites; two copper binding sites (CuA and CuB) and one transmembrane (TM) domain followed by a relatively short carboxyl-tail that contains the essential signals for sorting and targeting to the melanosomes [1].
Based on previous studies, it has been implicated that OCA1 (OCA: Oculocutaneous Albinism, here Type 1), caused by mutations in TYR (TYR encoding human gene, OMIM #606933), is an ER-retention disorder [2–5] and different degrees of retentions in ER was evident for different TYR variants precipitating either OCA1A or OCA1B [2, 3, 6, 7]. It is important to mention here that any un/misfolded protein enters and re-enters the chaperone CNX/CRT (Calnexin/Calreticulin) cycle via UGGT (UDP-glucose: glycoprotein glucosyl transferase) pathway and if still fails to fold correctly even after repeated cycles of CNX/CRT binding, is sorted out to the cytosol by EDEM (ER degradation enhancing—mannosidase-like protein) for proteasomal degradation [1]. Thus, until proteasomal degradation, the misfolded mutant protein remains bound to ER chaperones, as reflected by Endoglycosidase H (Endo-H) sensitive bands post Endo-H assay. OCA1 causing missense mutations like p.L9P, p.D42N, p.R52I, p.K131E, p.H202R, p.R239W, p.G295R were observed to be completely ER retained post Endo-H assays and having zero TYR activities; in contrast, p.G41R, p.S192Y, p.Y433C were found to be partially retained within ER and having residual TYR enzyme activities, while few other TYR variants like p.L6F, p.S79P, p.T325A, p.A490G were found retain wild type (WT) like enzyme activity, besides not being retained at-all within the ER lumen. Interestingly, p.S192Y and p.H202R residing within the same Copper A domain were found to be partially and completely retained within ER lumen, respectively, having different degrees of TYR enzyme activities as already mentioned [2, 8]. In view of all this information, we thought variant wise modulation of TYR’s structural attributes might be the contributory factor towards different degrees of ER retentions for the different mutants. We thought of checking if the solvent accessibility of the mutant residue i.e if the position of the variant residue within TYR protein are buried deep inside the TYR protein structure or are more of surface exposed kind and if that can be correlated with different degrees of ER retentions. Interestingly, previous studies [9, 10] have shown that changes in hydrogen bonds with neighbouring residues, introduction of steric clash or weak bonds, changes in overall electrostatic potential maps are important parameters for a mutation to exert different degrees of “deleterious” effects on protein structure and/or functions, which may correlate with different degrees of severity of disease outcomes. Again, alteration of overall protein stability and torsional property have long been observed to contribute differential functions on mutated protein variants [11–13]. We thought these parameters too ought to be checked for TYR protein variants in understanding the molecular basis of differential degrees of ER retentions. In accordance, we modelled TYR WT and mutated protein variants and compared the above-mentioned structural attributes between WT and the mutated TYR protein variant models.