{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T02:06:00Z","timestamp":1780452360289,"version":"3.54.1"},"reference-count":29,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2021,9,22]],"date-time":"2021-09-22T00:00:00Z","timestamp":1632268800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000053","name":"National Eye Institute","doi-asserted-by":"publisher","award":["ZIA EY000476-10"],"award-info":[{"award-number":["ZIA EY000476-10"]}],"id":[{"id":"10.13039\/100000053","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally to understand Tyrp1 protein stability and catalytic activity. 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 confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. Further, the mutant variant structures were built and simulated for 100 ns to generate free energy landscapes and perform docking experiments. Free energy landscapes formed by Y362, N378, and T391 indicate that the binding clefts of C30R and H215Y mutants are larger than the wild-type Tyrp1. In docking simulations, the hydrogen bond and salt bridge interactions that stabilize DHICA in the active site remain similar among Tyrp1, D308N, and R326H. However, the strengths of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined natures of the binding clefts in mutants. Mutational perturbations in mutants that are not unfolded may result in allosteric alterations to the active site, reducing the stability of protein-ligand interactions.<\/jats:p>","DOI":"10.3390\/ijms221910203","type":"journal-article","created":{"date-parts":[[2021,9,22]],"date-time":"2021-09-22T22:50:48Z","timestamp":1632351048000},"page":"10203","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Tyrp1 Mutant Variants Associated with OCA3: Computational Characterization of Protein Stability and Ligand Binding"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0938-2805","authenticated-orcid":false,"given":"Milan H.","family":"Patel","sequence":"first","affiliation":[{"name":"National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monika B.","family":"Dolinska","sequence":"additional","affiliation":[{"name":"National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7204-6572","authenticated-orcid":false,"given":"Yuri V.","family":"Sergeev","sequence":"additional","affiliation":[{"name":"National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,9,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"9812","DOI":"10.1002\/anie.201704616","article-title":"Structure of Human Tyrosinase Related Protein 1 Reveals a Binuclear Zinc Active Site Important for Melanogenesis","volume":"56","author":"Lai","year":"2017","journal-title":"Angew. Chem. Int. Ed. Engl."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"34913","DOI":"10.1074\/jbc.M102874200","article-title":"Crystal Structure of Human Epidermal Growth Factor and Its Dimerization","volume":"276","author":"Lu","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"81","DOI":"10.15406\/japlr.2020.09.00356","article-title":"Functional in silico analysis of human tyrosinase and OCA1 associated mutations","volume":"9","author":"Patel","year":"2020","journal-title":"J. Anal. Pharm. Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/j.jdermsci.2011.09.005","article-title":"Oculocutaneous albinism type 3: A Japanese girl with novel mutations in TYRP1 gene","volume":"64","author":"Yamada","year":"2011","journal-title":"J. Dermatol. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"726","DOI":"10.1080\/14756366.2020.1740691","article-title":"Histidine residues at the copper-binding site in human tyrosinase are essential for its catalytic activities","volume":"35","author":"Noh","year":"2020","journal-title":"J. Enzym. Inhib. Med. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1111\/pcmr.12546","article-title":"Oculocutaneous albinism type 1: A link between mutations, tyrosinase conformational stability, and enzymatic activity","volume":"30","author":"Dolinska","year":"2017","journal-title":"Pigment. Cell Melanoma Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1007\/s12013-011-9234-0","article-title":"Oculocutaneous albinism type 3 (OCA3): Analysis of two novel mutations in TYRP1 gene in two Chinese patients","volume":"61","author":"Zhang","year":"2011","journal-title":"Cell Biochem. Biophys."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"466","DOI":"10.1111\/pcmr.12688","article-title":"Molecular characterization of a series of 990 index patients with albinism","volume":"31","author":"Lasseaux","year":"2018","journal-title":"Pigment. Cell Melanoma Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"8169","DOI":"10.1074\/jbc.275.11.8169","article-title":"Mutations at Critical N-Glycosylation Sites Reduce Tyrosinase Activity by Altering Folding and Quality Control","volume":"275","author":"Negroiu","year":"2000","journal-title":"J. Biol. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4697","DOI":"10.1021\/cr990291y","article-title":"N-Glycosylation Processing and Glycoprotein Folding\u2212Lessons from the Tyrosinase-Related Proteins","volume":"100","author":"Petrescu","year":"2000","journal-title":"Chem. Rev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"37298","DOI":"10.1038\/srep37298","article-title":"In silico Mapping of Protein Unfolding Mutations for Inherited Disease","volume":"6","author":"McCafferty","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_12","unstructured":"NEI Commons Website (2021, July 02). Ocular Proteome Webpage, National Eye Institute, Available online: https:\/\/neicommons.nei.nih.gov\/#\/proteomeData."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"W382","DOI":"10.1093\/nar\/gki387","article-title":"The FoldX web server: An online force field","volume":"33","author":"Schymkowitz","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"McCafferty, C.L., and Sergeev, Y.V. (2017). Global computational mutagenesis provides a critical stability framework in protein structures. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0189064"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3676","DOI":"10.1038\/s41598-019-39905-9","article-title":"Global computational mutagenesis of domain structures associated with inherited eye disease","volume":"9","author":"Ortiz","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1002\/jcc.20084","article-title":"UCSF Chimera\u2014A visualization system for exploratory research and analysis","volume":"25","author":"Pettersen","year":"2004","journal-title":"J. Comput. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1011","DOI":"10.1002\/jcc.540130812","article-title":"The Weighted Histogram Analysis Method for Free-Energy Calculations on Biomolecules. I. The Method","volume":"13","author":"Kumar","year":"1992","journal-title":"J. Comput. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1339","DOI":"10.1002\/jcc.540161104","article-title":"Multidimensional Free-Energy Calculations Using the Weighted Histogram Analysis Method","volume":"16","author":"Kumar","year":"1995","journal-title":"J. Comput. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1016\/0010-4655(95)00053-I","article-title":"The calculation of the potential of mean force using computer simulations","volume":"91","author":"Roux","year":"1995","journal-title":"Comput. Phys. Commun."},{"key":"ref_20","unstructured":"Grossfield, A. (2021, September 21). WHAM: The Weighted Histogram Analysis Method, 2.0.10.2 ed. Available online: http:\/\/membrane.urmc.rochester.edu\/wordpress\/?page_id=126\/."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1719","DOI":"10.1038\/s41598-017-19135-7","article-title":"Effects of Distal Mutations on the Structure, Dynamics and Catalysis of Human Monoacylglycerol Lipase","volume":"8","author":"Tyukhtenko","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.sbi.2018.09.004","article-title":"Modulation of allosteric coupling by mutations: From protein dynamics and packing to altered native ensembles and function","volume":"54","author":"Naganathan","year":"2018","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1002\/humu.22315","article-title":"DNA variations in oculocutaneous albinism: An updated mutation list and current outstanding issues in molecular diagnostics","volume":"34","author":"Simeonov","year":"2013","journal-title":"Hum. Mutat."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1007\/s10822-013-9644-8","article-title":"Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments","volume":"27","author":"Sastry","year":"2013","journal-title":"J. Comput. Aided Mol. Des."},{"key":"ref_25","first-page":"33","article-title":"Open Babel: An open chemical toolbox","volume":"3","author":"Banck","year":"2011","journal-title":"J. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"681","DOI":"10.1007\/s10822-007-9133-z","article-title":"Epik: A software program for pK(a) prediction and protonation state generation for drug-like molecules","volume":"21","author":"Shelley","year":"2007","journal-title":"J. Comput. Aided Mol. Des."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1007\/s10822-010-9349-1","article-title":"Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution","volume":"24","author":"Greenwood","year":"2010","journal-title":"J. Comput. Mol. Des."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Dolinska, M.B., Young, K.L., Kassouf, C., Dimitriadis, E.K., Wingfield, P.T., and Sergeev, Y.V. (2020). Protein Stability and Functional Characterization of Intra-Melanosomal Domain of Human Recombinant Tyrosinase-Related Protein 1. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21010331"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"753","DOI":"10.1111\/pcmr.12791","article-title":"The TYRP1-mediated protection of human tyrosinase activity does not involve stable interactions of tyrosinase domains","volume":"32","author":"Dolinska","year":"2019","journal-title":"Pigment. Cell Melanoma Res."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/19\/10203\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:03:21Z","timestamp":1760166201000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/19\/10203"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,9,22]]},"references-count":29,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2021,10]]}},"alternative-id":["ijms221910203"],"URL":"https:\/\/doi.org\/10.3390\/ijms221910203","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,9,22]]}}}