Biophysical Compatibility of a Heterotrimeric Tyrosinase-TYRP1-TYRP2 Metalloenzyme Complex

Abstract

Tyrosinase (TYR) is a copper-containing monooxygenase central to the function of melanocytes. Alterations in its expression or activity contribute to variations in skin, hair and eye color, and underlie a variety of pathogenic pigmentary phenotypes, including several forms of oculocutaneous albinism (OCA). Many of these phenotypes are linked to individual missense mutations causing single nucleotide variants and polymorphisms (SNVs) in TYR. We previously showed that two TYR homologues, TYRP1 and TYRP2, modulate TYR activity and stabilize the TYR protein. Accordingly, to investigate whether TYR, TYRP1, and TYRP2 are biophysically compatible with various heterocomplexes, we computationally docked a high-quality 3D model of TYR to the crystal structure of TYRP1 and to a high-quality 3D model of TYRP2. Remarkably, the resulting TYR-TYRP1 heterodimer was complementary in structure and energy with the TYR-TYRP2 heterodimer, with TYRP1 and TYRP2 docking to different adjacent surfaces on TYR that apposed a third realistic protein interface between TYRP1-TYRP2. Hence, the 3D models are compatible with a heterotrimeric TYR-TYRP1-TYRP2 complex. In addition, this heterotrimeric TYR-TYRP1-TYRP2 positioned the C-terminus of each folded enzymatic domain in an ideal position to allow their C-terminal transmembrane helices to form a putative membrane embedded three-helix bundle. Finally, pathogenic TYR mutations causing OCA1A, which also destabilize TYR biochemically, cluster on an unoccupied protein interface at the periphery of the heterotrimeric complex, suggesting that this may be a docking site for OCA2, an anion channel. Pathogenic OCA2 mutations result in similar phenotypes to those produced by OCA1A TYR mutations. While this complex may be difficult to detect in vitro, due to the complex environment of the vertebrate cellular membranous system, our results support the existence of a heterotrimeric complex in melanogenesis.

Keywords: computational molecular docking; melanosome; molecular modeling; oculocutaneous albinism; pigmentary disorders; protein-protein interface; tyrosinase.

FIGURE 1

Global Needleman and Wunsch sequence alignment between TYR and TYRP1 (PDB: 5m8n) showing 45% homology with a p-value (p = −log of pvalue) of 10^−40.2 for structural similarity. Residues highlighted in green represent conserved residues. Residues highlighted in yellow represent non-identical but homologous residues, which are expected to have little to no effect on the overall structure of the protein.

FIGURE 2

Protein-protein interaction surface between TYR and TYRP1. Residues making key contacts are displayed in ball-and-stick (radius represents contribution size). (A). Full size dimer with helix bundle. (B). 24 contact residues highlighted at the interface of the two proteins. H256 residue where mutation associated with OCA1A occurs, is shown in black. Two Zn atoms in the active site of TYRP1 are shown in gray. Cu atoms in the active site of TYR are shown in orange.

FIGURE 3

Protein-protein interaction surface between TYR and TYRP2. Residues making key contacts are displayed in ball-and-stick (radius represents contribution size). (A). Full size dimer with helix bundle. (B). 37 contact residues highlighted at the interface of the two proteins. Residues where mutation associated with OCA1A occurs, is shown in black. Two Zn atoms in the active site of TYRP2 are shown in gray. Cu atoms in the active site of TYR are shown in orange.

FIGURE 4

TYR-TYRP1-TYRP2 heterotrimeric complex. (A). Side view, highlighting compatibility of the docked locations of the three N-terminii and their linker lengths between intramelanosomal domains and N-terminal TM-helices with formation of three-helix TM bundle. (B). Top view. (C). Electrostatic surfaces of the TYR-TYRP1 interface is shown, revealing continuity of active sites of TYR and TYRP1 in heterotimer (zinc ions are green spheres marking active sites) (D). Electrostatic surface of heterotrimer surface facing the membrane (red = electronegative/acidic, blue = electropositive/basic).

FIGURE 5

Cluster of mutations associated with ER retention map to a contiguous surface of the protein, suggesting a new interaction site with an unknown binding partner. R403, R402, P406L, R422, D448, and G446 map onto the surface of the protein in the region located distant from the active site. Substitutions shown in red (R402Q, P406L, and R422Q) result in temperature-sensitive TYR mutants.

FIGURE 6

Schematic representation of the proposed site of binding between the melanogenic complex and a binding partner such as OCA2.