Lactoferrin-melanin interaction and its possible implications in melanin polymerization: crystal structure of the complex formed between mare lactoferrin and melanin monomers at 2.7-A resolution

Sharma, A.K.; Kumar, S.; Sharma, V.; Nagpal, A.; Singh, N.; Tamboli, I.; Mani, I.; Raman, G.; Singh, T.P.

Proteins 45(3): 229-236

2001


ISSN/ISBN: 0887-3585
PMID: 11599026
DOI: 10.1002/prot.1143
Accession: 003830224

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Abstract
The concentration of melanin determines the intensity of colors of the skin and hair of animals. Melanin pigments are tyrosine-based polymers formed in melanocytes within specialized organelles called melanosomes. In order to understand the mechanism of melanin polymerization, lactoferrin, a basic protein with a pI value of 9.0, has been used to produce melanin. Lactoferrin is a monomeric iron-binding protein with a molecular weight of 80 kDa. The crystals of lactoferrin were soaked in a solution containing dihydroxyphenylalanine (DOPA) and tyrosinase enzyme. These crystals were used for X-ray intensity data collection. The intensity data were collected to 2.7-ANG resolution to an overall completeness of 91% with an Rsym of 0.071. The crystals belong to orthorhombic space group P212121 with cell dimensions: a=85.0 ANG, b=99.8 ANG, c=103.4 ANG. The structure was determined by molecular replacement method, using the model of diferric mare lactoferrin, and refined to an R-factor 0.215 (Rfree=0.287) for all the data to 2.7-ANG resolution. The final model comprises 5,281 protein atoms from 689 amino acids, 2Fe3+, 2CO32- ions, 2 indole-5,6-quinone molecules (IQ), and 73 water molecules. Two IQ molecules, one in each lobe, bind to lactoferrin. In the C-lobe, the IQ binds in the iron-binding cleft, whereas in the N-lobe, it is located in the side pocket between two alpha-helices, filled with solvent molecules in the native iron-saturated mare lactoferrin. The IQ molecules interact with protein molecule mainly through glutamic acid in both lobes, without significant perturbation to the protein structure. The orientation of N- and C-lobes in the present structure is similar to that observed in the native iron-saturated protein. However, as a result of the binding of IQ molecules, the orientations of the domains N1, N2 and C1, C2 in the two cases differ slightly.

Lactoferrin-melanin interaction and its possible implications in melanin polymerization: crystal structure of the complex formed between mare lactoferrin and melanin monomers at 2.7-A resolution