Copper is an essential nutrient whose concentration is controlled by proteins that help in safe trafficking of the metal ion inside cells. Improper distribution of copper can cause severe metabolic disorders. ATP7B (also known as Wilson protein) is a copper transporting pump of the P1B-type ATPase found in the trans Golgi network of the cell, especially in hepatocytes. HAH1 copper metallochaperone is a single domain cytoplasmic protein while Wilson protein (WDP) is a multi-domain protein.  The cytosolic N-terminal domain of WDP has six metal binding domains (MBDs) which can receive copper form the cytoplasmic HAH1. Metal binding domain 4 (WLN4) is believed to interact directly with HAH1 during the copper ion transfer between the metal binding domains of WDP. HAH1 may form multiple interaction complexes with WLN4. However, the geometries of these complexes are not clearly known.  This information will help in understanding how HAH1 interacts with WDP or WLN4 during the metal transfer. Our laboratory has previously shown that WLN4 of ATP7B acquires the Cu1+ ion from the cytosolic HAH1.  In order to better understand the nature of the interaction between WLN4 and HAH1, we have linked them together in a protein chimera complex.  The chimera was constructed by cloning the gene encoding HAH1 into a plasmid that contains the gene for WLN4.  The chimera, called WLN4~HAH1, was expressed as a fusion protein linked to Thioredoxin followed by a His-tag and an engineered TEV protease site.  The protein sequence between WLN4 and HAH1 is identical to the native protein sequence that links WLN4 and WLN5.  The TEV protease removed the fusion tag from WLN4~HAH1.  The protein was purified by affinity chromatography and gel filtration. We characterized the protein by SDS-PAGE, mass spectrometry and circular dichroism spectroscopy. Experiments in progress include metal-dependent gel filtration and crystallization.

Chimera of metal-binding domain four of ATP7B with the copper chaperone HAH1

247th National Meeting (2014)

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