Abstract
Vesicle amine transport protein-1 (VAT-1) has been implicated in the regulation of vesicular transport, mitochondrial fusion, phospholipid transport and cell migration, and is a potential target of anticancer drugs. Little is known about the molecular function of VAT-1. The amino acid sequence indicates that VAT-1 belongs to the quinone oxidoreductase subfamily, suggesting that VAT-1 may possess enzymatic activity in unknown redox processes. To clarify the molecular function of VAT-1, we determined the three-dimensional structure of human VAT-1 in the free state at 2.3 Å resolution and found that VAT-1 forms a dimer with the conserved NADPH-binding cleft on each protomer. We also determined the structure of VAT-1 in the NADP-bound state at 2.6 Å resolution and found that NADP binds the binding cleft to create a putative active site with the nicotine ring. Substrate screening suggested that VAT-1 possesses oxidoreductase activity against quinones such as 1,2-naphthoquinone and 9,10-phenanthrenequinone.
Highlights
Vesicle amine transport protein-1 (VAT-1) was originally isolated as a synaptic vesicle membrane protein, abundantly found in cholinergic synaptic vesicles, and suggested to be involved in vesicular transport1
Part of the Switch segment is folded into an α-helix. (d) Topology of secondary structures found in the free form of VAT-1
We confirmed that NADPH binds VAT-1 with low affinity using low-c isothermal titration calorimetry (ITC) method16
Summary
Vesicle amine transport protein-1 (VAT-1) was originally isolated as a synaptic vesicle membrane protein, abundantly found in cholinergic synaptic vesicles, and suggested to be involved in vesicular transport. A recent study indicated that a natural polyenone, neocarzilin A (NCA), produced by Streptomyces carzinostaticus, functions as a potent inhibitor of cancer cell motility by targeting VAT-1-controlled p athways10 These investigations have suggested a variety of VAT-1 molecular functions in cell regulation, amino acid sequence analysis has indicated that VAT-1 belongs to the NAD(P)-dependent quinone oxidoreductase subfamily. The Switch segment contains two 310-helices (η3 and η4) in the free form, but in the NADP-bound form one 3 10-helix (η4) is transformed into a longer α-helix (αS), which is shown. Structures of free-form VAT-1 and NADP-bound VAT-1 complex and additional enzymatic studies, we revealed that VAT-1 contains a conserved NADPH binding cleft and binds NADPH to function as an oxidoreductase
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