Both P-glycoprotein nucleotide-binding sites are catalytically active

Area of Science:

• Biochemistry
• Molecular Biology
• Membrane Transport

Background:

• P-glycoprotein (Pgp) is a crucial efflux pump involved in multidrug resistance.
• Understanding Pgp's catalytic mechanism is key to developing strategies against multidrug resistance.
• The interaction of nucleotides with Pgp's catalytic sites remains incompletely understood.

Purpose of the Study:

• To investigate the functional properties of Pgp's nucleotide-binding sites using vanadate trapping.
• To elucidate the mechanism of Pgp ATPase activity inhibition.
• To determine the selectivity and catalytic roles of Pgp's N- and C-terminal nucleotide sites.

Main Methods:

• Vanadate trapping of Mg- or Co-8-azido-nucleotide with Pgp from multidrug-resistant cells.
• Measurement of Pgp ATPase activity and reactivation rates.
• Photolabeling of Pgp using UV irradiation after vanadate trapping.
• Limited proteolysis (trypsin digestion) to assess nucleotide site labeling.

Main Results:

• Vanadate trapping of nucleotide completely inhibited Pgp ATPase activity.
• UV irradiation of vanadate-trapped Pgp resulted in permanent inactivation and photolabeling.
• Both N- and C-terminal nucleotide sites were labeled equally, indicating non-selective trapping.
• Reactivation rates differed for Mg- and Co-nucleotide complexes, suggesting distinct transition states.

Conclusions:

• Both nucleotide sites in Pgp are capable of hydrolysis and contribute to catalysis.
• Vanadate trapping at one site allosterically inhibits hydrolysis at the other, implying coupled function.
• The Pgp-nucleotide-vanadate complex serves as a transition-state analog, supporting an alternating catalytic site model.