Structure-function relationships in an anion-translocating ATPase

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• The ArsAB ATPase is an inner membrane efflux pump in Escherichia coli.
• It confers resistance to arsenite and antimonite by extruding these toxic metalloids.
• The pump comprises the catalytic ArsA subunit and the membrane-bound ArsB subunit, functioning as an ATP-binding cassette (ABC) transporter.

Purpose of the Study:

• To elucidate the mechanism of allosteric activation of the ArsA ATPase.
• To understand how conformational changes in ArsAB contribute to its function as an efflux pump.
• To establish ArsAB as a model for studying the broader ABC transporter superfamily.

Main Methods:

• Molecular genetics
• Biochemical analyses
• Structural analyses
• Kinetic analyses
• Stopped-flow fluorescence measurements using single-tryptophan derivatives of ArsA

Main Results:

• The ArsB subunit contains 12 transmembrane segments and forms the substrate translocation pathway.
• The ArsA subunit is a substrate-activated ATPase with two nucleotide-binding domains (NBDs), resulting from gene duplication.
• Allosteric activation accelerates catalysis by increasing the rate of a slow isomerization between enzyme conformations, making product release rate-limiting.

Conclusions:

• The ArsAB pump's mechanism involves overcoming a rate-limiting isomerization step through conformational changes.
• ArsAB serves as a valuable model for understanding the mechanistic principles of ABC transporters.
• This study provides insights into substrate-activated ATPase mechanisms within the ABC transporter superfamily.