Investigation of Protonation Effects on ATP Binding in ABC Transporters

Abstract

ATP binding cassette (ABC) transporters consist of two characteristic nucleotide binding domains (NBD) and two transmembrane-spanning domains (TMD). Binding and hydrolysis of ATP at the NBDs controls substrate transport or, in rare cases, other physiological functions.Previous studies have identified residue motifs of functional importance, but our understanding of the hydrolysis process in ABC NBDs remains incomplete. An acidic residue of the Walker-B motif has been suggested to act as a general base that abstracts a proton from the hydrolytic water. Other work has suggested a greater role of a histidine (in the switch motif) in activating the hydrolytic water. Also the role of the Mg2+ ion in orienting hydrolytic residues and waters is poorly understood.One limitation in interpreting existing ABC NBD structures for their hydrolytic function is the assignment of protonation states to the relevant residues and the exact orientation of water molecules in the NBDs. The highly charged nature of the NBS renders protonation assignment particularly challenging.In this study, we vary protonation states at the NBDs of the multidrug ABC exporter Sav1866 and simulate the ATP-bound NBD dimers by molecular dynamics. We consider combinations of protonation of the Walker-B glutamate, the switch histidine and the ATP itself with and without Mg2+. The resulting 24 systems are simulated to at least 50 ns duration.We show that the Mg2+ and residue protonation affect protein dynamics. Crucially. we show that the local geometries of ATP-binding residues in many available ABC NBD crystal structures can potentially be rationalized by different protonation states. Conformational changes of the Glu and His upon protonation support the idea of the Glu acting as a general base. Furthermore, we discuss the coordination and dynamics of putative hydrolytic waters.