The human ATP-binding cassette (ABC)-transporter ABCB10 is essential for red blood cell development and protects mitochondria against oxidative stress. The transporter's basal ATPase activity is stimulated by the transported substrate (biliverdin), implying long-range communication between the transmembrane substrate binding site(s) and the nucleotide-binding domains that hydrolyze ATP. However, the molecular events that lead to stimulation by substrate are not fully understood. A recent Cryo-EM model of biliverdin-bound ABCB10 in GDN-micelles shows a substrate binding site that differs from a prior sequence alignment prediction, generating ambiguity. Therefore, we conducted a functional analysis to identify AB... More
The human ATP-binding cassette (ABC)-transporter ABCB10 is essential for red blood cell development and protects mitochondria against oxidative stress. The transporter's basal ATPase activity is stimulated by the transported substrate (biliverdin), implying long-range communication between the transmembrane substrate binding site(s) and the nucleotide-binding domains that hydrolyze ATP. However, the molecular events that lead to stimulation by substrate are not fully understood. A recent Cryo-EM model of biliverdin-bound ABCB10 in GDN-micelles shows a substrate binding site that differs from a prior sequence alignment prediction, generating ambiguity. Therefore, we conducted a functional analysis to identify ABCB10's residues necessary for substrate-induced ATPase stimulation, as well as relevant biliverdin functional groups. We found that mutation of the highly conserved R232 and R295 decreased the stimulation by biliverdin, suggesting a more relevant role for these residues than what was derived from the Cryo-EM model. GDN abolished ABCB10's stimulation by biliverdin, so it might also affect substrate binding in this model. We also found that ABCB10's stimulation is specific for biliverdin, as biliverdin dimethyl ester was not an effective stimulator, whereas mesobiliverdin inhibits instead of stimulating. Interestingly, some of the arginine mutants display elevated basal ATPase activity. Also, by using Luminescence Resonance Energy Transfer we have detected alterations in the conformational equilibrium of these arginine mutants and a lack of response to biliverdin. In general, our data suggest delicate complementarities between the substrate and its binding pocket on ABCB10, with small modifications largely impacting the basal ATPase activity and the transporter's ability to be stimulated by substrate.
