Interaction of asymmetric ABCC9-encoded nucleotide binding domains determines KATP channel SUR2A catalytic activity

Abstract

Nucleotide binding domains (NBDs) secure ATP-binding cassette (ABC) transporter function. Distinct from traditional ABC transporters, ABCC9-encoded sulfonylurea receptors (SUR2A) form, with Kir6.2 potassium channels, ATP-sensitive K+ (K ATP) channel complexes. SUR2A contains ATPase activity harbored within NBD2 and, to a lesser degree, NBD1, with catalytically driven conformations exerting determinate linkage on the Kir6.2 channel pore. While homodomain interactions typify NBDs of conventional ABC transporters, heterodomain NBD interactions and their functional consequence have not been resolved for the atypical SUR2A protein. Here, nanoscale protein topography mapped assembly of monodisperse purified recombinant SUR2A NBD1/NBD2 domains, precharacterized by dynamic light scattering. Heterodomain interaction produced conformational rearrangements inferred by secondary structural change in circular dichroism, and validated by atomic force and transmission electron microscopy. Physical engagement of NBD1 with NBD2 translated into enhanced intrinsic ATPase activity. Molecular modeling delineated a complemental asymmetry of NBD1/NBD2 ATP-binding sites. Mutation in the predicted catalytic base residue, D834E of NBD1, altered NBD1 ATPase activity disrupting potentiation of catalytic behavior in the NBD1/NBD2 interactome. Thus, NBD1/NBD2 assembly, resolved by a panel of proteomic approaches, provides a molecular substrate that determines the optimal catalytic activity in SUR2A, establishing a paradigm for the structure-function relationship within the K ATP channel complex.

Figure 1

Structure of SUR2A NBD1 and NBD2 proteins. Recombinant nucleotide binding domains, NBD1 and NBD2, were expressed using a solubility enhancing NusA tag, and purified from Ni-affinity and size exclusion chromatography. (A) SDS-PAGE of Coomassie blue-stained NBD1 (86 kDa) and NBD2 (89 kDa) fusion constructs or NusA (66 kDa) alone, loaded 5 μg/protein samples, migrating at corresponding molecular weights. (B) Homogeneous NBD1 and NBD2 display monodisperse size distribution profile on dynamic light scattering, distinct from the size of NusA. (C) NBD1 and NBD2 protein size independently determined by dynamic light scattering and atomic force microscopy. Structures of NBD1 (D) and NBD2 (E) mapped at nanoscale resolution by atomic force microscopy.

Figure 2

Interaction of NBD1 and NBD2. (A) Circular dichroism spectra reveal the distinctive α-helix profile of NBD1, NBD2, and NBD1 + NBD2. Of note, the circular dichroism spectrum measured from coincubated NBD1 and NBD2 demonstrated a profile distinct from the theoretically predicted sum Σ(NBD1 + NBD2), indicating structural change upon protein–protein interaction. Inset: Coincubation of NusA with an NBD did not produce a significant change in α-helicity beyond the predicted sum of the two coincubated proteins. Units of the x and y axis are identical as in panel A. (B) Paired nanostructures of NBD1/NBD2 complexes, after incubation at 37 °C for 10 min, resolved by atomic force microscopy. (C) The probability for NBD1 and NBD2 to assemble exceeded the likelihood of NBD1 or NBD2, when incubated alone, to form nonspecific aggregates even with increase in protein concentration as determined by atomic force microscopy. Enhanced formation of NBD1/NBD2 complexes at physiological temperature. + and − indicate presence or absence of an NBD; ++ indicates presence of an NBD at a concentration >2-fold higher than in +.

Figure 3

Electron micrographs of negatively stained isolated NBD1 (A) or NBD2 (B) versus the NBD1 + NBD2 mixture (C). Note that pairing occurs only when NBD1 and NBD2 are free to interact forming heterodomain complexes as shown in (C). All bars = 25 nm.

Figure 4

NBD1/NBD2 assembly promotes intrinsic ATPase activity. (A) The ATPase activity of NBD1 or NBD2 was significantly higher than background ascribed to the NusA tag. Upon NBD1/NBD2 interaction, ATPase activity significantly increased, exceeding the sum of individual NBD1 + NBD2 catalytic activities. *, #, and & indicate significantly greater (P < 0.05) than NusA, than NusA or NBD1, and NusA, NBD1, or NBD2, respectively. Inset: The D834E mutation increased NBD1 ATPase activity approximating wildtype NBD2 catalysis. NBD1 (D834E) and NBD2, free to interact, produced a total ATPase that did not exceed the arithmetic sum of individual catalytic activities. (B) Homology structural model of the NBD1/NBD2 heterodimer, developed in the “head to tail” orientation, indicated that Walker A (W_A) and Walker B (W_B) motifs along with a signature motif delineate nucleotide docking sites. NBD1 in yellow, NBD2 in purple, and the ATPase cofactor Mg²⁺ in white. Hydrogen bonds shown as dotted lines. N- and C-termini of individual NBDs are labeled. Close-up view of ATP-binding sites at the “head” (C) and “tail” (D) of the heterodimer.