Quaternary structure of KATP channel SUR2A nucleotide binding domains resolved by synchrotron radiation X-ray scattering

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Abstract

Heterodimeric nucleotide binding domains NBD1/NBD2 distinguish the ATP-binding cassette protein SUR2A, a recognized regulatory subunit of cardiac ATP-sensitive K+ (KATP) channels. The tandem function of these core domains ensures metabolism-dependent gating of the Kir6.2 channel pore, yet their structural arrangement has not been resolved. Here, purified monodisperse and interference-free recombinant particles were subjected to synchrotron radiation small-angle X-ray scattering (SAXS) in solution. Intensity function analysis of SAXS profiles resolved NBD1 and NBD2 as octamers. Implemented by ab initio simulated annealing, shape determination prioritized an oblong envelope wrapping NBD1 and NBD2 with respective dimensions of 168 × 80 × 37 Å3 and 175 × 81 × 37 Å3 based on symmetry constraints, validated by atomic force microscopy. Docking crystal structure homology models against SAXS data reconstructed the NBD ensemble surrounding an inner cleft suitable for Kir6.2 insertion. Human heart disease-associated mutations introduced in silico verified the criticality of the mapped protein–protein interface. The resolved quaternary structure delineates thereby a macromolecular arrangement of KATP channel SUR2A regulatory domains.

Introduction

The ATP-binding cassette (ABC) transporters are evolutionarily conserved transmembrane proteins, with canonical family members characterized by ATP hydrolysis-driven substrate translocation that accounts for diverse biological processes ranging from nutrient import to toxin efflux (Dean, 2005, Linton, 2007, Linton and Higgins, 2007, Rees et al., 2009). Atypical ABC proteins have also been identified, and account for substrate translocation-independent functions, such as ion channel gating (Aittoniemi et al., 2009, Burke et al., 2008, Higgins and Linton, 2004, Moreau et al., 2008). A case in point is the sulfonylurea receptor 2A (SUR2A), a distinctive ABC protein that regulates the operation of the ATP-sensitive potassium (KATP) channels in cardiac myocytes (Alekseev et al., 2005, Ashcroft, 2006, Inagaki et al., 1995, Inagaki et al., 1996, Nichols, 2006, Wheeler et al., 2008).

Encoded by ABCC9, SUR2A belongs to the ABCC subfamily along the cystic fibrosis transmembrane conductance regulator (CFTR or ABCC7) and the multidrug resistance-related protein (MRP or ABCC1) (Biemans-Oldehinkel et al., 2006, Chutkow et al., 1996, Oram and Vaughan, 2006, Solbach et al., 2006, Yamada and Kurachi, 2005). Through physical association with the potassium channel Kir6.2 pore, SUR2A endows KATP channel complexes with a unique metabolic decoding capacity that assures linkage of the cellular energetic state with membrane excitability (Bryan et al., 2006, Dupuis et al., 2008, Lorenz and Terzic, 1999, Zingman et al., 2007). SUR2A harbors essential nucleotide binding domains – NBD1 and NBD2. Each NBD encompasses Walker A (GX4GKS/T), Walker B (Ф4DD/E; Ф represents hydrophobic residues) and linker (LSGGQ) signature motifs (Walker et al., 1982). Responsible for adenine nucleotide recognition and processing, the SUR2A NBD1/2 tandem is integral in transduction of metabolic signals to the KATP channel pore (Bienengraeber et al., 2000, Karger et al., 2008, Zingman et al., 2001, Zingman et al., 2002). Genetic mutations in SUR2A NBDs alter channel function, and human KATP channelopathies have been implicated in cardiac disease susceptibility underscoring the structural integrity of regulatory domains in optimal channel performance (Bienengraeber et al., 2004, Kane et al., 2005, Olson et al., 2007, Reyes et al., 2009, Sattiraju et al., 2008).

To date, over 50 crystal structures of isolated NBDs from both bacterial and eukaryotic ABC transporters have been resolved (Hollenstein et al., 2007, Linton and Higgins, 2007, Moussatova et al., 2008, Rees et al., 2009). Despite the wealth of information pertinent to NBD structures of canonical ABC proteins, little is known regarding atypical ABC counterparts, including the mammalian SUR2A NBDs. In part, the lack of information is due to challenges in protein expression and chaotic orientation in solution that impede crystal formation. Although a molecular model of SUR2A NBD dimers has provided an initial discrete domain map (Park et al., 2008), the actual shape underlying structural arrangement remains uncertain.

Small-angle X-ray scattering (SAXS) offers an approach to delineate supramolecular conformations (Hura et al., 2009, Petoukhov and Svergun, 2007, Putnam et al., 2007). Here, SAXS, in tandem with ab initio and rigid body model reconstruction, was applied to decipher the molecular envelope of SUR2A nucleotide binding domains in solution. We report a quaternary structural portrait of NBD1/NBD2 that provides a blueprint of structural constraints within regulatory KATP channel domains.

Section snippets

Purified SUR2A NBD1 and NBD2

Murine cDNA SUR2A (GenBank D86037; kindly provided by Dr. Seino) encoding NBD1 (D666-890) and NBD2 (G1301-K1546) were amplified by PCR, and incorporated into a modified pET-15b vector (Novagen) containing the N-terminal (His)6-tag and TEV protease cleavage site. Plasmids were transformed in the Escherichia coli Rosetta(DE3)pLysS strain (Novagen), and NBD1 as well as NBD2 proteins purified (Park et al., 2008). Pelleted cells were suspended in buffer A (50 mmol/L Tris–HCl, 50 mmol/L NaCl, 1 mmol/L

Secondary and tertiary structures from purified SUR2A NBDs

The nucleotide binding domains, NBD1 and NBD2, of the KATP channel ABCC9-encoded SUR2A were individually expressed in E. coli, refolded and purified by gel filtration chromatography. Recombinant proteins, with purity >92%, migrated at corresponding molecular weights on SDS–PAGE (Fig. 1A), and formed oligomers detected on size-exclusion chromatography (Fig. 1B). Purified NBDs displayed a distinctive secondary structure in far-UV circular dichroism spectroscopy reflecting peptide backbone

Discussion

The atypical ABC protein SUR2A serves as a regulatory subunit of heteromultimeric ATP-sensitive K+ (KATP) channels in heart muscle, in particular within the ventricle (Du et al., 2006, Flagg et al., 2008, Inagaki et al., 1996, Li et al., 2000, Nichols, 2006, Shi et al., 2005). Coupling of the pore-forming Kir6.2 with cellular energetic signaling systems relies on cooperative interaction of the SUR2A nucleotide binding domains, NBD1 and NBD2, endowing high-fidelity metabolic sensing properties

Acknowledgments

We thank the SIBYLS beamline 12.3.1 staff at the Advance Light Source (ALS) at Lawrence Berkeley National Laboratories for assistance with synchrotron data collection. We acknowledge the organizers of SXS2008 at the Advanced Photon Source (APS) for support with experiments at beamline DND-CAT. Authors express their gratitude to Drs. Georges Mer and Christina Miranda D. Correia (Mayo Clinic) for expert discussions. This work was supported by the NIH (R01 HL064822), and Marriott Heart Disease

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