Quaternary structure of KATP channel SUR2A nucleotide binding domains resolved by synchrotron radiation X-ray scattering
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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2014, International Journal of CardiologyCitation Excerpt :Mutations that alter nucleotide binding or compromise the cooperative nucleotide-dependent NBD interaction within the regulatory channel subunit, such as the S1402C and V734I described in this study, are primary candidates for metabolic sensing deficits underlying cardiac. Using synchrotron radiation X-ray scattering, we recently demonstrated that V734I of SUR2A can disrupt protein–protein interaction critical for the structural integrity of the KATP channel complex [20]. ABCC9 mutations have been reported to be associated with atrial fibrillation, dilated cardiomyopathy with tachycardia, and to influence susceptibility to MI in the human heart [33].
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2013, International Journal of CardiologyCitation Excerpt :This implies that the mutation does not affect transduction of MgADP binding into channel opening but that it affects MgATP binding and/or hydrolysis. A recent study indicated that the region to which V734I maps is a site potentially critical for the interaction between NBD1 of adjacent SUR2 subunits [62]. In general, a number of KATP channel pathogenic mutations have been shown to cluster in regions involved in interaction between subunits [12,63].
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2013, Journal of Biological ChemistryCitation Excerpt :Low resolution three-dimensional reconstruction of a KATP channel complex suggested a compact architecture, with the central Kir6.2 tetramer intimately surrounded by four SUR1 subunits (13). Crystal structures of isolated NBDs of related ABC proteins indicate that they form dimers (14), and an x-ray scattering study suggested that the NBDs of SUR2A can themselves form tetramers of such dimers, with a maximum dimension of ∼18 nm, reasonably consistent with packing predicted by EM observations (13, 15). GFP can be fused to KATP channel subunits without compromising channel function; such subunits are visible in vivo within the secretory pathway of transfected cells and form functional KATP channels with characteristic properties (16–19).