Phosphorylation-dependent Changes in Nucleotide Binding, Conformation, and Dynamics of the First Nucleotide Binding Domain (NBD1) of the Sulfonylurea Receptor 2B (SUR2B)*

Background: Phosphorylation of SUR2B NBD1 activates ATP-sensitive K+ (KATP) channels. Results: Phosphorylation-dependent changes in NBD1 conformation and nucleotide binding are mimicked by removing the N-terminal tail that contains the phosphorylation sites. Conclusion: Phosphorylation disrupts interactions of the N-terminal tail with the NBD1 core, leading to increased nucleotide binding. Significance: These data provide insights into the molecular basis by which NBD1 phosphorylation activates KATP channels. The sulfonylurea receptor 2B (SUR2B) forms the regulatory subunit of ATP-sensitive potassium (KATP) channels in vascular smooth muscle. Phosphorylation of the SUR2B nucleotide binding domains (NBD1 and NBD2) by protein kinase A results in increased channel open probability. Here, we investigate the effects of phosphorylation on the structure and nucleotide binding properties of NBD1. Phosphorylation sites in SUR2B NBD1 are located in an N-terminal tail that is disordered. Nuclear magnetic resonance (NMR) data indicate that phosphorylation of the N-terminal tail affects multiple residues in NBD1, including residues in the NBD2-binding site, and results in altered conformation and dynamics of NBD1. NMR spectra of NBD1 lacking the N-terminal tail, NBD1-ΔN, suggest that phosphorylation disrupts interactions of the N-terminal tail with the core of NBD1, a model supported by dynamic light scattering. Increased nucleotide binding of phosphorylated NBD1 and NBD1-ΔN, compared with non-phosphorylated NBD1, suggests that by disrupting the interaction of the NBD core with the N-terminal tail, phosphorylation also exposes the MgATP-binding site on NBD1. These data provide insights into the molecular basis by which phosphorylation of SUR2B NBD1 activates KATP channels.


The sulfonylurea receptor 2B (SUR2B) forms the regulatory subunit of ATP-sensitive potassium (K ATP ) channels in vascular smooth muscle. Phosphorylation of the SUR2B nucleotide binding domains (NBD1 and NBD2) by protein kinase A results
in increased channel open probability. Here, we investigate the effects of phosphorylation on the structure and nucleotide binding properties of NBD1. Phosphorylation sites in SUR2B NBD1 are located in an N-terminal tail that is disordered. Nuclear magnetic resonance (NMR) data indicate that phosphorylation of the N-terminal tail affects multiple residues in NBD1, including residues in the NBD2-binding site, and results in altered conformation and dynamics of NBD1. NMR spectra of NBD1 lacking the N-terminal tail, NBD1-⌬N, suggest that phosphorylation disrupts interactions of the N-terminal tail with the core of NBD1, a model supported by dynamic light scattering. Increased nucleotide binding of phosphorylated NBD1 and NBD1-⌬N, compared with non-phosphorylated NBD1, suggests that by disrupting the interaction of the NBD core with the N-terminal tail, phosphorylation also exposes the MgATPbinding site on NBD1. These data provide insights into the molecular basis by which phosphorylation of SUR2B NBD1 activates K ATP channels.
ATP-sensitive potassium (K ATP ) 4 channels are K ϩ -selective channels present in many tissues (1). Because gating of K ATP channels depends on the cellular concentrations of ATP and ADP, K ATP channels couple cellular metabolism to membrane potential (2). Thus, K ATP channels play critical roles in several biological processes, such as insulin secretion in the pancreas (3), neurotransmitter release in the brain (3), cardiac function, and regulation of blood pressure (1).
K ATP channels are multimeric protein complexes composed of four copies of a pore-forming inward-rectifying potassium channel (Kir6.2 or Kir6.1) and four copies of a sulfonylurea receptor (SUR1, SUR2A, or SUR2B) that surround the pore (1,4). Different combinations of SUR and Kir6.x proteins combine to form K ATP channels in different tissues. The SUR proteins are members of the ATP-binding cassette (ABC) superfamily of proteins (5), which consist of a minimum of two membrane spanning domains (MSD1 and MSD2) and two nucleotide binding domains (NBD1 and NBD2) (Fig. 1A). Binding of MgATP leads to dimerization of the NBD1 and NBD2, which dissociate upon MgATP hydrolysis (6). In addition to the minimum ABC protein structure, the SUR proteins contain a third membrane spanning domain (MSD0) that is linked to the N terminus of MSD1 by the cytoplasmic L0 linker (7)(8)(9)(10). Unlike most members of the family, SUR proteins possess no known transporter activity but instead regulate gating of the Kir6.x pore. ATP binding at the Kir6.x pore results in K ATP channel inhibition, whereas nucleotide binding and/or hydrolysis at the SUR NBDs results in channel opening (11). Data on full-length channels indicate that K ATP channels open in response to MgATP binding at NBD1 and MgADP binding at NBD2 (12). The interaction of NBD2 with MgADP may result from direct nucleotide binding or hydrolysis of MgATP to MgADP at NBD2 (2,12). The SUR NBDs are critical in regulating K ATP channel gating. Mutations in the NBDs of SUR1 cause neonatal type II diabetes or hyperinsulinism (13), whereas mutations in the NBDs of SUR2A and SUR2B result in various cardiovascular disorders (14 -18).
Although nucleotide binding and/or hydrolysis at the NBDs are sufficient to induce channel currents, K ATP channel activity is also affected by phosphorylation of SUR and/or Kir6.x subunits (19 -27). In the case of vascular K ATP channels, monophosphorylation of murine SUR2B at three sites (Thr-633 and Ser-1465 (24) or Ser-1387 (26)) by protein kinase A (PKA) results in K ATP channel activation, with di-phosphorylation of Thr-633 and Ser-1465 further stimulating channel activity (24). Notably, the phosphorylation sites are conserved among species (Thr-635, Ser-1390, and Thr-1547, respectively, in human SUR2B), highlighting their importance in regulating the activity of the SUR NBDs and their subsequent control of K ATP channel function. A molecular level understanding of how phosphorylation activates K ATP channels is not available but may involve altered MgATP binding and hydrolysis and/or intraand intermolecular interactions of the NBDs, as seen for the cystic fibrosis transmembrane conductance regulator (CFTR) (28 -31).
Here, we present experiments detailing the effect of phosphorylation on the conformation and nucleotide binding of rat SUR2B NBD1. The sequence of rat SUR2B NBD1 is ϳ96% identical to human SUR2B NBD1, including the phosphorylation site Thr-632 (Thr-635 in human SUR2B), with most amino acid changes occurring in unstructured loops (32). The studies presented here employed four different SUR2B NBD1-containing constructs (Fig. 1B). 1) The complete SUR2B NBD1 (NBD1) consists of residues Ser-615-Leu-933 and is a folded and functional protein domain that possesses a predicted disordered N-terminal tail, which contains the Thr-632 phosphorylation site and a predicted disordered C-terminal tail (32). 2) SUR2B NBD1 from Ser-615-Asp-914 (NBD1-⌬C) lacks the C-terminal tail. 3) SUR2B NBD1 from Asp-665-Leu-933 (NBD1-⌬N) lacks the N-terminal tail. 4) SUR2B NBD1 from Asp-665-Asp-914 (NBD1-⌬N⌬C) lacks the N-and C-terminal tails and includes only the canonical NBD1 fold. We also used a SUR2B construct composed of the isolated N-terminal tail (Ser-615-Glu-664), herein referred to as the N-tail. We have obtained resonance assignments for NBD1 and have probed conformational changes in NBD1 with phosphorylation. Data from NMR spectroscopy and dynamic light scattering suggest that the N-terminal tail interacts with multiple residues in NBD1, including residues in the NBD2-binding site, and that phosphorylation of the N-terminal tail disrupts its interactions with the core of NBD1. Because the N-terminal tail is ϳ50 residues long ( Fig. 1), it has the potential to adopt multiple conformations, including conformations that are bound to NBD1. Fluores-cence studies demonstrate increased nucleotide binding affinity of phosphorylated NBD1 (phospho-NBD1) and NBD1-⌬N. These findings provide insights into the molecular basis by which phosphorylation of NBD1 activates K ATP channel gating.

Experimental Procedures
Protein Expression and Purification-Wild type rat SUR2B NBD1 proteins comprising residues Ser-615-Leu-933 (NBD1), Ser-615-Asp-914 (NBD1-⌬C), Asp-665-Leu-933 (NBD1-⌬N), Asp-665-Asp-914 (NBD1-⌬N⌬C), and Ser-615-Glu-664 (Ntail) were prepared as described previously (32,33). We also prepared samples of NBD1 and NBD1-⌬N with mutations of Trp residues. Briefly, the SUR2B NBD1, NBD1-⌬C, NBD1-⌬N, NBD1-⌬N⌬C, and N-tail proteins were expressed as fusions with a cleavable N-terminal His 6 15 N-labeled M9 minimal media. All protein purification steps were conducted at 4°C. The His 6 -SUMO fusion proteins were isolated using a Ni 2ϩnitrilotriacetic acid (NTA) affinity column (GE Healthcare), and the His 6 -SUMO tag was cleaved with His 6 -Ulp1 protease. The SUR2B NBD1, NBD1-⌬C, NBD1-⌬N, NBD1-⌬N⌬C, and N-tail proteins were next purified to homogeneity by size exclusion chromatography (Superdex 75, GE Healthcare), followed by a reverse Ni 2ϩ -NTA affinity column to remove small amounts of the His 6 -SUMO and/or His 6 -Ulp1 proteins that co-elute with the NBD and N-tail proteins from the size exclusion column. For the NMR phosphorylation and fluorescence studies, the SUR2B NBD1, NBD1-⌬C, NBD1-⌬N, and NBD1-⌬N⌬C proteins were exchanged into NBD1 buffer (20 mM sodium phosphate, pH 7.3, 150 mM NaCl, 2% (v/v) glycerol, 2 mM DTT) with and without ATP and MgCl 2 , as required. Samples were exchanged by dialysis against the same stock of NBD1 buffer to ensure the solution conditions of the samples were identical. For the resonance assignment experiments, the NBD1-⌬N sample was exchanged into the NBD1 buffer containing 5 mM MgATP and 5 mM DTT and lacking NaCl, as the protein is more stable without salt (33). The N-tail was exchanged into a buffer identical to the NBD1 buffer but which lacked MgATP and contained 2 mM tris(2-carboxyethyl)phosphine-HCl rather than DTT.
Backbone 1 H, 15 N, 13 C, and 13 C␣, and side chain 13 C␤ resonance assignments for NBD1-⌬N were obtained from standard triple resonance TROSY-based experiments (39,40) recorded on samples of 0.5 mM NBD1-⌬N that were uniformly 15 N-and 13 C-labeled and fractionally 2 H-labeled to ϳ50%. The triple resonance assignment data were run on an 800 MHz spectrometer equipped with a cryoprobe (QANUC NMR Facility, McGill University) or on a 600 MHz spectrometer equipped with an H(F)CN room temperature probe at 25°C. These data were supplemented with 15 N-1 H TROSY HSQC spectra recorded on samples that were either 15 N-labeled only on specific amino acids (Gly, Leu, Ser, and Val) (31,41,42) or 14 N-labeled on specific amino acids (Ala, Arg, His, Lys, Met, and Thr) and 15 N-labeled at all other positions (43). Spectra of the specifically labeled NBD1-⌬N samples were recorded at 25°C at 600 MHz. Resonance assignments of NBD1-⌬N spectra at 25°C were transferred to NBD1-⌬N and NBD1-⌬N⌬C spectra at 30°C and then subsequently to phospho-NBD1 and phospho-NBD1-⌬C spectra at 30°C. The similarity of the NBD1-⌬N spectrum to the spectra of other NBD1-containing proteins allowed for the straightforward transfer of resonance assignments for most residues. We have obtained assignments for 58 -63% of backbone HN resonances of the NBD1-containing proteins. The combination of TROSY-based triple resonance data and specifically labeled samples has allowed for the level of resonance assignment obtained for other NBDs (31). Assignments of Trp indole HN resonances were obtained by site-directed mutagenesis of NBD1 or NBD1-⌬N.
Phosphorylation of NBD1 (Ser-615-Leu-933), NBD1-⌬C (Ser-615-Asp-914), and N-tail-Phosphorylation reactions were carried out at 30°C on purified samples of NBD1, NBD1-⌬C, and N-tail (ϳ200 M) in the NBD1 buffer with 15 mM MgATP rather than 5 mM MgATP. The phosphorylation reaction was initiated by the addition of 750 units (or 0.6 M) of the catalytic subunit of protein kinase A (PKA, Promega) and was monitored by recording a series of two-dimensional 1 H-15 N TROSY-HSQC spectra at 30°C. The NBD1 phosphorylation reaction was allowed to proceed for 16 h, at which point the NBD1 sample was almost fully phosphorylated (Ͼ97%), as determined by the peak intensities of a resonance corresponding to the indole HN group of Trp-616 in the non-phosphorylated and phosphorylated states. Phosphorylation of N-tail was allowed to proceed for 6 h. Phosphorylation sites were identified by mass spectrometry following in-gel tryptic digestion (Advanced Protein Technology Centre, Hospital for Sick Children). The phosphorylated NBD1 and NBD1-⌬C were exchanged into the NBD1 buffer, which contained 5 mM MgATP, by size exclusion chromatography. The phosphorylated N-tail was exchanged into the N-tail buffer.
Fluorescence Nucleotide Binding Experiments-The K d value for binding of the fluorescent ATP analogue TNP-ATP (Molecular Probes) to phosphorylated NBD1 and NBD1-⌬N was determined and compared with binding to NBD1, as done previously (33,44). The fluorescence nucleotide binding studies were performed on a Fluoromax-4 spectrofluorimeter (Horiba-Jovin, Inc.) equipped with a Peltier unit for precise temperature control. Mg 2ϩ and ATP were removed from the NBD1 samples using size exclusion chromatography and replaced with 2.5 M MgCl 2 and 2.5 M TNP-ATP. Because of the limited solubility of nucleotide-free NBD1 samples, binding experiments were conducted using 10% (v/v) glycerol at 15°C. Binding experiments were performed by serial dilutions of the protein from 50 to 70 M, depending on the concentration eluted from the size exclusion column, to 0.8 to 2.0 M while keeping the concentrations of the MgCl 2 and TNP-ATP constant at 2.5 M each. A separate sample was generated containing MgCl 2 , TNP-ATP, and buffer only for the 0 mM NBD1 sample. Fluorescence emission spectra (from 485 to 600 nm) of TNP-ATP were recorded immediately after each sample was generated using an excitation wavelength of 465 nm, an excitation slit width of 5 nm, and an emission slit width of 7 nm. The K d value for the NBD1nucleotide complex was determined by monitoring the ratio between the fluorescence intensity at 533 nm, which corresponds to the wavelength where the fluorescence difference of free and bound TNP-ATP is at a maximum, and 600 nm to account for any nonspecific fluorescence from the protein (45). The titration data were fit to the equation where I is the fluorescence intensity ratio at a given total concentration of TNP-ATP ([TNP total ]); I ∞ is the fluorescence intensity ratio at saturation; I 0 is the fluorescence intensity ratio in the absence of ligand; K d is the dissociation constant, and [NBD1 total ] is the total concentration of SUR2B NBD1 in the reaction. Equation 1 assumes a 1:1 complex of NBD1 with TNP-ATP (46,47). Thermal Stability Measurements-The thermal stabilities of the NBD1 proteins were measured using intrinsic Trp fluorescence spectroscopy, as described previously (33). Briefly, thermal denaturation was monitored by following the change in the emission spectrum at 350 nm, the wavelength at which the difference in the fluorescence spectra of the folded and denatured proteins is at a maximum. The excitation wavelength was 295 nm, and the excitation and emission slit widths were 1 and 3 nm, respectively. The NBD1 proteins were heated from 10 to 60°C in 1°C increments, with a 1-min equilibration time at each temperature. The fluorescence emission at 350 nm was recorded at each temperature in a 0.5-ml cuvette. Thermal denaturation studies were done with 2 M NBD1 in the absence and presence of 2 mM MgATP.
Dynamic Light Scattering Studies-Dynamic light scattering experiments were performed on a Malvern Zetasizer NanoZS instrument with 100 M samples of non-phospho-NBD1, phospho-NBD1, and NBD1-⌬N in the NBD1 buffer containing 2 mM MgATP. Samples were centrifuged for 10 min at 15,000 rpm prior to each experiment. The hydrodynamic radius was determined from the average of the frequency distribution of particle sizes in three separate experiments for each sample.

Results
Identification of Phosphorylation Sites in SUR2B NBD1-Phosphorylation of SUR2B NBD1, NBD1-⌬C, and N-tail in vitro was achieved by incubating purified proteins with the catalytic subunit of PKA, the in vivo kinase for the SUR proteins (24,26). Deletion of residues Gln-915-Leu-933 from NBD1 to form NBD1-⌬C results in the disappearance of 15 intense resonances centered about 8.2 ppm in the 1 H dimension ( Fig. 2A, highlighted with a ϩ), confirming previous predictions that the C-terminal tail is disordered (32). Removal of the C-terminal tail thereby results in reduced resonance overlap in the NMR spectrum, making it possible to see additional chemical shift changes upon phosphorylation with NBD1-⌬C compared with NBD1 (see below). Although disordered, the C-terminal tail residues Gln-915-Leu-933 significantly enhance the solubility of NBD1 (32). Furthermore, residues outside the canonical NBD fold are often involved in regulation of the NBD function (28,48,49).
NMR spectra of the isolated N-tail indicate that, as predicted (32), residues Ser-615-Glu-664 are also disordered (Fig. 2B). The N-tail used here is part of the intracellular linker (Gln-600 -Glu-664) connecting transmembrane helix 11 to NBD1 (Fig. 1A). Transmembrane helix predictions (50), sequence alignments (38,44), and homology models (44) of SUR2B based on structures of other ABC proteins (51-57) indicate that transmembrane helix 11 ends at Val-599, that residues Gln-600 -Leu-607 extend transmembrane helix 11 into the cytoplasm, and that NBD1 begins at Asp-665 (Fig. 1A). Furthermore, the overlay of resonances in the N-tail spectrum with resonances in the NBD1 spectrum from residues Ser-615-Glu-664 ( Fig. 2B and see below) indicates that the presence of the folded NBD1 core does not induce folding of the N-tail. Notably, residues Leu-607-Glu-664 in SUR2B are primarily predicted as being disordered (32) by PONDR analysis (58 -60). We have also recorded spectra on longer N-tail proteins (Gln-600 -Glu-664 and Ser-608 -Glu-664). Resonances in the spectra of the N-tail from Ser-615-Glu-664 overlay with resonances in the longer N-tail proteins, indicating that extension of N-tail does not induce folding of these residues. However, NBD1containing proteins starting at residues Gln-600 or Ser-608 aggregate at the high concentrations required for NMR studies (32), as is often seen when unstructured residues are included in constructs that contain folded domains (61). Thus, for comparison with NBD1 proteins, we are using an N-tail protein beginning at Ser-615.
Phosphorylation of NBD1, NBD1-⌬C, and N-tail is monitored by changes in the chemical shift of the indole HN resonance of Trp-616 (Fig. 3, W616 indole versus W616 indole -phospho). The identity of the Trp-616 indole HN resonance in NBD1 and NBD1-⌬C is known from two-dimensional 15 N-1 H NMR spectra of a deletion mutant of NBD1 in which residues Ser-615-Arg-617 were removed (32). The overlay of the indole HN resonance from Trp-616, the only Trp in the N-terminal tail, in the spectra of NBD1 and the N-tail (Fig. 2) confirms assignment of the Trp-616 indole HN resonance. The combined chemical shift change (⌬␦ tot of 37 Hz) for the Trp-616 indole HN resonance and its location in a sparsely populated region of the spectrum allow for monitoring of the phosphorylation reaction. Chemical shift changes in the Trp-616 indole HN resonance indicate that it takes ϳ16 h to completely phosphorylate an NMR sample containing the 200 M NBD1 at 30°C (Fig. 2) and ϳ6 h to completely phosphorylate the 200 M N-tail at 30°C. Samples of NBD1-⌬C were also phosphorylated using the same protocol, but to a lower extent. According to peak intensities of the Trp-616 indole HN resonance from the non-phosphorylated and phosphorylated states, NBD1-⌬C is ϳ85% phosphorylated. We could not achieve uniform phosphorylation of NBD1-⌬C with higher temperatures due to compromised sample stability. Nonetheless, chemical shift changes observed with phosphorylation of NBD1 are also seen with phosphorylation of NBD1-⌬C (see below). Thus, information gleaned from phosphorylation of NBD1-⌬C can be applied to NBD1.
The identity of the phosphorylation sites was determined using mass spectrometry following in-gel trypsin digestion (Fig.  4A). Previous studies determined that NBD1 is phosphorylated at the PKA consensus site Thr-632, as well as at another unidentified site, as a peptide comprising residues Cys-628 -Ile-639 with a T632A mutation can still be phosphorylated by PKA (24). Our mass spectrometry data comparing non-phosphorylated and phosphorylated forms of NBD1, NBD1-⌬C, and N-tail confirm phosphorylation of Thr-632 and identified the second site as Ser-636. Residues Thr-632 and Ser-636 are located in the N-tail of NBD1 (Fig. 1). No phosphorylation sites were detected in the core of the NBD1. These data are consistent with control NMR phosphorylation experiments that show no chemical shift changes in the spectra of NBD1-⌬N and NBD1-⌬N⌬C proteins, which lack Thr-632 and Ser-636.
The phosphorylation site Thr-632 conforms to the PKA consensus site ((R/K)(R/K)X(pS/pT)) (62, 63), whereas Ser-636 comprises a non-consensus PKA phosphorylation site (Fig. 4B). Phosphorylation of non-canonical sites by PKA has been demonstrated to modulate the function of other proteins (64 -66). Furthermore, work with model peptides indicates that PKA can phosphorylate a Ser or Thr residue even if the dibasic motif is located up to eight residues N-terminal to the phosphorylation site, albeit with varying efficiency (67). Thus, the proximity of the two phosphorylation sites in SUR2B NBD1 may allow for the dibasic residues comprising the con-sensus site at Thr-632 to serve in PKA recognition of the non-consensus site Ser-636.
Phosphorylation-dependent Spectral Changes in SUR2B NBD1-In addition to the Trp-616 indole HN group, phosphorylation-dependent chemical shift changes are observed for two additional Trp indole HN and 40 backbone HN resonances in NBD1 (Fig. 5A) and for two additional Trp indole resonance and 48 backbone resonances in NBD1-⌬C (Fig. 6A). Chemical shift changes are expected for residues close in space to the phosphorylation sites. Phosphorylation of Thr-632 and Ser-636 Together, these regions form the intracellular domains. NBD1 and NBD2 are shown in blue and green, respectively. The N-terminal tail connecting MSD1 to NBD1 is shown in red, and the C-terminal tail connecting NBD1 to MSD2 is shown in yellow. Phosphorylation sites in the N-terminal tail are indicated as red circles. Scale bars on the right indicate the lengths throughout the protein as determined from homology models of SUR2B based on crystal structures of other ABC transporters (51-54, 56, 57). The length of the lines representing the L0 linker, N-terminal tail, and C-terminal tail were determined assuming that these linkers are completely disordered. Notice that because the intracellular domains are only 30 Å in length, the N-terminal tail is drawn with multiple bends to fit it into the space allocated in the two-dimensional representation. B, schematic diagram of NBD1 (residues Ser-615-Leu-933), NBD1-⌬C (residues Ser-615-Asp-914), NBD1-⌬N (residues Asp-665-Leu-933), NBD1-⌬N⌬C (residues Asp-665-Asp-914), and N-tail (residues Ser-615-Glu-664). As in A, the NBD1 core is in blue, the N-terminal extension that is missing in NBD1-⌬N is in red, and the C-terminal extension that is missing in NBD1-⌬C is in yellow. C, schematic ribbon diagram of the homology model of SUR2B NBD1 is based on the crystal structure of MRP1 NBD1 (Protein Data Bank code 2CBZ (95)). The coloring is as in A and B, with the exception that the different subdomains in the NBD1 core are colored in different shades of blue. The ␣/␤ subdomain, which contains the MgATP-binding site, is in dark blue; the ␤-sheet subdomain is in cyan, and the ␣-helical subdomain is in light blue. The side chain of Trp-616 is shown in cyan, and the Q-loop (also known as the ␥-phosphate loop) is in magenta. The N-terminal tail is shown in an extended conformation because there were no restraints placed on these residues in the modeling. All structure figures were created using MOLMOL (96). SEPTEMBER 11, 2015 • VOLUME 290 • NUMBER 37

Phosphorylation-dependent Changes in SUR2B NBD1
introduces two negative charges each into otherwise polar but uncharged side chains, and thus it significantly changes the chemical environment for residues close to Thr-632 and Ser-636. The cyan circles in Figs. 5A and 6A highlight phosphorylation-dependent chemical shift changes common to NBD1 and NBD1-⌬C, and the magenta squares highlight phosphorylation-dependent chemical shift changes that are specific to each protein.
Analysis of the NMR spectra indicates that phosphorylation of the N-terminal tail affects both disordered and structured residues in NBD1 and NBD1-⌬C. Of the backbone HN resonances that exhibit chemical shift changes, 6 in NBD1 and 10 in NBD1-⌬C have intense signals and are centered about 8.2 ppm in the 1 H dimension (Figs. 5A and 6A, respectively). Comparison of spectra of NBD1, NBD1-⌬N, NBD1-⌬C, NBD1-⌬N⌬C, and N-tail indicates that these intense HN resonances are from the residues in the N-terminal tail (highlighted with * in Figs. 5 and 6), as is expected considering the location of the phosphorylation sites, and not the C-terminal tail. Furthermore, the quality of the NBD1-⌬N⌬C spectrum indicates that the struc-tural boundaries of the NBD1 core in SUR2B are from residues Asp-665-Asp-914 (Fig. 6B). Some of the disordered resonances that exhibit chemical shift changes are seen only with phosphorylation of NBD1-⌬C (Fig. 6A, magenta squares). However, these resonances may also be present in the spectra of NBD1 and phospho-NBD1 but may be overlapped by the additional disordered residues from the C-terminal tail in NBD1. We cannot assign the remaining disordered resonances that change with phosphorylation to the N-terminal tail due to resonance overlap in this region of the spectrum. The chemical shift change of the indole HN resonance from Trp-616, which is also located in the disordered N-terminal tail (Fig. 1C), demonstrates that phosphorylation affects residues far from Thr-632 and Ser-636 in the primary sequence. Chemical shift changes of the Trp-616 indole would not be expected if the N-terminal tail is completely disordered and residues Trp-616, Thr-632, and Ser-636 were independent of one another.
In keeping with the long range effects seen with the Trp-616 indole HN resonance change, phosphorylation of the disordered N-terminal tail also affects multiple structured residues    for several residues that are located at the predicted NBD1/ NBD2 dimer interface (Gln-753, Trp-756, Gly-811, His-841, Glu-904, and His-905). Thus, the phosphorylation-dependent changes of the functional NBD1 residues may be coupled to altered NBD1 activity. Similar changes are seen upon phosphorylation of NBD1-⌬C. However, the decreased overlap afforded by the nonphospho-and phospho-NBD1-⌬C spectra allow identification of additional residues that exhibit phosphorylation-dependent changes. These include Asn-729 and Val-730, which are located in a loop connecting the ␤-sheet and ␣/␤ subdomains, and Lys-907 located NBD1/NBD2 dimer interface. Phosphorylation also causes chemical shift changes in the indole HN resonances of Trp-727 and Trp-756 in NBD1 (Fig. 5A) and of Trp-756 and Trp-906 in NBD1-⌬C (Fig. 6A).
Analysis of the homology model of SUR2B NBD1 indicates that phosphorylation affects residues that are up to ϳ40 Å away

Phosphorylation-dependent Changes in SUR2B NBD1
SEPTEMBER 11, 2015 • VOLUME 290 • NUMBER 37 from the end (i.e. residue Glu-664) of the N-terminal tail (Figs. 5A and 6A). One hypothesis for the long range chemical shift changes is that the N-terminal tail possesses residual structure and interacts with multiple regions in the core of NBD1 and that phosphorylation alters the structure of the N-terminal tail and/or its interactions with the NBD core. The interaction of   Fig. 2. The spectrum of non-phospho-NBD1 is in the foreground and is colored as in Fig. 2. The spectrum of phospho-NBD1 is shown in the background. The red and green peaks in the phospho-NBD1 spectrum correspond to the black and blue resonances, respectively, in the non-phospho-NBD1 spectrum. A, panel ii, selected region of the overlaid spectra shown in A. Resonances highlighted by cyan circles highlight phosphorylation-dependent chemical shift changes that also occur in NBD1-⌬C. Magenta squares represent chemical shift changes seen with phosphorylation of NBD1 only. Assignment of selected resonances that exhibit phosphorylation-dependent chemical shift changes is shown. A, panel iii, chemical shift mapping of phosphorylation-dependent changes to specific residues in NBD1. The ribbon diagram is colored cyan for residues with resonance assignments, white for residues with no resonance assignments, and gray for residues that have assignments in NBD1-⌬N but not phospho-NBD1. The location of Pro residues is also indicated in gray, as these residues do not give signals in the NH-based NMR experiments, such as the two-dimensional 15 N-TROSY-HSQC. C␣ atoms of residues that exhibit phosphorylation-dependent chemical shift changes are shown as spheres colored from light pink, to highlight the smallest changes (⌬␦ tot ϭ 8 -10 Hz), to magenta for the largest changes (⌬␦ tot Ն25 Hz). The C␣ atoms of Thr-632 and Ser-636 are colored yellow. B, panel i, comparison of two-dimensional 15 N-TROSY-HSQC spectra of non-phospho-NBD1 and NBD1-⌬N (250 M) The spectrum of non-phospho-NBD1 is shown in the background in black and blue, as in A. The spectrum of NBD1-⌬N is in the foreground. The magenta and cyan peaks in the NBD1-⌬N spectrum correspond to the black and blue resonances, respectively, in the non-phospho-NBD1 spectrum. B, panel ii, selected region of the overlaid spectra shown in A. Green circles highlight resonances that also display chemical shift changes with phosphorylation. Assignment of selected resonances that exhibit chemical shift changes with deletion of the N-terminal tail only are shown. B, panel iii, chemical shift mapping of N-terminal tail deletion to specific residues in NBD1. The ribbon diagram is colored as in A, except that residues colored in gray represent residues that have been assigned in spectra of NBD1-⌬N recorded at 800 MHz at 25°C that cannot be transferred to NBD1-⌬N spectra recorded at 600 MHz at 30°C. Selected residues that exhibit changes with removal of the N-terminal tail, only, are highlighted. disordered proteins or disordered residues of a folded protein with multiple binding sites has been observed for many intrinsically disordered proteins (30,72).
Chemical shift mapping of phosphorylation-dependent changes in NBD1 and NBD1-⌬C is possible because most back-bone and Trp indole HN chemical shift assignments of NBD1-⌬N can be transferred to spectra of phospho-NBD1 and phospho-NBD1-⌬C. The remaining backbone resonances (2 in NBD1 and 11 in NBD1-⌬C) that display chemical shift changes are not mapped to specific residues either because they have  Fig. 5A. The solution conditions for each sample are identical to those described in Fig. 5. A, panel ii, selected region of the overlaid spectra shown in A. Cyan circles highlight resonances that also display chemical shift changes with phosphorylation of NBD1. Magenta squares highlight resonances that display phosphorylation-dependent chemical shift changes in NBD1-⌬C only. Phosphorylation-dependent chemical shift changes are seen for backbone resonances (black peaks) and resonances from N⑀H⑀ groups in Arg side chains (green peaks). A, panel iii, chemical shift mapping of phosphorylation-dependent changes to specific residues in NBD1-⌬C. The ribbon diagram is colored based on available resonance assignments for phospho-NBD1-⌬C and chemical shift changes. The C␣ atoms for selected residues that show phosphorylation-dependent chemical shift changes in NBD1-⌬C, but not NBD1, are highlighted. B, panel i, comparison of two-dimensional 15 N-TROSY-HSQC spectra of non-phospho-NBD1-⌬C and NBD1-⌬N⌬C (320 M) The spectrum of non-phospho-NBD1-⌬C is shown in the background in black and blue, as in A. The spectrum of NBD1-⌬N⌬C is in the foreground. The magenta and cyan peaks in the NBD1-⌬N⌬C spectrum correspond to the black and blue resonances, respectively, in the non-phospho-NBD1-⌬C spectrum. B, panel ii, selected region of the overlaid spectra shown in A. Green circles highlight resonances that also display chemical shift changes with phosphorylation. B, panel iii, chemical shift mapping of N-terminal tail deletion to specific residues in NBD1-⌬C. Residues are colored based on available resonance assignments for NBD1-⌬N⌬C and chemical shift changes. The C␣ atoms for selected residues that exhibit chemical shift changes with deletion of the N-terminal tail in NBD1-⌬C, but not NBD1, are highlighted. SEPTEMBER 11, 2015 • VOLUME 290 • NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 22707 not been assigned in the spectra of NBD1-⌬N or because assignments could not be transferred from the spectra of NBD1-⌬N to the spectra of phospho-NBD1 and/or phospho-NBD1-⌬C.

Phosphorylation-dependent Changes in SUR2B NBD1
Removal of the N-terminal Region Mimics Phosphorylation of NBD1-To determine how phosphorylation alters the conformation of NBD1, we compared spectra of NBD1, phospho-NBD1, and NBD1-⌬N (Fig. 5) as well as NBD1-⌬C, phospho-NBD1-⌬C, and NBD1-⌬N⌬C (Fig. 6). Almost every residue in NBD1 and NBD1-⌬C that displays phosphorylation-dependent chemical shift changes also shows chemical shift changes upon removal of the N-terminal tail. Chemical shift changes common to phosphorylation and N-terminal tail removal are highlighted by green circles in spectra in Figs. 5B and 6B, respectively, and are also mapped onto the NBD1 model. Additional chemical shift changes in NBD1 and NBD1-⌬C with removal of the N-terminal tail (e.g. Ala-667-Thr-671 and Asp-688 -Arg-690 resonances in Fig. 5B) arise from changing the N-terminal residue in the protein from Ser-615 to Asp-665. This change brings the charged N terminus closer to the NBD1 core and, as expected, causes multiple chemical shift changes for residues in the ␤-sheet subdomain. In some cases, these chemical shift changes observed with removal of the N-terminal tail were also seen with phosphorylation, but the ⌬␦ tot value calculated for these residues was not statistically significant. Additional differences observed between phosphorylation and removal of the N terminus may result from overlap in spectra of NBD1 and phospho-NBD1 versus NBD1 and NBD1-⌬N (and their NBD1-⌬C counterparts), which limits our ability to detect those chemical shift changes. For example, more chemical shift changes are seen with removal of the N-terminal tail compared with phosphorylation for residues in the loop composed of residues Asn-729 to Ser-747.
Notably, a few resonances show linear chemical shift changes (73) when comparing non-phospho-NBD1, phospho-NBD1, and NBD1-⌬N (Fig. 7). Linear chemical shift changes result from fast exchange on the NMR time scale between two distinct states. In the case of SUR2B NBD1, one state consists of the N-terminal tail bound to the NBD1 core, as in non-phospho-NBD1, and the second state has the N-terminal tail fully displaced from the NBD1 core, as in NBD1-⌬N. The intermediate position of phospho-NBD1 resonances indicates that phosphorylation only partly displaces the N-terminal tail from the NBD1 core.
Further differences between non-phospho-NBD1 versus phospho-NBD1 and NBD1-⌬N are observed when comparing spectra of the proteins at different temperatures (Fig. 8).
Although the spectra of non-phospho-NBD1 show evidence of broadening in backbone and Trp side chain resonances as the temperature is decreased from 30 to 20°C, spectra of phospho-NBD1 and NBD1-⌬N change very little with temperature. A similar trend is seen for non-phospho-NBD1-⌬C, phospho-NBD1-⌬C, and NBD1-⌬N⌬C. For clarity, only a subset of resonances affected by temperature in spectra of non-phospho-NBD1 are highlighted. For example, a number of backbone and Trp indole HN resonances that are observable in spectra of non-phospho-NBD1 at 30°C (Fig. 8, solid cyan circles) are missing in the spectra of non-phospho-NBD1 at 25°C and/or 20°C (Fig. 8, dashed cyan circles highlight the position of these missing resonances). Notably, some of the non-phospho-NBD1 resonances that completely disappear at 25°C and/or 20°C have strong intensities at 30°C. In contrast, most of these resonances are observable in the spectra of phospho-NBD1 at lower temperatures, and all are observable in NBD1-⌬N in spectra recorded at 25 and 20°C. Furthermore, phosphorylation of NBD1 and removal of the N-terminal tail from NBD1 results in the appearance of additional peaks (Fig. 8, magenta circles). These resonances are likely also present in the spectra of nonphospho-NBD1, but their low intensities do not allow them to be unambiguously distinguished from background noise.
The temperature-induced broadening of non-phospho-NBD1 is reversible, and we have already determined that NMR samples of non-phospho-NBD1 at 250 M concentration are monomeric (44). Thus, we do not attribute the broadening to NBD1 dimerization or aggregation. Because broadening in NMR spectra can arise from microsecond to millisecond time scale protein motions, these temperature-dependent spectral changes imply differences in NBD1 dynamics, possibly caused by differential interactions of the N-terminal tail with the NBD1 core in the different proteins. In fact, many of the nonphospho-NBD1 resonances that experience temperature-induced broadening also exhibit chemical shift changes with phosphorylation and removal of the N-terminal tail. These include residues in the ␤-sheet subdomain (Ile-691, Tyr-726, and Trp-727), the ATP binding ␣/␤ subdomain (Gly-694, Leu-696, Ile-714, Ala-751, Trp-756, Leu-758, Phe-830, Leu-31, Lys-859, and Asp-876), and the ␣-helical subdomain (Thr-767, Gly-769, Tyr-777, and Cys-784). Interactions of the N-terminal tail in nonphospho-NBD1 result in multiple NBD1 core residues experiencing s-ms motions, leading to overall broadening of the spectra. These microsecond-millisecond motions are quenched by the limited interactions of the phosphorylated N-terminal tail and NBD1 core, resulting in reduced broadening in the spectra of phospho-NBD1. The absence of the N-terminal tail binding to the NBD1 core in NBD1-⌬N eliminates broadening in spectra of NBD1-⌬N.
Disruption of the N-terminal tail interactions with the NBD1 core by phosphorylation of Thr-632 and Ser-636 would lead to a less compact protein. To test this model, we measured the hydrodynamic radius of non-phospho-and phospho-NBD1 using dynamic light scattering (Fig. 9). Phosphorylation of Thr-632 and Ser-636 increased the hydrodynamic radius of NBD1 by ϳ20% from 3.07 Ϯ 0.06 to 3.66 Ϯ 0.07 nm, consistent with our hypothesis. For comparison, NBD1-⌬N has a hydrodynamic radius of 2.35 Ϯ 0.07 nm. The hydrodynamic radius of NBD1-⌬N (30 kDa) is slightly smaller than the hydrodynamic radius of ovalbumin (45 kDa) of 2.81 Ϯ 0.01 nm. Together, the NMR and the light scattering data suggest that phosphorylation of Thr-632 and Ser-636 disrupts interactions of the N-terminal segment with the core of NBD1.
Phosphorylation Increases the Nucleotide Binding Affinity of NBD1-PKA phosphorylation of SUR2B at NBD1 and NBD2 results in increased K ATP channel activity (24,26). Because K ATP channel gating is also activated by MgATP binding and hydrolysis at the NBDs, we sought to determine whether phosphorylation alters the nucleotide binding properties of NBD1. We used a fluorescently labeled ATP analogue, TNP-ATP, to probe binding of nucleotide to NBD1 (33,44). TNP-ATP exhibits very low fluorescence in the absence of NBD1. Addition of increasing amounts of non-phosphorylated NBD1, phosphorylated NBD1, and NBD1-⌬N resulted in a concentration-dependent and saturable increase in TNP-ATP fluorescence (Fig.  10A). Fitting of the binding curves reveals a K d value of 8.4 Ϯ 1.1 M for the interaction of non-phospho-NBD1 with TNP-ATP, as measured previously (33,44). In comparison, there is a modest increase in the nucleotide binding affinity for phospho-NBD1 (K d 6.3 Ϯ 0.2 M) and a 2-fold increase in the nucleotide binding affinity of NBD1-⌬N (K d 4.4 Ϯ 0.7 M). Mg 2ϩ was required for these experiments, as no nucleotide binding could be detected without Mg 2ϩ , as assessed by TNP-ATP fluorescence binding experiments and NMR titrations with ATP. Note that NBD1 samples for these Mg 2ϩ -free studies were dialyzed against a buffer lacking Mg 2ϩ and containing Chelex 100 resin to remove any residual metals.
The intermediate increase in the MgATP binding affinity of phospho-NBD1 is consistent with a model in which phosphorylation of Thr-632 and Ser-636 disrupt interactions of the N-terminal region with the NBD1 core to partly expose the ATP-binding site, with removal of these residues in NBD1-⌬N fully exposing the ATP-binding site. The increased nucleotide binding observed for the isolated and monomeric phospho-NBD1 and NBD1 in which the N-terminal tail is completely displaced from binding the protein core (NBD1-⌬N) likely has a greater effect in the intact channel, which contains four SUR2B proteins. Increased MgATP binding at NBD1 will likely also increase binding of NBD1 and NBD2, which ultimately increases K ATP channel gating (74).
We used intrinsic tryptophan fluorescence to monitor thermal unfolding of the NBD1 proteins (Fig. 10B). In the absence of MgATP, phospho-NBD1 and NBD1-⌬N have melting profiles and melting temperatures (T m ϳ30°C) that are similar to that previously seen for NBD1 (33). However, in the presence of MgATP, the proteins have different thermal stabilities. MgATP

Discussion
Our NMR studies demonstrate that phosphorylation of Thr-632 and Ser-636 affects residues in the disordered N-terminal tail and the structured NBD1 core. Residue Thr-632 corresponds to a canonical PKA phosphorylation site that is conserved among species, and residue Ser-636 is a variable noncanonical PKA phosphorylation site. Residue Ser-636 in rat and murine SUR2B corresponds to a Pro residue (Pro-639) in human SUR2B, which would promote alternative conformations of the N-terminal tail (77,78). Furthermore, Pro-639 is part of exon 14 (residues Gln-638 -Lys-673) that is missing in a naturally occurring splice isoform, known as human SUR2C (79), which further illustrates the regulatory nature of the N-terminal tail. Our NMR data showing that removal of the N-terminal tail mimics phosphorylation of NBD1 suggest that phosphorylation of Thr-632 and Ser-636 disrupts some of the transient interactions of the N-terminal tail with NBD1, with complete disruption achieved by removal of the N-terminal tail in NBD1-⌬N (Fig. 11). Chemical shift mapping indicates that these transient interactions occur with residues that exhibit conformational changes with MgATP binding, such as residues in the Q-loop and at the interface between the ␣/␤ and ␣-helical subdomains (68 -71). The more "open" state of NBD1 achieved by phosphorylation and N-terminal tail deletion would result in increased exposure of the MgATP-binding site, thereby explaining the increased nucleotide binding affinity of phospho-NBD1 and NBD1-⌬N. Chemical shift mapping also indicates that transient interactions occur with NBD1 residues that bind NBD2. Phosphorylation promotes stabilization of NBD1 into a conformation that allows NBD1 to make productive interactions (80), such as with MgATP and/or NBD2, leading to increased K ATP channel opening (12). Thus, the model (Fig.  11) is consistent with the demonstrated phosphoryla-tion-mediated activation of K ATP channels (24,27). Because SUR2B NBD2 is also phosphorylated, additional studies using NBD2 are necessary. Furthermore, phosphorylation of the NBDs may affect their interactions with other regions of SUR2B, such as the coupling helices, and/or the Kir6.2 channel pore. Therefore, a full understanding of the molecular basis of the mechanism by which multisite phosphorylation regulates K ATP channels will require additional studies to address these questions. Notably, phosphorylation of SUR1 NBD2 also regulates K ATP channel gating (20,21), suggesting that SUR subunit phosphorylation is a common regulatory mechanism to control K ATP channel gating.
Phosphorylation has been observed for many ABC transporters across all subfamilies, which implies that phosphorylation is a general mechanism for regulating ABC transporter function. ABC transporters are phosphorylated at specific sites in the NBDs and/or in the NBD1-MSD2 linker by various kinases, with the effects of phosphorylation depending on the specific site phosphorylated (81). For example, phosphorylation of ABCA1 at NBD2 is required for full cholesterol transport activity, whereas phosphorylation of the NBD1-MSD2 linker affects protein stability (81)(82)(83). ABCB1, which is also known as P-glycoprotein and is associated with multidrug resistance in cancers (84), is also phosphorylated at a number of sites in the NBD1-MSD2 linker by PKA, protein kinase C, and casein kinase 2 (81). PKC phosphorylation increases the ATPase activity of ABCB1 and regulates efflux of anions.
The best characterized ABC protein in terms of phosphorylation is CFTR. Like the SUR proteins, CFTR is a member of the C-subfamily of ABC transporters (5). CFTR is phosphorylated by PKA and PKC at multiple sites in the disordered R region, which links NBD1 to MSD2, and by PKA at one site in the regulatory insert located within NBD1 (85)(86)(87)(88). As with SUR2B, phosphorylation of CFTR disrupts transient interactions of the regulatory insert and R region with the NBD1 core (29,31) to promote a more open CFTR NBD1 conformation (31,89,90), which then permits binding of NBD1 with coupling helix 1 (31). Furthermore, as with SUR2B, phosphorylation of CFTR leads to increased MgATP binding and hydrolysis, and subsequent channel activation (28,48).
Studies of CFTR highlight additional functional consequences of phosphorylation, which may also be relevant for SUR2B. There are differences in the conformation of phosphorylated CFTR with the severe cystic fibrosis-causing mutation ⌬F508 (31, 91), which partly explain the molecular defects of mutant CFTR compared with the wild type protein (91)(92)(93)(94). Furthermore, deletion of the regulatory insert, which mimics phosphorylation (31), increases the response of mutant CFTR to small molecule correctors (91). Phosphorylation also disrupts interactions of the R region with NBD2, but it enhances interactions of the R region with the C terminus of CFTR and the accessory proteins STAS (sulfate transporters and antisigma factor) and 14-3-3 (30). As observed for CFTR, phosphorylated SUR NBDs bearing disease-causing mutations may also possess a different conformation from phosphorylated wild type NBDs. In addition, phosphorylation may affect interactions of the SUR2B NBDs with other proteins, such as the Kir6.2 pore, or with therapeutics, such as pinacidil which binds NBD1 The NBD1 core is shown as a rounded blue square, and the N-terminal tail that contains Thr-632 and Ser-636 is shown as a red line with phosphorylation indicated by open red circles labeled with a P as applicable. Different interactions of the N-terminal tail with NBD1 are displayed schematically on a gradient from a "closed state," in which the N-terminal tail is bound to the NBD1 core, to an "open state," where the N-terminal tail is removed and cannot bind the NBD1 core. Phosphorylation of the N-terminal tail results in an intermediate state in which the N-terminal tail makes limited interactions with the NBD core. (44). Thus, data presented here on the conformation of wild type SUR2B NBD1 provide a platform to address these additional questions.
Author Contributions-E. D. A. generated all non-phospho-NBD1, phospho-NBD1, and NBD1-⌬N samples and conducted the nucleotide binding and dynamic light scattering experiments. E. D. A. and J. P. L.-A. recorded NMR data on non-phospho-NBD1, phospho-NBD1, and NBD1-⌬N samples. C. P. A. generated the non-phospho-NBD1-⌬C, phospho-NBD1-⌬C, and NBD1-⌬N⌬C samples and recorded all NMR spectra of these proteins. C. R. S. generated the N-tail samples and together with E. D. A. recorded NMR spectra of these proteins. M. S. generated initial NBD1-⌬N proteins and performed the initial feasibility studies. V. K. and E. D. A. analyzed the NMR data. Figures were generated by V. K. and E. D. A. The manuscript was written by V. K., with contributions from E. D. A., and edited by V. K., E. D. A., and C. P. A.