Phosphorylation Alters the Residual Structure and Interactions of the

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Phosphorylation alters the residual structure and interactions of the regulatory L1 linker connecting NBD1 to the membrane-bound domain in SUR2B Clarissa Rana Sooklal, Jorge Pedro Lopez-Alonso, Natalia Papp, and Voula Kanelis Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00503 • Publication Date (Web): 01 Oct 2018 Downloaded from http://pubs.acs.org on October 16, 2018

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Biochemistry

Phosphorylation alters the residual structure and interactions of the regulatory L1 linker connecting NBD1 to the membrane-bound domain in SUR2B Clarissa R. Sooklal,1,2 Jorge López-Alonso,1,2,* Natalia Papp,1 and Voula Kanelis1,2,4 1

Department of Chemistry, University of Toronto, Toronto, ON, Canada, M5S 3H8

2

Department of Chemical and Physical Sciences, University of Toronto Mississauga,

Mississauga, ON, Canada, L5L 1C6 3

Department of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada

M5S 3G5 *

Current address: Structural Biology Unit, CIC bioGUNE, Parque Tecnológico de Bizkaia,

48160 Derio, Bizkaia, Spain

Corresponding Author *Voula Kanelis DV 4042 Department of Chemical and Physical Sciences University of Toronto Mississauga 3359 Mississauga Road N. Mississauga, ON, Canada, L5L 1C6 Tel: 1-905-569-4542 Fax: 1-905-828-5425 Email: [email protected]

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Abstract ATP-sensitive potassium (KATP) channels in vascular smooth muscle are comprised of four poreforming Kir6.1 subunits and four copies of the sulfonylurea receptor 2B (SUR2B), which acts as a regulator of channel gating. Recent cryo-EM structures of the pancreatic KATP channel show a central Kir6.2 pore that is surrounded by the SUR1 subunits. Mutations in the L1 linker connecting the first membrane spanning domain and the first nucleotide binding domain (NBD1) in SUR2B cause cardiac disease, however this part of the protein is not resolved in the cryo-EM structures. Phosphorylation of the L1 linker, by protein kinase A, disrupts its interactions with NBD1, which increases the MgATP affinity of NBD1 and KATP channel gating. To elucidate the mode by which the L1 linker regulates KATP channels, we have probed the effects of phosphorylation on its structure and interactions using NMR spectroscopy and other techniques. We demonstrate that the L1 linker is an intrinsically disordered region of SUR2B, but possesses residual secondary and compact structure, both of which are disrupted with phosphorylation. NMR binding studies demonstrate that phosphorylation alters the mode by which the L1 linker interacts with NBD1. The data show that L1 linker residues with the greatest α-helical propensity also form the most stable interaction with NBD1, highlighting a hot-spot within the L1 linker. This hot-spot is the site of disease-causing mutations and is associated with other processes that regulate KATP channel gating. These data provide insights into the mode by which the phospho-regulatory L1 linker regulates KATP channels.

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Introduction ATP sensitive potassium (KATP) channels are K+ selective channels that link the metabolic activity of cells to their membrane potential (1). KATP channels regulate many biological processes, including insulin secretion in the pancreas (2-7), neurotransmitter release in the brain (1, 8), as well as cardiac function and blood pressure (1, 6, 9, 10). KATP channels consist of four pore-forming inward-rectifying K+ channel (Kir6.1 or Kir6.2) proteins and four regulatory sulfonylurea receptors (SUR1 or SUR2 isoforms) (11-20). Different combinations of Kir6 and SUR proteins form KATP channels in different tissues (1, 22). For example, KATP channels in the pancreas are formed by Kir6.2 and SUR1 subunits, while KATP channels in cardiac tissues vary in subunit composition, depending on the cell type. KATP channels in vascular smooth muscle are formed predominantly by SUR2B and Kir6.1 subunits, whereas KATP channels in the cardiac ventricle are formed from SUR2A and Kir6.2. SUR proteins are members of the ATP binding cassette (ABC) superfamily of transporters (23) and contain the hallmark ABC core structure of two membrane-spanning domains (MSD1, MSD2) and two nucleotide binding domains (NBD1, NBD2) (Supplementary Figure 1). In addition to the core 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 (24-27). As in other C subfamily ABC proteins, such as the cystic fibrosis conductance regulator (CFTR) and multidrug resistance proteins (MRPs) (23), SUR proteins contain asymmetric nucleotide binding sites (28). The canonical catalytic Glu in the Walker B motif is an Asp in NBD1. Thus, NBD1 is capable only of binding MgATP, while NBD2 is catalytically active and both binds and hydrolyzes ATP. Unlike most members of the family, SUR proteins possess no known transporter activity but instead regulate gating of the Kir6 pore (2, 5, 13, 29-33). KATP channels are closed when ATP binds the Kir6 pore (30, 33) and are open when MgATP is bound to NBD1 and MgADP is bound to NBD2 (30, 32, 33). The interaction of NBD2 with MgADP may result from direct nucleotide binding or hydrolysis of MgATP to MgADP at NBD2. KATP channel activity is also affected by phosphorylation of the SUR and/or Kir6 subunits (34-41). In the case of vascular KATP channels, SUR2B is phosphorylated by protein kinase A (PKA) at T632, S636, S1387, and S1465 (38, 40, 42), leading to increased channel activity (38, 40). Residues T632 and S636 are located in the linker connecting MSD1 to NBD1 (Supplementary Figure 1), which we refer to as

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the L1 linker, while residues S1387 and S1465 are located within NBD2. The L1 linker plays an important role in KATP channel activity. In addition to phosphorylation, the L1 linker is the site of heme binding, which increases KATP channel gating (43), and possesses disease-causing mutations (10, 44, 45). Recent cryo-EM structures of the pancreatic KATP channel (16-20), in the closed state, have been determined with ATP bound to the Kir6.2 pore, MgATP and MgADP bound to the SUR1 NBDs, and/or the sulfonylurea drug glibenclamide, which closes the channel (46, 47), bound to transmembrane helices in core ABC structure of SUR1. As expected, the four Kir6.2 subunits form the pore and are surrounded by the SUR1 subunits (Supplementary Figure 2). The KATP channel adopts either propeller or quatrefoil structures (16-20). The propeller form (Supplementary Figure 2A) is observed when samples of the KATP channel are formed by four individual Kir6.2 and four individual SUR1 subunits (17-19), or by four Kir6.2-SUR1 fusions in which the C terminus of SUR1 is connected to the N terminus of Kir6.2 by a 6-residue (16) or 39-residue linker (20). The propeller structures of the KATP channels formed from SUR1-Kir6.2 fusions were solved in the presence of Mg nucleotides so that the NBDs are dimerized (16, 20), whereas the NBDs are separated in other propeller structures (17-19). The quatrefoil structure (Supplementary Figure 2B) has only been observed for KATP channels formed from the Kir6.2SUR1 fusions containing the 6-residue linker, and also solved in the presence of Mg nucleotides so that the NBDs are dimerized (16). In all structures, interactions are observed between SUR1 MSD0 and the outer transmembrane helix in Kir6.2. The propeller and quatrefoil structures differ in the orientation of the SUR1 subunits with respect to the Kir6.2 pore. In the propeller form (Supplementary Figure 2A), there are no interactions between the ABC core of SUR1, including the NBDs, with the Kir6.2 pore (16-20). The SUR1 L0 linker, which adopts a helical structure, connects the SUR1 ABC core to the Kir6.2 cytoplasmic domains. In contrast, in the quatrefoil form of the KATP channel (Supplementary Figure 2B), interactions are observed between SUR1 NBD2 and the cytoplasmic domains of the Kir6.2 subunits (16). Further, the SUR1 L0 linker in the quatrefoil form is disordered and not observed in the cryo-EM structure. Structural changes in the L0 linker may lead to KATP channel activation (16). The SUR1 L1 linker is also disordered and not resolved in any of the structures (16-20) (Supplementary Figure 3). Notably, the linker connecting MSD1 and NBD1 in SUR1 and SUR2 proteins is much longer than that for other proteins in the C subfamily of ABC proteins, including CFTR and MRP1. The

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Biochemistry

longer SUR L1 linker may be reflective of its involvement in KATP channel regulation. In addition to phosphorylation sites found in the SUR2B L1 linker, the SUR1 L1 linker possesses endoplasmic reticulum recognition sequences that control trafficking of pancreatic KATP channels (48). Intrinsically disordered regions (IDRs) in proteins containing folded domains, such as the SUR L1 linker, are common regulatory sites in ABC proteins. Further, as with the SUR2B L1 linker, phosphorylation of other IDRs regulates the ATPase and/or transport activity of the parent ABC protein, including CFTR and the cholesterol transporter ABCA1 (49-53). IDRs, as well as intrinsically disorder proteins (IDPs), do not adopt a single structure, but exist as ensembles of conformations from states with residual structure to those that are fully unstructured (54-56). IDPs and IDRs also possess different conformational states in complex with their binding partners, with some IDPs and IDRs adopting one conformation upon binding a folded protein while other IDPs and IDRs form dynamic complexes with their binding partners (57). IDPs and IDRs are also sites of post-translational modifications that regulate the function of the disordered protein (58). The dynamic nature of IDPs and IDRs precludes their structural characterization by X-ray crystallography and cryo-EM, requiring solution-state techniques such as NMR and other spectroscopies (59). Because the L1 linker is a 50-residue long IDR in SUR2B, (Supplementary Figure 1), it has the potential to adopt multiple conformations, some of which are bound to the SUR NBDs (42). Note that, although the L1 linker is identical in SUR2A and SUR2B, regulation of KATP channel activity by phosphorylation of the L1 linker has only been shown for SUR2Bcontaining channels and has not been studied in SUR2A-containing channels (38, 40, 41). Hence we will discuss our results in the context of SUR2B. Previous NMR studies on a protein comprised of the L1 linker and NBD1 (L1-NBD1, Supplementary Figure 1) showed that phosphorylation of the L1 linker disrupted its transient interactions with multiple sites on NBD1, leading to increased MgATP binding (42). However, because NMR resonance assignments were obtained on a protein comprised of SUR2B NBD1 alone, no residue-level information about the L1 linker was available. Thus, the effect of phosphorylation on the structural properties of the L1 linker and its interaction with NBD1 could not be addressed. In that previous study, the L1 linker was referred to as the N-terminal tail of NBD1 (Supplementary Figure 1C). However, we now prefer the term L1 linker in order to

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reflect its location in the full length SUR protein and because data in this paper show that it retains its structural and NBD1 interaction properties when removed from NBD1. To address the lack of information on the L1 linker, we have conducted NMR spectroscopy and other solution-state studies on the SUR2B L1 linker in the absence and presence of NBD1. As in previous studies, we are using rat SUR2B in our experiments, as the sequence of rat SUR2B is 96% identical to human SUR2B, including the canonical PKA phosphorylation site T632 (T635 in human SUR2B). In addition, rat SUR2B is 99% identical to murine SUR2B, which was the SUR2B species for which phosphorylation was studied in cells. Our NMR studies indicate that phosphorylation affects L1 linker residues near and far from the phosphorylation sites in the isolated L1 linker and in L1-NBD1. Spectroscopic and biochemical studies indicate that the L1 linker possesses residual secondary and compact structure that is disrupted with phosphorylation, which leads to differential interactions of the nonphosphorylated and phosphorylated L1 linker with NBD1. Notably, there are cardiac diseasecausing mutations in or near the region of the L1 linker that possesses the greatest α-helical propensity and forms the most stable interactions with NBD1, defining this region as a hot-spot in KATP channel regulation. NMR studies show that these mutations have differing effects on the L1 linker. Together, the studies of the isolated L1 linker and L1 linker in the presence of NBD1 provide the foundation for understanding the mode by which the L1 linker regulates KATP channel function.

Materials and Methods Protein Expression and Purification SUR2B L1 linker (residues S615-D664) was expressed as an N-terminal 6xHis-SUMO fusion protein (60) using a pET26b-derived expression vector (61). The 6xHis-SUMO-L1 linker fusion was expressed in E. coli BL21(DE3) Codon Plus cells grown in M9 minimal media with 15

N-NH4Cl or with 15N-NH4Cl and 13C-glucose to generate 15N-labelled or 15N/13C-labelled

proteins, respectively. Cells were grown at 37 ºC until an OD600 of 0.8 was reached, at which point the incubating temperature was reduced to 18 ºC. After 30 min of incubation at 18 ºC, gene expression was induced with 0.75 mM isopropyl β-D-thiogalactoside (IPTG). After 16 hours, cells were harvested by centrifugation and pellets were stored at -20 ºC.

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Biochemistry

Protein purification was conducted at 4 ºC. The SUR2B L1 linker was purified using a procedure similar to that used for purification of SUR1 and SUR2 NBD1 proteins (42, 62-64). Cell pellets from a 1 L culture were resuspended in 15 mL of lysis buffer (20 mM Tris HCl, pH 7.6, 100 mM arginine, 150 mM NaCl, 2 mM β-mercaptoethanol, 5% (v/v) glycerol, 0.2% (v/v) Triton X-100, 2 mg/mL deoxycholic acid, 1 mg/mL lysozyme, 5 mM 6-aminocaproic acid, 5 mM benzamidine, and 1 mM PMSF). The cells were lysed by sonication and centrifuged at 18, 000 g for 40 min to remove cellular debris. The cell lysate, which contained the soluble 6xHis-SUMO-L1 linker, was passed through a 0.45 µM syringe filter and loaded onto a 5 mL High Performance Ni2+-NTA affinity column (GE Healthcare) that was pre-equilibrated with equilibration buffer (20 mM Tris HCl pH 7.6, 150 mM NaCl, 5 mM imidazole, 5% (v/v) glycerol, and 2 mM β-mercaptoethanol). The Ni2+-NTA column was washed with 5-6 column volumes of equilibration buffer and the 6xHis-SUMO-L1 linker was eluted in 5 mL fractions with elution buffer (20 mM Tris HCl pH 7.6, 150 mM NaCl, 400 mM imidazole, 5% (v/v) glycerol, and 2 mM β-mercaptoethanol). Eluents containing the 6xHis-SUMO-L1 linker fusion protein were combined and diluted 3-fold to reduce the final imidazole concentration to less than 150 mM. The 6xHis-SUMO tag was removed from the L1 linker with the addition of 6xHisUlp1 protease. The resultant mixture was concentrated using a 1 kDa molecular weight cutoff (MWCO) centrifugal filter (Pall) to 3 mL and loaded onto a size exclusion column (Superdex 75 10/300, GE Healthcare) that was pre-equilibrated with size exclusion buffer (20 mM Tris HCl, pH 7.6, 150 mM NaCl, 5% (v/v) glycerol, and 2 mM β-mercaptoethanol). Fractions containing the L1 linker were collected, combined, and purified to homogeneity by a reverse Ni2+-NTA affinity column in 20 mM Tris HCl, pH 7.6, 150 mM NaCl, 25 mM imidazole, 5% (v/v) glycerol, and 2 mM β-mercaptoethanol. The reverse Ni2+-NTA affinity step is necessary to remove remaining 6xHis-SUMO contaminants that were not separated in the gel filtration step. Confirmation of the isolation and purity of the L1 linker was determined by SDS-PAGE, mass spectrometry, and amino acid analysis. Mass spectrometry and amino acid analysis were conducted by the SPARC facility at the Hospital for Sick Children. For NMR studies, the purified L1 linker was buffer exchanged into NMR buffer (20 mM Na+ phosphate, pH 7.2, 150 mM NaCl, 2% (v/v) glycerol, and 2 mM DTT) using a 1 kDa MWCO centrifugal filter (Pall) or by size exclusion chromatography (Superdex 75). Protein concentration was determined by A280 readings of samples in 6 M guandinium HCl using an

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extinction coefficient of 8380 M-1cm-1. The extinction coefficient was calculated using ε280 nm = (# Trp)(5500) + (# Tyr)(1490) + (# Cys)(125) (65). SUR2B NBD1 (D665-L933) samples were generated following established protocols (42).

Phosphorylation of SUR2B L1 linker Phosphorylation reactions were carried out at 30 °C on purified L1 linker (200 µM) in the L1 linker NMR buffer containing 15 mM ATP and 10 mM MgCl2. As done for SUR2B L1NBD1 (42), phosphorylation reactions were initiated by the addition of 1000 units (0.8 µM) of the catalytic subunit of protein kinase A (PKA, Promega) and was monitored by recording a series of 2 hour 2D 1H-15N HSQC spectra at 30 °C. Phosphorylation was confirmed by mass spectrometry on tryptic fragments of non-phosphorylated and phosphorylated L1 linker (SPARC, Sick Kids Hospital). The phosphorylated L1 linker was isolated from ATP and PKA, and exchanged into the NMR buffer using size exclusion chromatography.

Circular dichroism Circular dichroism (CD) spectra of the non-phosphorylated and phosphorylated L1 linker proteins were recorded at 12 °C on an Aviv 250 CD spectrometer (Aviv Biomedical Inc., Lakeview, NJ) with a bandwidth of 1 nm and using a 1 mm path length quartz cell. Each spectrum was averaged from a total of five scans and was blank corrected. Samples contained 50 µM non-phosphorylated or phosphorylated L1 linker in 20 mM Na+ phosphate pH 7.2, 150 mM NaCl, 2% (v/v) glycerol, and 2 mM TCEP, with increasing concentrations of 2,2,2trifluoroethanol (TFE).

Analytical size exclusion chromatography Purified non-phosphorylated and phosphorylated L1 linker (100 µl, 200 µM) were applied to a size exclusion column (Superdex 75) that was pre-equilibrated in the NMR buffer. The column was run at a flow rate of 0.5 ml/min. The column was calibrated with blue dextran and the proteins conalbumin, carbonic anhydrase, ovalbumin, ribonuclease A and aprotinin (Gel Filtration Calibration Kit LMW, GE Healthcare). Samples of non-phosphorylated and

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Biochemistry

phosphorylated L1 linker (100 µL, 200 µM) were also run on the size exclusion column in NMR buffer containing 6 M guanidinium HCl and a flow rate of 0.5 ml/min.

Dynamic light scattering Dynamic light scattering (DLS) experiments were performed in triplicate on a Zetasizer Nano Series Nano-2S (Malvern Instruments, UK) at 12 °C. Samples contained 50 µM of nonphosphorylated and phosphorylated L1 linker in NMR buffer. Samples were centrifuged for 10 minutes at 21,000g 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.

NMR Spectroscopy 15

N-HSQC spectra (66) of non-phosphorylated and phosphorylated L1 linker were

recorded at 12 °C or 30 °C on a 600 MHz Varian VNMRS spectrometer equipped with a H(F)CN room temperature probe and actively-shielded z-gradients, whereas 15N-TROSY-HSQC spectra (67) were recorded on a 600 MHz Varian INOVA equipped with a H(F)CN cryo-probe and actively-shielded z-gradients . Chemical shift changes with phosphorylation of L1 linker were determined by calculating the combined chemical shift difference in Hz, ∆δtot, from the equation ∆δtot = (∆δHN2 + ∆δN2)0.5 (62, 64, 68). When assessing chemical shift differences, only resonances exhibiting a significant combined chemical shift difference (∆δtot), which is greater than the average of all ∆δtot values plus one standard deviation are considered. For NMR data presented in this paper, only ∆δtot ≥ 7 Hz are considered. Backbone 1H, 15N, 13C, and 13Cα, and side chain 13Cβ resonance assignments for nonphosphorylated L1 linker were obtained from standard triple resonance experiments (69, 70) recorded on samples of 0.2 mM L1 linker that were uniformly 15N- and 13C-labeled. The triple resonance assignment data were run on an 700 MHz Agilent DD2 700 spectrometer equipped with a 5 mm Xsens cryoprobe at 10 °C. The NMR resonance assignment data were supplemented with 15N-1H HSQC spectra recorded on samples that were either 15N-labeled only on Gly and Ser (42, 71) or 14N-labeled on Lys and 15N-labeled at all other positions (71). Chemical shifts were referenced to DSS for all spectra (72). NMR spectral data were processed using NMRPipe/NMRDraw (73) and were analyzed with NMRView (74).

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Results The SUR2B L1 linker is an intrinsically disordered region The available cryo-EM structures of the pancreatic KATP channel demonstrate that the L1 linker in SUR1 is an intrinsically disordered region (IDR) that lacks a stable, folded structure (16-20). Although the sequence identity between SUR1 and SUR2B is high in the folded regions of the proteins (e.g. the membrane-spanning domains and NBDs), there is variability in the sequences of the IDRs such as the L1 linker. However, the SUR2B L1 linker is expected to also be disordered, considering that 60 % of the residues in the SUR2B L1 linker are charged or polar. Further, analysis of the SUR2B sequence by PONDR (75, 76) demonstrates that the SUR2B L1 linker is disordered, and that the disordered residues in SUR2B are analogous to the disordered residues in the SUR1 L1 linker (Supplementary Figure 1B).

SUR2B L1 linker is disordered in non-phosphorylated and phosphorylated states Phosphorylation of the L1 linker in vitro was achieved by incubating the purified protein with the catalytic subunit of protein kinase A (PKA), the in vivo kinase for SUR2B (38, 40). Mass spectrometry analysis following in gel trypsin digestion indicated that phosphorylation was achieved at T632 and S636, as previously determined from mass spectrometry data of SUR2B NBD1 proteins containing the L1 linker (L1-NBD1) (42). Analysis of resonance intensities in NMR spectra indicates that we routinely obtain complete phosphorylation of T632 and almost complete (~80-90 %) phosphorylation of S636. Residue S636 does not conform to a canonical PKA phosphorylation site, consistent with its sub-stoichiometric phosphorylation (42). Substochiometric phosphorylation has also been observed for other disordered proteins (77, 78). Two-dimensional 15N-HSQC spectra of the non-phosphorylated and phosphorylated L1 linker have limited dispersion in the 1H dimension, with almost all backbone amide 1H resonances having chemical shifts of 7.9 to 8.6 ppm, and sharp resonances (Figure 1). Resonances for L1 linker residues in spectra of L1-NBD1 also display limited dispersion and sharp line shapes (42). The limited dispersion of resonances in the 1H dimension is a characteristic feature in the NMR spectra of disordered proteins and IDRs, in which each amide 1

H nucleus experiences on average the same chemical environment. In contrast, backbone amide

1

H resonances in folded proteins have chemical shifts from ~7.0 to 10.0 ppm (79). The narrow

line shape (or sharpness) of the resonances is attributed to the rapid and nearly independent

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motion for each residue in a disordered protein so that each HN bond vector experiences a short rotational correlation time (59). Additionally, the L1 linker elutes from a size exclusion column at the volume expected for elution of globular proteins of ~14 kDa, rather than proteins of ~6 kDa that are the size of the L1 linker (Supplementary Figure 4). The earlier elution of the L1 linker is also consistent with its disordered nature and larger hydrodynamic radii of the ensemble of the L1 linker conformers compared to globular proteins of the same molecular weight (54-56, 59). Notably, the presence of the L1 linker causes SUR2B and SUR2A NBD1 proteins to elute earlier than expected for a 36 kDa protein (63), the molecular weight of SUR2 L1-NBD1 proteins, in keeping with the disordered nature of the L1 linker in the L1-NBD1 (42). In order to map the chemical shift changes in the L1 linker upon phosphorylation, we obtained resonance assignments for 91% of 1HN, 84% of 15N, 98% of 13Cα, and 98% of 13Cβ nuclei in non-phosphorylated L1 linker using standard triple resonance experiments and specifically-labeled samples (see Materials and Methods). Phosphorylation of the L1 linker results in the appearance of resonances at 8.79 and 9.13 ppm in the 1H dimension, in addition to the complete loss of the signal for T632 and nearly complete loss in the signal for S636, described above (Figure 1). PKA phosphorylation of Ser and Thr residues C-terminal to the consensus sequence has been shown to modulate the activity of other proteins (80-83). Even partial phosphorylation of S636 may be important to impart changes in structure and interactions to the L1 linker as NMR spectra of an L1-NBD1 containing a Glu at position 632 to mimic phosphorylation of T632 did not show any chemical shift changes common to phosphorylated L1-NBD1 (42). Residue S636 is conserved between rat and murine SUR2B, but is altered to a Pro (Pro 639) in human SUR2B. However, there is a Thr residue at position 641 that may serve as a second PKA phosphorylation site in human SUR2B, as PKA can phosphorylate a Ser or Thr residue that is up to eight residues C-terminal of the canonical site (83). The downfield shifted resonances (Figure 1A, cyan circles) may be derived from the phosphorylated T632 and S636 residues, as seen for other phosphorylated proteins (77, 78, 8486). The phosphorylation-induced downfield shift for these resonances is due to presence of the phosphate group, which adds two negative charges per residue (87). Unfortunately, the phosphorylation-dependent resonances and most other resonances in the phosphorylated L1 linker spectra could not be assigned because of low NMR signals in the triple resonance assignment experiments, likely due to increased amide exchange in the phosphorylated protein

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(see below). The lower intensity of the resonance at 9.13 ppm is also likely due to amide exchange. Nonetheless, the available non-phosphorylated L1 linker resonances allow for mapping of phosphorylation-dependent chemical shift changes to specific L1 linker residues in two dimensional 15N-HSQC spectra. Figure 1. The SUR2B L1 linker is disordered in non-phosphorylated and phosphorylated states. (A) Comparison of the 1H-15N-HSQC spectra of the non-phosphorylated (250 µM) and phosphorylated L1 linker (100 µM). The spectrum of the nonphosphorylated L1 linker is in black and in the background, while that of the phosphorylated L1 linker is in red and in the foreground. Chemical shifts for each spectrum were referenced to 4,4dimethyl-4-silapentane-1-sulfonic acid (DSS) (72). The resonance labeled g614 is associated with a Gly from the vector that remains after removal of the 6xHisSUMO fusion partner. The two downfield shifted resonances that appear with phosphorylation are highlighted with cyan circles. (B) Combined chemical shift difference between the non-phosphorylated and phosphorylated L1 linker as a function of residue number. The red line shows significant chemical shift changes, which are defined as values higher than the average of all ∆δ(tot) plus one standard deviation. ∆δ(tot) values for residues T632 and S636 are depicted as greater than for other residues, as they experience the largest phosphorylationdependent chemical shift changes. Considering the ∆δ(tot) value for the indole HN for W616, which is smaller than for the phosphorylation sites, the ∆δ(tot) values for T632 and S636 are at least 40 Hz. The absence of a bar indicates that data could not be analyzed for those residues.

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Biochemistry

Phosphorylation results in multiple chemical shift changes in the spectrum of the L1 linker (Figure 1). As expected, phosphorylation-dependent chemical shift changes are observed for residues near the phosphorylation sites, such as K630, in addition to T632 and S636. Changes can not be determined for K629, H631, G633, K635, and I639 because of lack of resonance assignments or spectral overlap. Chemical shift changes are also seen for the backbone HN resonances of the N- and C-terminal residues S615-E620, L649, T663, and the indole HN resonance of W616. Phosphorylation-dependent chemical shift changes of the W616 indole HN resonance were also observed for L1-NBD1 (42). Note that resonances for residues S627-T632, and S636, which encompass the phosphorylation sites, suffer from loss of signal intensity and increased width of the NMR resonance, termed resonance broadening, and thus assignments are missing for some of these backbone 1HN and 15N resonances (e.g. C628, H631, G633). Resonance broadening in the spectra may result from sampling of stabilized conformations of the non-phosphorylated L1 linker, at low populations, on a µs-ms timescale, as seen for other intrinsically disordered proteins (77, 78). However, it is unlikely that these SUR2B residues adopt a stable structure in solution, as they are part of a larger disordered region in the L1 linker. As described earlier, the absence of the corresponding SUR1 L1 linker in the cryo-EM structures (16-19), also suggests that these regions are disordered in the context of intact cardiovascular KATP channels. There are many phosphorylation-dependent chemical shift changes that are common to the isolated L1 linker and to the L1 linker within the context of the L1-NBD1 protein, in addition to the indole HN resonance of W616 reported previously (42). Comparison of L1 linker and L1NBD1 spectra at 30 °C indicates that phosphorylation of L1-NBD1 results in the appearance of a peak at 8.67 ppm in the 1H dimension, which corresponds to the peak at 8.79 ppm in the spectrum of the phosphorylated L1 linker at 12 °C (Supplementary Figure 5, labeled a). The weaker resonance at 9.13 ppm in the phosphorylated L1 linker spectrum at 12 °C is not visible at 30 °C, likely due to the increased amide exchange of this resonance at the higher temperature. L1 linker and L1-NBD1 spectra recorded at 30 °C are compared, as 30 °C is the temperature at which high quality spectra of L1-NBD1 are acquired (42). Phosphorylation of L1-NBD1 also causes chemical shift changes in the backbone HN resonances of L1 linker residues W616, R617, and L649. Identification of the additional phosphorylation-dependent chemical shift changes in the L1-NBD1 spectrum is possible because

13


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of the L1 linker resonance assignments reported here. Notably, while resonances for W616 and R617 display similar phosphorylation-dependent chemical shift changes for the isolated L1 linker and L1-NBD1, the resonance for L649 displays a much larger chemical shift change in phosphorylated L1-NBD1. The close proximity of L649 to residues in the structured NBD1 core may be the cause for the greater chemical shift for L649 in L1-NBD1 compared to the isolated L1 linker. The other phosphorylation-dependent changes in the L1 linker and L1-NBD1 spectra could not be assigned to specific L1 linker residues on account of the difference in the spectra of the L1 linker at 30 °C and 12 °C, or because of overlap in the L1-NBD1 spectrum (Supplementary Figure 5 and reference (42)). Nonetheless, the similarity of changes in spectra of L1 linker and L1-NBD1 spectra indicates that studies of the isolated L1 linker will provide insights into how phosphorylation affects this regulatory region in SUR2B. As with other IDPs and IDRs (58, 77, 78), the residual structure of the L1 linker may change with phosphorylation and/or disease-causing mutations.

The intrinsically disordered L1 linker possesses residual secondary structure The availability of NMR resonance assignments allowed for determination of the fraction of secondary structure elements sampled by non-phosphorylated L1 linker using the secondary structure propensity (SSP) program (88). NMR resonances provide a direct measure of the secondary structure of individual residues in proteins. 13Cα and 13Cβ chemical shifts were used as inputs for the SSP program, as they are the most useful resonances for assessing residual secondary structure in disordered proteins (88). In folded proteins, SSP scores of +1 and -1 for consecutive residues indicate the presence of stable α-helices and β-strands, respectively. For IDRs and IDPs, the magnitudes of the positive and negative SSP scores for consecutive residues indicate the percentage of conformers in the disordered state that are in α-helical or β-strand conformations. SSP analysis shows that many regions of the L1 linker possess residual secondary structure (Figure 2A). Non-phosphorylated L1 linker residues S615-R617, E620, E626, S627, and H648-R657 have greater than 5% α-helical conformations, while residues L649-R657 possess as much as 11% - 35% α-helical structure. In comparison, regions with secondary structure propensity of 5 % and greater in other IDRs and IDPs mediate their interactions with other proteins (77, 89-91). Notably, the region of the L1 linker sequence from L649-R657 contains a consecutive stretch of five residues comprised of E, Q, A, and R, which

14


Page 14 of 50

Page 15 of 50

score high on the helix propensity score (92). Further, residues L649-L658 are also predicted to have significant α-helical propensity by the program AGADIR (93-95) (Figure 2B). The lower values from AGADIR compared to SSP scores have also been observed for regions with αhelical propensity in other disordered proteins (86). In addition to residues with significant αhelical conformations, there are some residues in the non-phosphorylated L1 linker (H631, S636P644) that have β-strand conformations of 8-18%. Note that the available data indicates that the phosphorylation sites T632 and S636 sample different secondary structures in nonphosphorylated L1 linker. While S636 possesses 8% β-strand conformations, T632 does not significantly sample any secondary structure conformations.

0.3

0.2

0.1

0

-0.1

-0.2

W616 T618 E620 T622 P624 E626 C628 K630 T632 V634 S636 P638 N640 K642 P644 R646 H648 D650 Y652 Q654 R656 L658 P660 E662 E664

Secondary structural propensity β-strand propensity α-helical propensity

A

Residue

B 12 10

8 6

4

2


% helix (AGADIR)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Residue

15


Figure 2. The SUR2B L1 linker possesses secondary structure propensity. (A) Secondary structure propensity (SSP) scores, which were calculated using 13Cα and 13Cβ chemical shifts and averaged over a sliding window of five residues, are shown as a function of residue number for the nonphosphorylated L1 linker. The black open circles on the bottom of the panel indicate the phosphorylation sites at T632 and S636. The absence of a bar indicates that data could not be analyzed for those residues. Schematic representation of the secondary structure propensity of the L1 linker as derived from the SSP values is shown below the plot. Consecutive residues with α-helical propensity are depicted as cylinders while consecutive residues with β-strand propensity are shown as arrows. The cylinders and arrows are shaded according to the SSP values for residual secondary structure elements. (B) AGADIR calculation (93-95) depicting the helical behavior of nonphosphorylated L1 linker residues.

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Few 13C resonance assignments were obtained for the phosphorylated L1 linker due to poor signal in the triple resonance data, possibly because of greater amide exchange of resonances in the phosphorylated protein at pH 7.2. A pH of 7.2 was chosen so that data on the isolated L1 linker proteins could be compared to data on L1-NBD1 proteins (42). It is possible that greater signal intensity could be achieved by recording NMR spectra at pH values < 7.2 that would enable additional 13C resonances assignments in both non-phosphorylated and phosphorylated L1 linker to be obtained. However, the available 13Cα and 13Cβ resonance assignments for the phosphorylated L1 linker indicate that residues L649-R657 retain their helical propensity upon phosphorylation, although SSP scores decrease by 2% - 6 % for residues at the ends of this residual α-helix. Note that residues L649-R657 possess the greatest α-helical propensity in non-phosphorylated L1 linker. The significant α-helical propensity of L649-R657 in phosphorylated L1 linker allowed for decreased amide exchange, and assignment of these resonances.

Phosphorylation reduces the 2°° structure propensity and compact structure of L1 linker Because only partial 13Cα and 13Cβ resonance assignments of the phosphorylated L1 linker were obtained, we decided to use circular dichroism (CD) spectroscopy to determine the effect of phosphorylation on the residual structure of the L1 linker, as has been done for other IDRs (96, 97). The CD spectra of the non-phosphorylated and phosphorylated L1 linker are typical of what is observed for primarily disordered proteins (Figure 3), consistent with the NMR data. The small negative values for the mean residue ellipticity at wavelengths < 220 nm is also consistent with the residual structure of the L1 linker. CD spectra of the non-phosphorylated L1 linker in the presence of increasing amounts of trifluoroethanol (TFE) have increasingly negative ellipticity values at 206 and 222 nm, indicating an increase in α-helical and β-strand structure in the protein (Figure 3A). TFE induces the formation of secondary structures in regions of proteins with secondary structure propensity (98, 99). Notably, although CD spectra of the nonphosphorylated and phosphorylated L1 linker in the absence of TFE are similar, there is no change in the CD spectra of the phosphorylated L1 linker with addition of TFE (Figure 3B), suggesting that the phosphorylated L1 linker possesses lower secondary structure propensity compared with the non-phosphorylated L1 linker.

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mean residue ellipticity (deg cm2 dmol-1)

A non-phosphorylated L1 linker

4000 2000 0 -2000 -4000 -6000

Figure 3. Phosphorylation decreases secondary structure propensity of the L1 linker. Circular dichroism (CD) spectra of (A) non-phosphorylated L1 linker and (B) phosphorylated L1 linker with increasing concentrations of trifluoroethanol (TFE). The protein concentration was 50 µM. Each spectrum is the average of 5 scans and is blank corrected against a buffer containing the same amount of TFE used for the protein sample.

0% TFE 20% TFE 30% TFE 40% TFE 50% TFE

-8000 -10000 -12000 190

200

210

220 230 wavelength (nm)

240

250

260

B mean residue ellipticity (deg cm2 dmol-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

phosphorylated L1 linker

4000 2000 0 -2000 -4000 -6000 -8000


-10000 -12000 190

200

210


240

250

260

The combination of the change in secondary structure propensity and long-range chemical shift changes observed with phosphorylation prompted us to examine the effect of phosphorylation on the tertiary conformation of the L1 linker. Analytical size exclusion chromatography shows a difference in elution profile of the non-phosphorylated and phosphorylated L1 linker (Figure 4A). The earlier elution of the phosphorylated L1 linker (12.8 mL) compared to that of the non-phosphorylated L1 linker (13.1 mL) indicates that the phosphorylated protein is less compact than the non-phosphorylated protein. We also measured the hydrodynamic radii of the two proteins using dynamic light scattering. Phosphorylation increases the hydrodynamic radius of the L1 linker by greater than 2-fold, from 0.86 nm for the non-phosphorylated L1 linker to 2.15 for the phosphorylated L1 linker. Notably, under

17


Biochemistry

denaturing conditions both the non-phosphorylated and phosphorylated proteins elute from the size exclusion column at 10.5 ml (Figure 4B), which is significantly earlier than the elution of the non-phosphorylated or phosphorylated L1 linker under native conditions, indicating that neither state adopts a random coil conformation in the native state. Further, confirmation of the residual structure of the L1 linker comes from Trp fluorescence quenching studies that probe exposure of W616. The Stern-Volmer constant for acrylamide quenching of the single Trp in the non-phosphorylated L1 linker is much lower (9.7 ± 0.3 M-1) than for N-acetyl-Ltryptophanamide (23.7 ± 1.7 M-1), which mimics a free Trp residue, indicating that the residual structure of the isolated L1 linker partly buries residue W616. Thus, the size exclusion, dynamic light scattering, and fluorescence quenching data indicate that the L1 linker possesses residual

mAU (at 280 nm)

35.0 30.0 25.0 20.0

a

kD

a

kD

a kD

6. 5

13 .7

A

29 .0

75 .0

kD

a

and compact tertiary structure that is altered, but not eliminated with phosphorylation.

Native analytical SEC non-phosphorylated L1 linker phosphorylated L1 linker VC = 23.56 ml

15.0 10.0 5.0 0.0

B

45.0

Denaturing analytical SEC

40.0

non-phosphorylated L1 linker phosphorylated L1 linker

35.0 mAU (at 280 nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 50

VC = 23.56 ml

30.0 25.0 20.0 15.0 10.0 5.0 0.0 9.0

9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 Elution volume (ml)

18


Figure 4. Phosphorylation reduces the compactness of the L1 linker. Analytical gel filtration chromatography of non-phosphorylated L1 linker (light grey lines) and phosphorylated L1 linker (dark grey lines) in native buffer (A) and in 6M Gdm HCl (B). For each trace, 100 µl sample of 200 µM protein was applied to a 24 ml Superdex 75 column. The lines at the top of the upper panel indicate the elution volume of standards.

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Biochemistry

Phosphorylation alters the mode by which the L1 linker binds NBD1 Previous data on NBD1 containing the L1 linker (termed L1-NBD1, see above) demonstrated that phosphorylation partly disrupted the interaction of the L1 linker with NBD1 (42). However, because resonance assignments were not previously available for the L1 linker residues in L1-NBD1, that study could not determine which L1 linker residues mediate the interaction with NBD1. Thus, here we added unlabeled NBD1 (lacking L1 linker residues) to 15

N-labeled L1 linker to determine which residues in the L1 linker bind NBD1. Addition of the

unlabeled NBD1 results in peak intensity changes and small chemical shift changes for specific resonances in L1 linker spectra (Figure 5 and Supplementary Figure 6). Note that the dispersion in the 1H dimension of the spectrum of L1 linker bound to NBD1 does not change from that seen for the spectrum of apo L1 linker (from 7.9 to 8.6 ppm), demonstrating that the L1 linker is also disordered when bound to NBD1. If binding of NBD1 induced a disorder-to-order transition in the L1 linker, the peak intensities of all L1 linker resonances would broaden and possess similar intensities (see below). Further, the spectrum of the L1 linker bound to NBD1 would show increased dispersion in the 1H dimension that is typical of folded proteins (58, 91, 100). Small chemical shift changes are expected for dynamic complexes, such as those between IDPs or IDRs with folded proteins (57, 77, 91) like the L1 linker with NBD1. However, the intensities (i.e. peak heights) of disordered protein NMR resonances change when an IDP or IDR binds a folded protein (59, 77, 90, 91). Whether intensities of L1 linker resonances increase or decrease upon NBD1 binding depends on whether specific L1 linker residues directly bind NBD1 or interact with other L1 linker residues that bind NBD1. Decreased resonance intensity (termed resonance broadening) will be observed for L1 linker residues that interact with NBD1 directly, as these tumble more slowly from their association with the much larger NBD1. L1 linker residues that do not bind NBD1 directly but that interact with L1 linker residues that do bind NBD1 may also appear to tumble more slowly, and hence these resonances will also broaden. The overall L1 linker resonance line shape can also change if NBD1 binding alters interactions within the L1 linker and by dissociation of the L1 linker residues from NBD1. For example, if two residues in the free L1 linker interact and NBD1 binding to one of the L1 linker residues disrupts the interaction between the L1 linker residues, different intensity changes will be seen for the NMR resonances of those two L1 linker residues. The NMR resonances for the L1 residue that binds NBD1 will decrease, as explained above, while the resonance of the L1

19


Biochemistry

residue that does not bind NBD1 may increase in intensity. In contrast, L1 linker resonances that are not affected by NBD1 binding will not experience any changes in intensity. A

B

Figure 5. Phosphorylation of the L1 linker alters its 119 R656 interactions with NBD1. (A) Comparison of V634 120 E662 120.0 selected region of the R646 spectra of non121 D650 R657 E662 phosphorylated L1 linker 120.5 E662 Y674 D650 D650 (40 µM) in the absence 122 W616 L658 and presence of NBD1 R617 121.0 (393 µM) with 5 mM 123 MgCl N640 2, 5 mM ATP, and 5 8.55 8.50 8.55 8.50 L649 A655 mM DTT in NMR buffer A661 124 1 1H (ppm) H (ppm) at 12 °C. The spectrum of K637 Q635 the L1 linker in absence 125 of NBD1 is in black and 8.6 8.4 8.2 8.0 1H (ppm) in the background, while the spectrum of the L1 linker in the presence of C NBD1 is in blue and in 2.0 non-phosphorylated L1 +/- NBD1 the foreground. (B) phosphorylated L1 +/- NBD1 1.8 Traces through the 1.6 approximate center of 1.4 two L1 linker resonances 1.2 is shown at the top of each spectrum, 1.0 illustrating the line shape 0.8 and demonstrating the 0.6 difference between L1 0.4 linker resonances that do 0.2 not broaden with addition 0.0 of NBD1 (E662, left) compared to L1 linker resonances that do broaden (D650, right). HN resonances (backbone or side chain) (C) Plot of the ratio of the peak intensities for 15Nlabeled nonphosphorylated (grey) and phosphorylated L1 linker (red) with and without NBD1. The schematic showing the secondary structure propensity from Figure 2 is shown at the bottom of the plot for comparison. Peak intensities for apo and NBD1bound L1 linker were calculated at the chemical shifts of each peak, as NBD1 binding causes chemical shift changes in some resonances. Error bars for each peak intensity ratio are determined by error propagation of the noise level in the spectra. The resonance intensities of sequential L1 linker residues that bind NBD1 broaden to the same extent, even if the intensities of the individual resonances in the free and NBD1-bound L1 linker spectra differ (e.g. R646 and L649). The absence of a bar indicates that data could not be analyzed for those residues. Note that the intensity ratio of g614 is 0.82 +/- 0.1, indicating that the g614 and the remaining vector derived amino acids do not interact with NBD1. Data is not available for H648 in the phosphorylated L1 linker due to resonance overlap. In addition, changes in L649 are ambiguous. It is possible that the L649 resonance in the phosphorylated L1 linker is completely broadened with NBD1 binding, as no signal is seen for this resonance in the NBD1-bound state. It is also possible that the L649 resonance shifts with NBD1 binding, as there is an unassigned resonance in spectra of phosphorylated L1 linker with NBD1 (Supplementary Figure 6A, labeled i). Using the greatest decrease in the peak intensity ratio 15N (ppm)

15N (ppm)

non-phosphorylated L1 non-phosphorylated L1 + NBD1

W616 T618 E620 T622 P624 E626 C628 K630 T632 V634 S636 P638 N640 K642 P644 R646 H648 D650 Y652 Q654 R656 L658 P660 E662 E664 W616indole

Bound L1 linker peak intensity / free L1 linker peak intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

20


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Biochemistry

allows us to estimate the Kd for the L1/NBD1 interaction, at least for the residues that form the most stable complex with NBD1. For this calculation, we assume that the peak intensity ratio is a measure of the free L1 linker, as the NBD1-bound L1 linker is likely not observable in the experiment on account of the size of NBD1 (31.0 kDa) and low concentration of the L1 linker (40 µM). The smallest peak intensity ratio is ~0.4. Thus, the concentration of free L1 linker in the experiment is 16 µM and the concentration of the L1 linker/NBD1 complex is 24 µM, which gives rise to a minimum Kd value of ~250 µM for the region of L1 linker that forms the most stable interaction with NBD1. The remaining L1 linker regions would have Kd values that are greater than 250 µM.

Figure 5A,B highlights how different non-phosphorylated L1 linker resonances are affected by NBD1 binding. For example, the backbone HN resonance for D650 exhibits significant resonance broadening upon addition of NBD1 (Figure 5B), showing that D650 either directly binds NBD1 or interacts with other residues that bind NBD1. In contrast, the peak intensity of the E662 backbone HN resonance does not change, indicating that this residue is not involved in binding NBD1. Multiple non-phosphorylated L1 linker resonances display decreased signal intensity upon binding NBD1 (Figure 5C, dark grey), including backbone HN resonances from N-terminal residues (W616-S627), residues near the phosphorylation sites (V634, Q635, N640), and residues near the C terminus (G645-R659) of the L1 linker. Note that we can not determine the broadening of the weak T632 backbone HN resonance because it is not observed in the L1 linker spectra at the sample concentration (40 µM) used in this experiment. We do not observe a decrease in the intensity for the S636 resonance. This resonance is very weak, and thus the error for the intensity ratio is high. Although it is unknown whether we completely saturated binding, we refer to the L1 linker samples in the presence of added NBD1 as NBD1-bound L1 linker. We also added unlabeled NBD1 to phosphorylated L1 linker (Figure 5C and Supplementary Figure 6). As expected considering that phosphorylation only partly displaces the L1 linker from the NBD1 core in the L1-NBD1 protein (42), phosphorylated L1 linker also binds NBD1. As with non-phosphorylated L1 linker, residues near the C terminus of the phosphorylated L1 linker (D650-R659) bind NBD1 and these backbone resonances broaden to the same degree as those for non-phosphorylated L1 linker upon NBD1 addition. However, there are differences in the extent and mode of NBD1 binding to the phosphorylated L1 linker compared to the non-phosphorylated protein. First, there is little broadening of backbone HN resonances of N-terminal residues (W616-S627) and residues near the phosphorylation sites (Q635, K637, N640) (Figure 5C), indicating that these residues in the phosphorylated L1 linker do not interact with NBD1. In addition, binding of NBD1 to the phosphorylated L1 linker results

21


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

in chemical shift changes for some L1 linker resonances (Supplementary Figure 6), including the backbone resonance of F625, the downfield resonance specific to the phosphorylated protein (labeled a), and two unassigned backbone resonances (labeled g and h). Because these resonances do not broaden with NBD1 addition, it is possible that the chemical shift changes reflect conformational changes at these sites caused by NBD1 binding to other L1 linker residues. For example, the phosphorylated site (either T632 or S636) may interact with other L1 linker residues in the free phosphorylated L1 linker. Recall that phosphorylated L1 linker still possesses some residual compact structure (Figure 4). Binding of NBD1 to these interacting residues would alter the chemical environment of T632 and/or S636 and thus affect their chemical shift. In support of this hypothesis, the phosphorylation-specific resonance (labeled a) has slightly different chemical shift in spectra of phosphorylated L1-NBD1 and phosphorylated L1 linker at 30 °C (Supplementary Figure 5). The upfield chemical shift change of resonance “a” in phosphorylated L1 associated with binding to NBD1 is consistent with the position of resonance “a” in the spectrum of phosphorylated L1-NBD1 (Supplementary Figures 5, 6). Note that L1 linker residues with fractional β-strand and α-helical propensity are the ones that respond to NBD1 addition (Figures 2, 5). Further, non-phosphorylated and phosphorylated L1 linker residues with the greatest fractional α-helical propensity (Figure 2, residues L649R657) display the greatest amount of broadening (Figure 5C), suggesting they bind NBD1 more stably than other L1 linker residues. Differences in affinities for binding of different regions of an IDP or IDR to a folded protein have been observed in other systems (58, 59). We assume that the binding of isolated L1 linker and NBD1 proteins to be very weak, considering that they are connected in the full length protein. Binding of other IDRs with NBDs involves residues in the IDR with fractional α-helical structure, such as the interaction of the disordered regulatory (R) region with the NBDs in CFTR (77, 91), an ABC protein in the same subfamily as the SUR proteins (23). Note that resonance broadening upon NBD1 addition is also seen for residues outside the L659-R657 α-helical region in both non-phosphorylated (G645-H648, L658, R659) and phosphorylated (L658, R659) L1 linker. Thus it is possible that NBD1 binding further stabilizes and extends the α-helical structure of the L1 linker in this region, as seen for interaction of the R region with NBD1 in CFTR (91). If residual secondary structure in the SUR2B L1 linker is a requirement for binding NBD1, the decreased interaction observed for phosphorylated L1 linker and NBD1 (Figure 5,

22


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Biochemistry

and reference (42)) is consistent with the reduced secondary structure propensity in the L1 linker upon phosphorylation (Figure 3).

Cardiac disease-causing mutations have differential effects on the L1 linker The region in the L1 linker that possesses the greatest secondary structure propensity (Figure 2) and makes the most stable interactions with NBD1 (Figure 5) is the site of two cardiac disease-causing mutations, R659C and A661T (45). Thus, we compared NMR spectra of the wild type L1 linker to NMR spectra of the L1 linker containing the R659C (Figure 6) and A661T (Supplementary Figure 7) mutations. As expected, the R659C and A661T mutations cause chemical shift changes of L1 resonances derived from residues near the site of the mutation. For example, the A661T mutation results in the loss of a peak for A661 and large chemical shift changes for the resonances of R659, E662 and T663 (Supplementary Figure 7). Note that residue 660 is a Pro, and hence is not visible in the 15N-1H HSQC spectrum. The R659C mutation also causes chemical shift changes in the 659 resonance and the A661, E662, and T633 resonances. However, the R659C mutation causes chemical shift changes in residues at least 10 amino acids N-terminal to R659, including K629, K630, and L649 (Figure 6). Residues K629, K630, and L649 either possess significant secondary structure propensity or are adjacent to regions with residual secondary structure (Figures 2, 6B). The longer-range affect of the R659C mutation suggests that this mutation may alter the global structure of the L1 linker and its interactions with NBD1. Note the altered L1/NBD1 interactions may be the reason that L1-NBD1 possessing the R659C mutation is prone to aggregation, precluding the acquisition of high quality NMR spectra of L1-NBD1-R659C.

23


Biochemistry

A 110

L1-WT L1-R659C

E662

120 R657

114

15N (ppm)

112

T663

116

122

124

R659

A661

L658

K630 L649

15N (ppm)

K629

118

8.4 1H (ppm)

120

8.0

E662 R657

122 L649

A661

124

K629

126 128 130 10

9

8

7

1H (ppm)

30.0

20.0

10.0

0.0


B ∆δ (ppm) for HN resonances

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 50

HN resonances (backbone or side chain)

Figure 6. Spectral changes in L1 linker with the R659C disease-causing mutation. (A) The spectrum of the wild type L1 linker (L1-WT, 40 µM) with that of the L1 linker containing the R659C (L1-R659C, 21 µM) are overlaid. The spectrum of the L1-WT is in black and in the background while spectra of the L1-R659C mutant is in magenta and in the foreground. The full spectrum is on the left and a selected region of the overlaid spectra is shown on the right. The R659C mutation also causes chemical shift changes in the 659 resonance, as well as the A661, E662, and T633 resonances. The smaller shift observed for the 659 resonance when R659 is mutated to a Cys, compared to the shift observed for the 661 resonance when A661 is altered to a Thr (Supplementary Figure 7), is due to the similarities of the average chemical shifts observed for Arg and Cys residues in disordered proteins, according to statistics in the BioMagResBank (BMRB) (101). (B) Combined chemical shift difference between L1-WT and L1-

24


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Biochemistry

R659C as a function of residue number. The red line shows significant chemical shift changes, which are defined as values higher than the average of all ∆δ(tot) plus one standard deviation. The schematic showing the secondary structure propensity from Figure 2 is shown at the bottom of the plot for comparison.

Discussion In this study, we examined how phosphorylation alters the structural properties of the phospho-regulatory L1 linker of SUR2B and its interactions with NBD1 (Figure 7). Our studies demonstrate significant residual secondary structure in the L1 linker, which is reduced with phosphorylation. Although both the non-phosphorylated and phosphorylated L1 linker are more compact than a random coil, phosphorylation makes the L1 linker less compact. We also show that L1 linker residues with secondary structure propensity mediate interactions with NBD1. Phosphorylation partly disrupts binding of the L1 linker to NBD1, consistent with data on the L1-NBD1 protein (42), possibly because of the decreased secondary structure in phosphorylated L1 linker. In the context of the full length SUR2B, disrupted L1 linker/NBD1 interactions result in NBD1 adopting a more “open” state, leading to increased MgATP binding (33, 42) and KATP channel activation (38, 40). The R659C mutation affects residues near and far from the mutation. Thus, mutation of R659, which is located in the region of the L1 linker with the greatest secondary structure propensity and that makes the most stable interaction with NBD1, may alter the L1 linker/NBD1 interactions and formation of the “open” state.

MgATP P

Closed

P

MgATP

Open

Figure 7. Model depicting changes in L1 linker structure and NBD1 interactions with phosphorylation. In the non-phosphorylated form, the L1 linker possesses regions of significant secondary structure propensity and compact structure. Further, many residues in the non-phosphorylated L1 linker bind NBD1. Previous data showed nonphosphorylated L1 linker interacts with residues of the Walker A and Walker B motifs in NBD1 to inhibit MgATP binding (42). Phosphorylation decreases the compactness and secondary structure propensity of the L1 linker, with the exception of residues at the C terminus of the L1 linker that are connected to NBD1 in the full SUR2B. Phosphorylation also reduces binding of most L1 linker residues to NBD1, again with the exception of residues G645-Y647, D650-R659.

The L1 linker is a regulatory region in SUR2. In addition to possessing phosphorylation sites, the L1 linker is also the site of alternative splicing. Removal of residues encoded by exon 14 (Q635-K669) forms the SUR2C isoform (102, 103). SUR2C lacks the non-consensus

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phosphorylation site, which is S636 in mouse and rat SUR2B and possibly at T641 in human SUR2B. Studies in cells show that mutation of the consensus T632 site inhibits the PKAmediated stimulation of KATP channel activity (38). Because the S636 site is a non-consensus PKA site, it likely can only be phosphorylated in the presence of the consensus site at T632. Our data showing that a phosphomimetic mutant at T632 does not reproduce the phosphorylationdependent changes in the NMR spectrum of L1-NBD1 implies that both sites need to be phosphorylated for maximal structural changes in the L1 linker. Whether having both T632 and S636 phosphorylated leads to maximal increase in KATP channel gating remains to be tested. The study presented here allows for comparison of the structure and interactions of the L1 linker in the non-phosphorylated and fully phosphorylated states. KATP channels in a cell are likely heterogeneous with respect to phosphorylation. It is possible that some KATP channels are phosphorylated at both T632 and S636, while others are phosphorylated at only T632. These sites are in addition to a site in Kir6.1 and to two sites in SUR2B NBD2 (38, 40, 41). Having multiple phosphorylation sites, which may or may not be phosphorylated simultaneously, may allow for fine-tuning of KATP channel gating. The SUR2C isoform also lacks L1 linker residues with significant secondary structure propensity (S636-P644, L649-R657) and that interact with NBD1 (H637, N640, G645-R659). Removal of the strongest NBD1 interacting residues may promote greater interactions of other regions of the L1 linker (e.g. W616-S627) with NBD1 in SUR2C. Further, the L1 linker is also the site of heme binding in SUR2, which increases KATP channel activity (43). Of the three residues (C628, H631, and H648) shown to be involved in the heme regulatory response (43), residue H648 is part of the L1 linker region with significant α-helical propensity and that forms the most stable interaction with NBD1. Mutagenesis studies indicate that H648 has a major effect on the heme-mediated increase in KATP channel gating and likely binds the heme (43). Thus, it is possible that heme binding disrupts binding of residues G645-R659 to the NBD1 core, again creating an “open” state that results in KATP channel activation. The R659C and A661T mutations, which are associated with early repolarization syndrome (45), a life threatening condition (104, 105), and also located in the L1 linker regulatory hot-spot formed by G645R659. Phosphorylation is a common mechanism for controlling the activity of ABC proteins, and many of the phosphorylation sites are in the IDRs (49). Phosphorylation sites are found in

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large cytoplasmic linkers of many ABC proteins, such as the MSD1-NBD1 linker represented by the SUR2B L1 linker, the NBD1-MSD2 linker, and the L0 linker. The effect of phosphorylation depends on the specific ABC protein and the site phosphorylated (49). For example, phosphorylation of the NBD1-MSD2 linker affects protein stability, ATPase activity, and/or transport activity of specific ABC proteins (49, 50, 53, 106, 107), whereas phosphorylation of the L0 linker can lead to inhibition of ABC protein activity (108, 109). Complex interaction networks have been observed for the IDRs of CFTR in response to phosphorylation. CFTR is phosphorylated by PKA and PKC at multiple sites in R region, which is equivalent to the NBD1-MSD2 linker, and at one site in the disordered regulatory insert (RI) located within NBD1 (51, 61, 110, 111). As seen for the SUR2B L1 linker, NMR and cryo-EM studies show that phosphorylation of the R region and RI disrupts the transient structure and reduces interaction of these disordered elements with CFTR NBD1 (77, 91, 112, 113). The effect of phosphorylation on the interaction of R region with accessory proteins depends on the specific interacting partner. Whereas phosphorylation of R region increases its interactions with STAS (sulfate transporters and anti- sigma factor) and 14-3-3 proteins (91), phosphorylation of R region decreases binding to calmodulin (90). Further, and again similar to SUR2B L1 linker, regions in R region with α-helical propensity mediate interactions to the CFTR NBDs and accessory proteins (77, 90, 91). It is possible that the SUR2B L1 linker binds other sites in the KATP channels, such as SUR2B NBD2 or the Kir6.2 cytoplasmic domains, and that these interactions are altered with phosphorylation. These additional interactions may be mediated by the regulatory hot-spot of the L1 linker, and would be affected by the disease-causing mutations, or may involve other L1 linker residues. Thus, the studies presented here on the phosphoregulatory L1 linker of SUR2B provide the platform to address these questions.

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Author Contributions C.R.S. recorded the NMR, CD, and dynamic light scattering data, whereas N.P. conducted the fluorescence quenching data. C.R.S. and J. P. L.-A. determined the purification protocol for the L1 linker and conducted initial NMR feasibility studies. N.P. conducted Trp fluorescence studies. V. K. and C.R.S. analyzed the NMR and CD data. Figures were generated by V. K. The manuscript was written by V. K.

Acknowledgements We acknowledge Dr. John L. Rubinstein for critically reading the manuscript and for useful suggestions. Dr. Claudia Alvarez is also acknowledged for useful suggestions. Tetyana Murdza is acknowledged for help with purification of the R659C mutant. Triple resonance assignment data was acquired at the Centre for Spectroscopic Investigation of Complex Organic Molecules and Polymers (Department of Chemistry NMR Facility, University of Toronto), which is funded by the Canadian Foundation for Innovation (project number 19119) and the Ontario Research Fund. This work was funded by a grant from the Natural Sciences and Engineering Council of Canada (RGPIN-2015-05372) to V.K. V.K. acknowledges support from a CIHR New Investigator Award.

Supporting Information

Schematic diagram of SUR2B, comparison of PONDR prediction and structure of L1-NBD1, schematic diagrams of L1 and NBD1 proteins used in this study (Supplementary Figure 1); schematic diagrams of cryo-EM structures of pancreatic KATP channel (Supplementary Figure 2); SUR1 density map from pancreatic KATP channel cryo-EM data (Supplementary Figure 3); size exclusion chromatography purification of the L1 linker (Supplementary Figure 4); spectra of the L1 linker and L1-NBD1 in nonphosphorylated and phosphorylated states at 30 °C (Supplementary Figure 5); spectra of phosphorylated L1 linker in absence and presence of NBD1 (Supplementary Figure 6); spectra of L1 linker in the wild type state and with the A661T mutation (Supplementary Figure 7)

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41


Biochemistry

A

G645

110 non-phosphorylated L1 phosphorylated L1

g614

G621

112 T622

114

T663

116

T618 T632 S615

S627

15N (ppm)

118

S636

120

N651 E620

E662 D650

122 N640

124

A661

126

R656 V634 R646 R657 Y652

R617 L649 W616 R659

K630 K637

L623

128

E664

W616indole

130 10

9

8

7

1H (ppm)

B 40.0

30.0

20.0

10.0

0.0


Δδ (ppm) for HN resonances

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 1 Sooklal, Lopez-Alonso, Papp, Kanelis



% helix (AGADIR)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Secondary structural propensity β-strand propensity α-helical propensity -0.2

B W616 T618 E620 T622 P624 E626 C628 K630 T632 V634 S636 P638 N640 K642 P644 R646 H648 D650 Y652 Q654 R656 L658 P660 E662 E664

Page 43 of 50 Biochemistry

A 0.3

0.2

0.1

0

-0.1

Residue

12

10

8

6

4

2

Residue



Biochemistry

mean residue ellipticity (deg cm2 dmol-1)

A non-phosphorylated L1 linker

4000 2000 0 -2000 -4000 -6000 -8000


-10000 -12000 190

200

210


240

250

260

B mean residue ellipticity (deg cm2 dmol-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 50

phosphorylated L1 linker

4000 2000 0 -2000 -4000 -6000


-8000 -10000 -12000 190

200

210


240

250

260



30.0 25.0 20.0

a

a

6. 5

kD

kD 13 .7

kD 29 .0

kD 75 .0 mAU (at 280 nm)

35.0

Native analytical SEC non-phosphorylated L1 linker phosphorylated L1 linker VC = 23.56 ml

15.0 10.0 5.0 0.0 45.0

Denaturing analytical SEC

40.0

non-phosphorylated L1 linker phosphorylated L1 linker

35.0 mAU (at 280 nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

Biochemistry

a

Page 45 of 50

30.0

VC = 23.56 ml

25.0 20.0 15.0 10.0 5.0 0.0 9.0

9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 Elution volume (ml)



Biochemistry

A

non-phosphorylated L1 non-phosphorylated L1 + NBD1

119

V634

121

D650

R657 Y674

122

W616 L658 R617

120.0 15N (ppm)

E662

R646

15N (ppm)

B

R656

120

120.5

E662

E662 D650

D650

121.0

123 N640

124 125

A655

A661 Q635

8.6

L649

8.55 8.50 1H (ppm)

8.55 8.50 1H (ppm)

K637

8.4 8.2 1H (ppm)

8.0

C 2.0

non-phosphorylated L1 +/- NBD1 phosphorylated L1 +/- NBD1

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0


Bound N-tail peak intensity / free N-tail peak intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 50




Page 47 of 50

Biochemistry

A 110

L1-WT L1-R659C

120

E662 R657

114

15N (ppm)

112

T663

116

122

124

R659

A661

15N (ppm)

118 120

8.4 1H (ppm)

E662 R657

122 A661

124

L649 K629

126 128 130

B

10

9

1H (ppm)

8

7

30.0

20.0

0.0


10.0


Figure 6 Sooklal, Lopez-Alonso, Papp, Kanelis ACS Paragon Plus Environment

L658

K630 K629

Δδ (ppm) for HN resonances

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

L649

8.0

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

MgATP P

Closed

P

Page 48 of 50

MgATP

Open



Page 49 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

82x34mm (300 x 300 DPI)


Biochemistry 1 2 3 4

Page 50 of 50


Phosphorylation Alters the Residual Structure and Interactions of the

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