Hyperinsulinism-Causing Mutations Cause Multiple Molecular Defects

University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S ... In contrast, SUR1 mutations that disrupt trafficking and/or decre...
0 downloads 0 Views 5MB Size
Download PDF
Article pubs.acs.org/biochemistry

Hyperinsulinism-Causing Mutations Cause Multiple Molecular Defects in SUR1 NBD1 Claudia P. Alvarez,†,‡ Marijana Stagljar,†,‡,§ D. Ranjith Muhandiram,∥ and Voula Kanelis*,†,‡,§ †

Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6 ‡ Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, Canada M5S 3H6 § Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario, Canada M5S 3G5 ∥ Department of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8 S Supporting Information *

ABSTRACT: The sulfonylurea receptor 1 (SUR1) protein forms the regulatory subunit in ATP sensitive K+ (KATP) channels in the pancreas. SUR proteins are members of the ATP binding cassette (ABC) superfamily of proteins. Binding and hydrolysis of MgATP at the SUR nucleotide binding domains (NBDs) lead to channel opening. Pancreatic KATP channels play an important role in insulin secretion. SUR1 mutations that result in increased levels of channel opening ultimately inhibit insulin secretion and lead to neonatal diabetes. In contrast, SUR1 mutations that disrupt trafficking and/or decrease gating of KATP channels cause congenital hyperinsulinism, where oversecretion of insulin occurs even in the presence of low glucose levels. Here, we present data on the effects of specific congenital hyperinsulinism-causing mutations (G716V, R842G, and K890T) located in different regions of the first nucleotide binding domain (NBD1). Nuclear magnetic resonance (NMR) and fluorescence data indicate that the K890T mutation affects residues throughout NBD1, including residues that bind MgATP, NBD2, and coupling helices. The mutations also decrease the MgATP binding affinity of NBD1. Size exclusion and NMR data indicate that the G716V and R842G mutations cause aggregation of NBD1 in vitro, possibly because of destabilization of the domain. These data describe structural characterization of SUR1 NBD1 and shed light on the underlying molecular basis of mutations that cause congenital hyperinsulinism. ATP sensitive K+ channels (KATP channels) are K+ selective channels that link the cell’s metabolism and membrane potential. KATP channels are hetero-octameric protein complexes comprised of four copies of a pore-forming inwardrectifying K+ channel (Kir6.1 or Kir6.2) and four copies of a sulfonylurea receptor (SUR1 or isoforms of SUR2) that surround the pore.1−7 SUR proteins are members of the ATP binding cassette (ABC) family of proteins8 and, as such, contain the minimum ABC protein architecture of two membrane-spanning domains (MSD1 and MSD2) and two nucleotide binding domains (NBD1 and NBD2). The transmembrane segments in the MSDs extend into the cytoplasm.9−23 Short helices, known as coupling helices,13 link the cytoplasmic extensions of the transmembrane helices and contact the NBDs. Thus, the coupling helices link conformational changes that occur at the NBDs to the rest of the SUR protein. Binding and hydrolysis of MgATP lead to altered interactions between NBD1 and NBD2 and associated conformational changes in the NBDs.24 SUR proteins contain an additional membrane-spanning domain (MSD0) that is linked to the minimum ABC structure by the cytoplasmic L0 linker.25−28 As with other members of the C subfamily of ABC proteins (ABCC) that includes the cystic fibrosis transmembrane © 2017 American Chemical Society

conductance regulator (CFTR) and the multidrug resistant proteins 1 and 2 (MRP1 and MRP2, respectively), the SUR proteins contain asymmetric nucleotide binding sites.29 The NBD1 composite site, which is formed by the Walker A and Walker B motifs of NBD1 and the signature sequence of NBD2, contains a substitution of the catalytic Glu of the Walker B motif to an Asp. The substitution renders the NBD1 composite site catalytically inactive, and thus, the NBD1 composite site is termed the degenerate ATP binding site. In contrast, the motifs that form the NBD2 composite site bear consensus sequences. Thus, the NBD2 composite site, which is capable of MgATP binding and hydrolysis, is termed the consensus site. Unlike most members of the ABC protein family, the SUR proteins do not possess any transport activity but instead regulate gating of the KATP channel pore.1,2,4,30−35 ATP binding at the Kir6.x pore results in KATP channel inhibition. Data for full-length channels indicate that KATP channels open in response to MgADP binding at NBD2, which requires Received: July 5, 2016 Revised: March 7, 2017 Published: March 27, 2017 2400

DOI: 10.1021/acs.biochem.6b00681 Biochemistry 2017, 56, 2400−2416

Article

Biochemistry MgATP binding at NBD1.30,35 The interaction of NBD2 with MgADP may result from direct nucleotide binding or hydrolysis of MgATP to MgADP at NBD2.30,34,35 KATP channels in the pancreatic β-cells, which are composed of Kir6.2 and SUR1 proteins, regulate insulin secretion.1,2,36−39 Under high-glucose conditions, the ATP:ADP ratio in the cell is high and KATP channels are closed. The closure of KATP channels depolarizes the plasma membrane, which in turn opens voltage-gated Ca2+ channels, resulting in increased intracellular Ca2+ concentrations and subsequent insulin secretion. When the ATP:ADP ratio is low, the KATP channels are open and the plasma membrane is hyperpolarized. Hyperpolarization of the membrane keeps the voltage-gated Ca2+ channels closed, preventing insulin release. Mutations in SUR1 that disrupt the ability of KATP channels to close at high glucose concentrations cause neonatal diabetes.37,39−41 Conversely, mutations that decrease the level of KATP channel opening result in congenital hyperinsulinism,37,39−43 which is characterized by constant insulin secretion, despite low glucose concentrations.44 Congenital hyperinsulinism usually presents within the first few days of birth. Treatment of congenital hyperinsulinism, either with the drug diazoxide that promotes KATP channel opening or with partial or near-total pancreatectomy, is necessary to prevent brain damage from the associated hypoglycemia.45,46 Mutations that cause congenital hyperinsulinism affect many parts of the SUR1 protein, including the NBDs,40,41,45 and cause a decreased response of the channel to MgADP and/or reduce the level of expression of the number of KATP channels at the plasma membrane.37,47−53 These mutations can elicit their effects on KATP channel gating and trafficking by a number of molecular mechanisms. For example, NBD mutations that affect MgADP activation may reduce the nucleotide binding affnity of the domain, compromise NBD dimerization, or alter NBD/coupling helix interactions. Compromised interactions between NBD1 and NBD2 and betweeen the NBDs and coupling helices would also affect folding and assembly of the KATP channel, and thus trafficking of the channel to the plasma membrane. Mutations may also alter the structure and/or destabilize the indvidual NBDs, which would compromise the interactions necessary for KATP channel assembly and trafficking. An understanding of the molecular defects imparted by different mutations is essential for developing specific therapies, as has been done for diseases of other ABC proteins.54,55 During revision of this article, high-resolution structures of a pancreatic KATP channel in the closed state were published.56,57 The KATP channel adopts a propeller structure, with the center of the propeller formed by the Kir6.2 proteins and MSD0 of SUR1, and the blades of the propeller comprised of the minimum ABC structure of the SUR1 subunits. There are multiple interactions between the first transmembrane helix of SUR1 MSD0 and a Kir6.2 transmembrane helix. 56,57 Perturbation of these interactions may explain how hyperinsulinism-causing mutations in SUR1 MSD0 impair assembly and cell surface expression of pancreatic KATP channels.57,58 Notably, there are no interactions between the minimum ABC structure (or ABC core) of adjacent SUR1 subunits or between the SUR1 ABC core and Kir6.2. In addition, the SUR1 NBDs are not bound to Mg nucleotides, and thus, the ABC core of SUR1 adopts an inward-facing conformation in which the NBDs are separated from one another. Therefore, additional

structural information is needed to understand the molecular basis underlying disease-causing mutations in the SUR1 NBDs. To this end, we present studies describing how several congenital hyperinsulinism-causing mutations [G716V, R842G, and K890T (Figure 1)]39−41 affect the structure and activity of

Figure 1. Ribbon diagram of a homology model of the SUR1 NBD1/ NBD2 heterodimer. The NBD1 ribbon diagram is colored light blue, except for Walker A, Walker B, signature sequence, and Q loop residues that are colored magenta, green, orange, and blue, respectively. The NBD2 ribbon is colored gray. The Cα atom of G716 and side chain atoms of R842 and K890 are shown as red spheres, whereas the side chains of Trp residues are shown as yellow sticks.

SUR1 NBD1. These data constitute the first structural studies of wild type and mutant forms of SUR1 NBD1. We have obtained resonance assignments for NBD1 and have probed conformational changes in NBD1 imparted by disease-causing mutations. NMR and fluorescence studies indicate that the K890T mutation affects many residues throughout NBD1 and results in decreased MgATP binding affinity and thermodynamic stability of the domain. Data on NBD1 with the G716V and R842G mutations indicate that these two mutations cause aggregation of NBD1 in vitro, possibly because of destabilization of the domain structure. Size exclusion chromatography and NMR spectroscopy suggest that changes to NBD1 structure caused by the K890T mutation are not as severe as those imparted by the G716V and R842G mutations, consistent with the disease severity of the different mutations. Our data shed light on the underlying molecular basis by which specific SUR1 mutations cause congenital hyperinsulinism and provide a platform for assessing the effects of other mutations in SUR1.

■

EXPERIMENTAL PROCEDURES Protein Expression. The human SUR1 NBD1 proteins (D671−L956) in the wild type state, bearing hyperinsulinismcausing mutations (G716V, R842G, and K890T) or the diabetes-causing mutation H863T, or with mutations of specific Trp residues (W688F, W778F, and W928F) were expressed with an N-terminal His6-SUMO tag59 using a modified pET26b-derived expression vector.60 We also generated samples of human SUR1 NBD1 lacking residues encoded by exon 17 (S742−S753) and residues Q937−L956, which are homologous to the disordered C-terminal tail in SUR2A (and SUR2B) NBD1,61,62 that we term NBD1-WT-Δ17ΔC. For wild type NBD1 (NBD1-WT), constructs were generated from the cDNA of isoform 1 (NCBI accession code NP_001274103) and isoform 2 (NCBI accession code NP_000343) of SUR1. Isoforms 1 and 2 of SUR1 differ only 2401

DOI: 10.1021/acs.biochem.6b00681 Biochemistry 2017, 56, 2400−2416

Article

Biochemistry

ThermoFisher) pre-equilibrated in 20 mM Tris-HCl (pH 7.6), 5% (v/v) glycerol, 150 mM NaCl, and 25 mM imidazole. NMR Spectroscopy. Purified SUR1 NBD1 proteins were dialyzed against the NBD1 NMR buffer [20 mM Na + phosphate buffer (pH 7.2), 2% (v/v) glycerol, 50 mM NaCl, 2 mM DTT, 5 mM MgCl2, and 5 mM ATP]. 1H−15N TROSYHSQC64 and 15N R1ρ relaxation experiments were performed on a 600 MHz Varian Inova spectrometer equipped with a H(F)CN triple-resonance cryoprobe and actively shielded zgradients. 1H−15N TROSY-HSQC spectra of SUR1 NBD1 in the wild type state (NBD1-WT) and the forms bearing diseasecausing mutations (NBD1-G716V, NBD1-R842G, NBD1H863T, and NBD1-K890T) were recorded at 30 °C in NBD1 NMR buffer. 1H−15N TROSY-HSQC spectra were processed using NMRPipe/NMRDraw,65 and differences between wild type and mutant NBD1 spectra were analyzed using NMRView.66 Chemical shifts were referenced to 4,4dimethyl-4-silapentane-1-sulfonic acid (DSS).67 Chemical shift changes from the K890T mutation were determined by calculating the combined chemical shift difference in hertz, Δδtot, from the equation Δδtot = (ΔδHN2 + ΔδN2)0.5.63,68 Upon assessment of chemical shift differences, only resonances exhibiting a significant combined chemical shift difference (Δδtot), which is greater than the average of all Δδtot values plus one standard deviation, are considered. For NMR data presented in this paper, only Δδtot values of ≥8.5 Hz are considered. 15 N R1ρ relaxation experiments were performed at 30 °C using previously described pulse schemes.69,70 15N R1ρ values were measured from six different spectra recorded with delays of 2, 6, 12, 28, and 40 ms for a sample of 500 μM NBD1-WT. 15 N R2 values for each residue were obtained by correction of observed relaxation rate R1ρ for the offset Δν of the applied spin-lock rf field (ν1) to the resonance using the equation R1ρ = R2 sin2 θ, where θ = tan−1(ν1/Δν) and ν1 was 1865.7 Hz. All data sets were processed using NMRPipe. Peak intensities were obtained using the Rate Analysis tool in NMRView and used to fit a two-parameter function of the form I(t) = I0e−tR2 using a Matlab script.71 Errors in relaxation rates were estimated by Monte Carlo analysis. In total, 31 resolved peaks were analyzed. Backbone 1H, 15N, 13C, and 13Cα, and side chain 13Cβ resonance assignments for NBD1-WT-Δ17ΔC were obtained from standard triple-resonance TROSY-based experiments72,73 recorded on samples of 0.5 mM NBD1-WT-Δ17ΔC that were uniformly labeled with 15N and 13C and fractionally labeled with 2H to ∼50%. The triple-resonance assignment data were run on a 600 MHz spectrometer equipped with a HCN cryoprobe at 20 °C. These data were supplemented with 15 N−1H TROSY-HSQC spectra recorded on NBD1 samples that were labeled with 15N on only specific amino acids (Leu, Val, Gly, or Gly and Ser)74−76 or labeled with 14N on Lys and labeled with 15N at all other positions.77 We have obtained assignments for 88% of backbone HN resonances of NBD1WT-Δ17ΔC. The similarity of the NBD1-WT-Δ17ΔC and NBD1-WT spectra allowed for the straightforward transfer of resonance assignments for most residues. Resonance assignments of NBD1-WT-Δ17ΔC spectra at 20 °C were transferred to NBD1-Δ17ΔC spectra at 30 °C and subsequently to spectra of NBD1-WT at 30 °C. The combination of TROSY-based triple-resonance data and specifically labeled samples has allowed for the level of resonance assignment obtained for other NBDs.62,74 Assignments of Trp indole HN resonances were obtained by site-directed mutagenesis of NBD1.

by the insertion of a Ser residue at position 742 in isoform 1. Because wild type NBD1 from isoform 2 is more soluble than NBD1 from isoform 1, we conducted all studies with NBD1 from isoform 2 of SUR1. However, we are using residue numbers of isoform 1 in this paper to be consistent with the residue numbering of SUR1 in the literature. The domain boundaries of D671−L956 chosen correspond to those used to obtain soluble samples of SUR2B NBD1.62 The His6-SUMO-NBD1 fusion proteins were expressed in Escherichia coli BL21 (DE3) CodonPlusRIL cells (Stratagene) grown in M9 media that was supplemented with 5% LB and contained 15NH4Cl to allow isotopic enrichment with 15N for NMR studies. Cells expressing the His6-SUMO-NBD1-WTΔ17ΔC fusion protein for NMR resonance assignment experiments were grown in 97.5% M9 minimal media (containing 15NH4Cl, [13C]glucose, and 70% 2H2O) and 2.5% 15 N- and 13C-labeled E. coli OD2 media (Silantes) to achieve uniform 15N and 13C labeling and fractional 2H labeling. The cell cultures were incubated at 37 °C with constant agitation at 250 rpm until the OD600 reached 0.4, at which point the temperature was decreased gradually so that when the temperature was 18 °C the cell culture OD600 was 0.8. The cultures were then incubated at 18 °C for 30 min, at which point protein expression was induced with 750 μM isopropyl βD-thiogalactoside (IPTG). After ∼18 h, the cells were harvested by centrifugation at 4 °C and cell pellets were stored at −20 °C until protein purification. Protein Purification. The SUR1 NBD1 proteins were purified using the general protocol described previously for NBD1 from rat SUR2A and SUR2B,61−63 with a few modifications. All purification steps were conducted at 4 °C. Cell pellets from 2 L of culture with final OD600 values of ∼1.9 were resuspended in 60 mL of lysis buffer [20 mM Tris-HCl (pH 7.6), 10% (v/v) glycerol, 150 mM NaCl, 10 mM imidazole, 100 mM arginine, 2 mM β-mercaptoethanol, 15 mM MgCl2, 15 mM ATP, 0.2% (v/v) Triton X-100, 2 mg/mL deoxycholic acid, 1 mg/mL lyzozyme, 5 mM benzamidine, 5 mM ε-aminocaproic acid, and 1 mM PMSF]. The cells were lysed by sonication, and the insoluble cellular debris was removed by centrifugation at 17000g for 30 min. The lysate was filtered through a 0.45 μm filter (Pall) and loaded onto a 5 mL Ni2+-NTA affinity column (GE Healthcare) that was preequilibrated with 20 mM Tris-HCl (pH 7.6), 10% (v/v) glycerol, 150 mM NaCl, and 10 mM imidazole. The Ni2+-NTA affinity column was washed with 6 column volumes of wash buffer [20 mM Tri-HCl (pH 7.6), 10% (v/v) glycerol, 150 mM NaCl, 10 mM imidazole, 5 mM MgCl2, and 5 mM ATP], and the His6-SUMO-NBD1 protein was eluted in 20 mM Tris-HCl (pH 7.6), 10% (v/v) glycerol, 150 mM NaCl, 400 mM imidazole, 15 mM MgCl 2 , 15 mM ATP, 2 mM βmercaptoethanol, 1 mM benzamidine, and 1 mM ε-aminocaproic acid. The elution fractions were immediately diluted 2fold with a buffer containing 20 mM Tris-HCl (pH 7.6), 10% (v/v) glycerol, 2 mM β-mercaptoethanol, 15 mM MgCl2, 15 mM ATP, 1 mM benzamidine, and 1 mM ε-aminocaproic acid. The His6-SUMO tag was removed from NBD1 with His6-Ulp1 protease. The resulting mixture containing the His6-SUMO tag and SUR1 NBD1 was applied to a Superdex 75 size exclusion column (GE Healthcare) in 20 mM Tris-HCl (pH 7.6), 5% (v/ v) glycerol, 150 mM NaCl, 2 mM β-mercaptoethanol, 5 mM MgCl2, 5 mM ATP, 1 mM benzamidine, and 1 mM εaminocaproic acid. SUR1 NBD1 was purified to homogeneity with a reverse Co2+ affinity column (HisPur Cobalt Resin, 2402

DOI: 10.1021/acs.biochem.6b00681 Biochemistry 2017, 56, 2400−2416

Article

Biochemistry

Fcorrected/Fdilution. The normalized fluorescence data were analyzed using the Stern−Volmer equation81 Fo/F = 1 + KSV[Q], where Fo is the fluorescence in the absence of the quencher (Q) acrylamide, to obtain the Stern−Volmer quenching constant, KSV. The KI and CsCl quenching experiments were performed by adding increasing amounts of a solution of 0.8 M KI or 0.8 M CsCl dissolved in the NBD1 fluorescence buffer. All quenching solutions were prepared fresh, and 0.2 mM Na2S2O3 was added to the KI stock solution to prevent formation of I2 and I3− species.81 A separate titration with 0.8 M KCl was performed to account for NBD1 protein dilution and for the increase in ionic strength with addition of KI or CsCl. The initial fluorescence (Fo) was divided by the fluorescence (F) at each point in the KI and KCl titrations to give (Fo/F)KI and (Fo/F)KCl, respectively.82 The net change in fluorescence was determined by the equation Fo/F = (Fo/F)KI/(Fo/F)KCl and plotted against the concentration of the quencher KI to obtain the Stern−Volmer constant (KSV). A similar analysis was used to calculate Fo/F = (Fo/F)CsCl/(Fo/F)KCl. Nucleotide Binding Affinity. The nucleotide binding affinity of NBD1-WT and NBD1-K890T was measured using the ATP analogue 2′(3′)-O-(N-methylanthraniloyl)adenosine 5-O-triphosphate (MANT-ATP). The experiments were conducted at 15 °C with 0.05 or 0.25 μM NBD1-WT and 0.25 μM NBD1-K890T proteins that were freshly exchanged into the NBD1 fluorescence buffer containing 10% (v/v) glycerol and 1 mM MgCl2 using size exclusion chromatography, which also ensured that monomeric NBD1 proteins were used. MANT-ATP titration experiments with NBD1-WT and NBD1K890T were monitored at emission wavelengths of 345 and 440 nm with an emission slit width of 6 nm. The fluorescence excitation wavelength was 295 nm and the excitation slit width was 3 nm. The sample volume and protein concentration were kept constant throughout the titration, while the concentration of MANT-ATP was increased. The decreasing fluorescence at 345 nm with an increasing concentration of MANT-ATP was due to quenching of the Trp residues in the protein upon binding of MANT-ATP and was fit according to the following equation,83 assuming a 1:1 complex between NBD1 and MANT-ATP

Thermal Denaturation Experiments. The thermal stabilities of wild type NBD1 (NBD1-WT) and NBD1 with the K890T mutation (NBD1-K890T) were measured using intrinsic Trp fluorescence spectroscopy, as described previously.63 The NBD1 proteins were exchanged into fluorescence buffer [20 mM Na+ phosphate (pH 7.2), 150 mM NaCl, 2 mM DTT, and 2% (v/v) glycerol] by size exclusion chromatography (Superdex 75). The higher NaCl concentration (of 150 mM) used for the fluorescence buffer compared to that of the NMR buffer (of 50 mM) is due to the fact that NaCl concentrations of 150 mM are necessary in the fluorescence buffer to prevent nonspecific binding of the protein to the chromatography resin. However, lower NaCl concentrations (50 mM) produce higher-quality NMR spectra of SUR1 NBD1. To obtain MgATP-bound NBD1 proteins, varying amounts of a freshly prepared MgATP solution were added to NBD1 samples immediately upon elution from the size exclusion column. Only NBD1 samples that eluted at a volume consistent with the monomeric protein were used. Fluorescence spectra were recorded with a Fluoromax-4 spectrofluorimeter (HORIBA) equipped with a Peltier unit for temperature control. NBD1 samples (2 μM, 0.5 mL) were heated from 10 to 70 °C in 1 ± 0.3 °C increments with a 1 min equilibration time at each temperature. The fluorescence intensity at each temperature was monitored at 345 nm, which is the wavelength at which the difference in the fluorescence intensity of the folded and denatured NBD1 is maximal. The excitation wavelength was 295 nm, and the excitation and emission slit widths were 2 and 4 nm, respectively. Fitting fluorescence thermal denaturation curves was not possible because of the steep slopes of the folded and unfolded baselines, likely because there are five Trp residues in SUR1 NBD1. Tm values were obtained by determining the midpoint of the transition of the melting curves graphically. A total of three experiments were performed and analyzed for each MgATP concentration tested. Fluorescence Quenching Experiments. Fluorescence quenching experiments were performed on 2 μM NBD1 samples in the presence and absence of 5 mM MgATP. As described above, wild type and mutant NBD1 samples were exchanged into fluorescence buffer lacking MgATP by size exclusion chromatography. MgATP was added to NBD1 samples that eluted at a volume consistent with monomeric protein to a final concentration of 5 mM, as required. Apo NBD1 samples contained 5 mM EDTA in place of 5 mM MgATP, unless CsCl was used as the quencher, in which case no EDTA was added to the samples. Trp emission was recorded at 15 °C with an excitation wavelength of 295 nm, an excitation slit width of 2 nm, an emission wavelength of 345 nm, and an emission slit width of 4 nm. Acrylamide quenching experiments were performed by adding increasing amounts of a solution containing 1.6 M acrylamide in the NBD1 fluorescence buffer. The absorbances at 295 nm (A295) and 345 nm (A345) were recorded, and the observed fluorescence was corrected for the inner filter effect, according to the equation F c o r r e c t e d = F o b s e r v e d × 10(A295+A345)/2.78−80 The values of A295 and A345 at the highest acrylamide concentration used were 0.080 and 0.005, respectively. To correct for sample dilution upon addition of acrylamide, parallel titrations with buffer were performed. The normalized fluorescence (F) at each titration point was determined by dividing Fcorrected by the fluorescence of NBD1 obtained from dilution of the sample (Fdilution), so that F =

F = Fo +

F∞ − Fo × {([MANT‐ATPtotal ] 2[NBD1total ]

+ [NBD1total ] + Kd) − [([MANT‐ATPtotal ] + [NBD1total ] + Kd)2 − 4[MANT‐ATPtotal ][NBD1total ]]0.5 }

where F is the fluorescence at each point in the titration, Fo is fluorescence signal in the absence of MANT-ATP, F∞ is the fluorescence at the total concentration of MANT-ATP, [MANT-ATPtotal], and [NBD1total] is the total NBD1 concentration in the sample. The increasing fluorescence at 440 nm is partly due to fluorescence resonance energy transfer between the Trp residues in NBD1 and the bound MANT-ATP, as the excitation wavelength of the MANT fluorophore overlaps with the emission wavelength of the Trp residues (345 nm). However, there are other mechanisms that contribute to the fluorescence at 440 nm that are not related to nucleotide binding, including direct excitation of free MANT-ATP molecules in solution by either the excitation light (at 295 2403

DOI: 10.1021/acs.biochem.6b00681 Biochemistry 2017, 56, 2400−2416

Article

Biochemistry

Figure 2. The K890T mutation causes partial aggregation of NBD1. The size exclusion elution profiles of (A and D) NBD1-WT and (C, E, and F) NBD1-K890T are shown. The elution volume of standards is indicated at the top of each plot. Note the difference in total column volumes (Vc) between profiles for panels A and C and those of panels C, E, and F. Profiles for NBD1-WT and NBD1-K890T shown in panels D and E were obtained by adding 12.5% (v/v) glycerol and 15 mM MgATP to the samples prior to loading them on the size exclusion column. The size exclusion profile of NBD1-K890T shown in panel F was obtained upon re-injection of the NBD1-K890T sample that eluted as a monomer in panel E. For each sample, 200 μL of 100 μM protein was loaded onto the column. Panel B shows the SDS−PAGE gel corresponding to the elution profile of NBD1-WT in panel A.

Figure 3. SUR1 NBD1-WT and NBD1-WT-Δ17ΔC give high-quality protein NMR spectra, allowing for backbone NMR resonance assignment. (Ai) Overlay of the two-dimensional 15N−1H TROSY-HSQC64 NMR spectra of NBD1-WT (300 μM) and NBD1-WT-Δ17ΔC (300 μM) in NMR buffer at 30 °C at 600 MHz. (Aii) Selected region of the overlaid spectra shown in panel i. The spectrum of NBD1-WT is in the background with resonances of backbone nuclei, as well as those from side chain nuclei from Trp, Asn, and Gln residues are colored black. The green resonances are of opposite sign, caused by spectral aliasing, and are possibly from Arg NεHε side chain correlations. The spectrum of NBD1-WT-Δ17ΔC is shown in the foreground. The red and blue peaks in the NBD1-WT-Δ17ΔC spectrum correspond to the black and green resonances, respectively, in the NBD1-WT spectrum. Assignments for selected resonances in the NBD1-WT-Δ17ΔC spectrum are labeled. Resonance assignments for the indole HN resonances are indicated with a prime symbol (e.g., W778′ and W928′) to distinguish them from backbone HN resonances. (B) Schematic ribbon diagram of the homology model of hSUR1 NBD1, colored light blue for residues with resonance assignments and gray for residues with no resonance assignments. Residues encoded by exon 17 that are missing in NBD1-WT-Δ17ΔC are colored gray. Pro residues are also colored gray, as these residues do not give signals in the NH-based NMR experiments, such as the two-dimensional 1H−15N TROSY-HSQC experiments. 2404

DOI: 10.1021/acs.biochem.6b00681 Biochemistry 2017, 56, 2400−2416

Article

Biochemistry nm) or emission of the Trp residues in the unbound protein at 345 nm.84 To account for these additional mechanisms, we fit the increasing fluorescence data at 440 nm using the following equation derived by Kubala et al.85 F = Fo + Q {([MANT‐ATPtotal ] +

γ−1 2

{([MANT‐ATPtotal ] + [NBD1total ] + Kd) − [([MANT‐ATPtotal ] + [NBD1total ] + Kd)2 − 4[MANT‐ATPtotal ][NBD1total ]]0.5 }}

where γ corresponds to the enhancement of fluorescence of the bound MANT-ATP, which is determined from the initial part of the curve, and Q is the signal increase due to excess free MANT-ATP in solution. Our method of extracting Kd values differs slightly from that of Kubala et al.85 First, Kubala et al. divide the overall fluorescence intensity of samples containing protein and fluorescent ATP by Q (F/Q). Second, Kubala et al. determine the value of γ with two additional titration experiments. The first experiment measures the signal for increasing concentrations of the fluorescence probe (e.g., MANT-ATP) in buffer (in the absence of protein, so F = Q[MANT-ATPtotal]), while the second experiment monitors the fluorescence signal of increasing concentrations of the probe in the presence of enough excess protein so that all of the probe is bound (F = γQ[MANT-ATPtotal]). The ratio of the linear slopes obtained from these separate titrations is used to obtain the parameter γ. In our case, the Kd values for the interaction between MANT-ATP and NBD1-WT were small, and as a result, we were able to extract the parameters Q and γ directly from fitting the fluorescence at 440 nm. Because the values of A295, A345, and A440 at the highest MANT-ATP concentration used were very low (0.011, 0.009, and 0.001, respectively), which would affect the fluorescence emission intensity by only ≤2%, correction of fluorescence due to absorbance is not required. Decreasing and increasing fluorescence data were fit according to the equations given above using Origin Pro.

Figure 4. 15N R2 relaxation data for 0.5 mM NBD1-WT. (A) Selected resonances from the 15N−1H correlation spectrum recorded with a delay time of 2 ms and with the pulse sequence used to determine 15N R1ρ rates. (B) 15N R1ρ decay curves for peaks shown in panel A. The 15 N R1ρ rates were used to determine 15N R2 rates.

indicates that hSUR1 NBD1 possesses regions of disorder, as there are a number of intense resonances centered at 8.2 ppm in the 1H dimension. PONDR analysis87−89 indicates that residues Q937−L956, which comprise the C-terminal extension62 in SUR1 NBD1, and S741−R765 are disordered. Residues S742−S753 and P754−R765 are encoded by exons 17 and 18, respectively, and connect the β-sheet and α/β subdomains of NBD1. Removal of disordered residues Q937− L956 and S742−S753 in NBD1-WT-Δ17ΔC results in the disappearance of many of the intense resonances around 8.2 ppm in the 1H dimension. The high quality of the NBD1-WTΔ17ΔC spectra and decreased overlap due to the removal of many disordered resonances allowed resonance assignments of SUR1 NBD1 to be obtained. Resonance assignments were obtained for 88% of backbone HN resonances of NBD1-WTΔ17ΔC. The similarity between NBD1-WT and NBD1-WTΔ17ΔC spectra allowed for the straightforward transfer of most HN resonance assignments. Assignments were also obtained for two of the Trp indole resonances (labeled in the spectra as W778′ and W928′) through mutagenesis. The secondary structure propensity (SSP) scores90 derived from 13Cα and 13 Cβ chemical shifts (Figure S1) indicate that the secondary structure of SUR1 NBD1 is very similar to that observed for other NBD proteins91 and is also very similar to the secondary structure determined for SUR1 NBD1 in cryo-EM structures of the pancreatic KATP channel.56,57 Notably, isolated SUR1 NBD1 possesses all of the structural elements found in

■

RESULTS Characterization of Wild Type Human SUR1 NBD1. An understanding of the molecular defects imposed on NBD1 by disease-causing mutations first requires characterization of the wild type protein. Analytical size exclusion chromatography (Figure 2A,B) and NMR spectroscopy (Figures 3 and 4) demonstrate that samples of wild type SUR1 NBD1 (NBD1WT) are monomeric. NBD1-WT elutes at a volume of 10.12 mL (Figure 2A,B), which corresponds to a molecular mass of 44.3 kDa and is similar to the elution volume observed for monomeric rat SUR2A NBD1.61 The earlier elution volume for wild type SUR1 NBD1 (and SUR2A NBD1) compared to that expected for a 32 kDa globular protein is due to disordered residues in the protein that increase the hydrodynamic radius of NBD1.86 Disordered residues include the last 20 C-terminal residues of the NBD1 construct and the loop connecting the βsheet and α/β subdomains (discussed below). The two-dimensional (2D) 15N HSQC-TROSY spectra of NBD1-WT and NBD1-WT-Δ17ΔC are presented in Figure 3, showing resonance dispersion in the 1H dimension from 6.5 to 11 ppm that is typical of a folded protein. Of the 269 resonances expected from backbone NH groups for NBD1WT, 219 are visible in the spectrum. The spectrum also 2405

DOI: 10.1021/acs.biochem.6b00681 Biochemistry 2017, 56, 2400−2416

Article

Biochemistry

Figure 5. The K890T mutation decreases the thermal stability of NBD1. Intrinsic Trp fluorescence denaturation curves of 2 μM NBD1-WT (●) and NBD1-K890T (○) in the presence of (A) 0.1, (B) 0.5, and (C) 5 mM MgATP. The Tm values are 35.9 ± 0.5, 39.6 ± 0.4, and 44.3 ± 0.5 °C for NBD1-WT in the presence of 0.1 mM MgATP, 0.5 mM MgATP, and 5.0 mM MgATP, respectively. Tm values for NBD1-K890T are 31.7 ± 0.4, 36.0 ± 0.11, and 40.1 ± 0.3 °C in the presence of 0.1 mM MgATP, 0.5 mM MgATP, and 5.0 mM MgATP, respectively.

fraction of NBD1-K890T that elutes at the void volume is in part due to aggregation of the protein, as aggregates absorb more strongly because of light scattering. Notably, the level of aggregation of NBD1-K890T can be reduced by pretreating samples with 12.5% (v/v) glycerol and 15 mM MgATP prior to loading on the size exclusion column (Figure 2E). Glycerol is a protective osmolyte that stabilizes the folded state of proteins,95−98 including SUR2A NBD1.63 Addition of MgATP also stabilizes SUR2A NBD163 and SUR1 NBD1 (see below). Notably, pretreating samples of NBD1-WT with 12.5% (v/v) glycerol and 15 mM MgATP does not change the elution profile of the wild type protein (Figure 2D). Furthermore, the monomeric form of NBD1K890T is stable, as protein from the monomeric peak does not partition in multiple fractions upon being re-injected on the size exclusion column (Figure 2F). In addition, NMR spectra of SUR1 NBD1-WT and NBD1-K890T samples taken directly from the monomeric elution fraction from the size exclusion column (Figure 2D,E) without concentrating the samples further are identical to those of more concentrated samples of SUR1 NBD1-WT and NBD1-K890T, respectively (Figure S2). Note that all subsequent NBD1-K890T experiments were performed with monomeric NBD1-K890T. To further investigate the cause of aggregation of NBD1K890T, we performed thermal denaturation studies of wild type NBD1 and NBD1-K890T with varying concentrations of MgATP. The fluorescence denaturation curves for NBD1-WT and NBD1-K890T proteins show very steep baselines, possibly because human SUR1 NBD1 proteins contain five Trp residues that are located throughout the protein. The steep baselines preclude fitting the denaturation curves to obtain melting temperatures (Tm). However, comparison of the sigmoidal region of the curves shows that NBD1-K890T unfolds at temperatures lower than that of NBD1-WT at all MgATP concentrations, and particularly at low MgATP concentrations. In addition, estimation of the Tm values by determining the midpoint of the transition portion of the curve indicates that NBD1-K890T possesses Tm values that are 3.5−4.2 °C lower than those observed for NBD1-WT at all MgATP concentrations (Figure 5). Note that melting curves for NBD1 proteins in the absence of MgATP were not sigmoidal, precluding determination of Tm values. The decreased thermal stability imparted by the K890T mutation may cause partial unfolding of NBD1, leading to aggregation of NBD1-K890T in vitro.

NBD1 in the intact KATP channel, which is in contrast to isolated NBD1 from the TM287/TM288 transporter. Structural studies indicate that the β-sheet subdomain in isolated TM287 NBD1 is disordered and is folded only in the context of the full TM287/TM288 heterodimeric transporter.12,92 In contrast, isolated SUR1 NBD1 possesses all of the structural elements found for NBD1 in the intact KATP channel. Thus, structural changes observed for isolated NBD1 with the K890T mutation are also likely present in the KATP channel. To determine whether NBD1 was a monomeric protein at high concentrations, such as those used for NMR experiments (>0.2−0.5 mM), we determined 15N R2 relaxation rates for SUR1 NBD1-WT at 0.5 mM (Figure 4). 15N R2 relaxation rates were calculated for 31 isolated resonances that have 1H chemical shifts of 8.5 ppm and are derived from structured residues. Resonances with 1H chemical shifts between 7.7 and 8.5 ppm in the 1H dimension were excluded from the analysis because of resonance overlap, which would compromise analysis of the relaxation data. In addition, many resonances in this region of the spectrum are derived from disordered regions in the protein (Figure 3) and thus would give longer R2 relaxation rates that are typical of molecules smaller than NBD1. The correlation time (τc) was calculated from the 15N R2 relaxation rates using only the J(0) term from the spectral density function, as it dominates the 15N R2 relaxation rate for proteins.93 In this calculation, we assumed an order parameter (S2) of 0.85, which is the typical value for a folded protein.69 The τc for SUR1 NBD1-WT was determined to be 22.9 ± 3.0 ns, similar to the τc calculated for monomeric SUR2A NBD1.94 Thus, the 15N R2 relaxation data indicate that the wild type SUR1 NBD1 samples (at concentrations of ≤0.5 mM) used in these studies are monomeric. The high quality of the NMR data for SUR1 NBD1-WT indicates that we can use NMR spectroscopy to probe structural changes in NBD1 resulting from disease-causing mutations. The K890T Mutation Causes Aggregation and Conformational Changes in NBD1. The size exclusion column elution profile for NBD1 bearing the K890T mutation (NBD1-K890T) indicates that the mutation causes partial aggregation of the protein (Figure 2C). NBD1-K890T elutes from the size exclusion column in multiple peaks. The elution profile of NBD1-K890T possesses a peak at the void volume (7.8 mL). However, the NBD1-K890T elution profile also shows a peak at 10.2 mL that corresponds to monomeric NBD1 (Figure 2A,B). The high absorbance reading for the 2406

DOI: 10.1021/acs.biochem.6b00681 Biochemistry 2017, 56, 2400−2416

Article

Biochemistry

Figure 6. NMR data show that the K890T mutation affects residues throughout NBD1. (Ai) 2D 15N−1H TROSY-HSQC NMR spectra of NBD1WT (300 μM) and NBD1-K890T (150 μM) are overlaid. (Aii) Selected region of the overlaid spectra shown in panel Ai. The spectrum of NBD1WT is in the background colored as in Figure 3. The spectrum of NBD1-K890T is in the foreground. Resonances colored red and blue correspond to the black and green resonances, respectively, in the spectrum of NBD1-WT. Assignments for selected resonances in NBD1-WT that exhibit chemical shift changes with the K890T mutation are labeled. (B) Mapping of chemical shift changes with the K890T mutation to specific residues in NBD1. Cα atoms of residues that show chemical shift changes are shown as spheres colored from light pink, to highlight the smallest changes (Δδtot = 8−10 Hz), to dark pink, for the largest changes (Δδtot ≥ 25 Hz). The side chain of K890 is colored yellow. The side chain of F857 is also shown, in dark pink, to highlight the potential cation−π interaction between K890 and F857.

The K890T mutation also affects the conformation of many residues in NBD1, which could affect interaction of NBD1 with other regions in SUR1, such as the coupling helices and NBD2. Comparison of NMR spectra of NBD1-WT and NBD1-K890T in the presence of MgATP shows that the K890T mutation results in chemical shift changes for many (>100) backbone resonances, the majority of which are derived from structured residues in the protein (Figure 6A,B). As expected, large-scale chemical shift changes are observed for resonances from backbone NH groups of V887−L894, all of which are close (within 6 Å) to the site of the mutation (Figure 6B). The K890T mutation also results in chemical shift changes for residues D855−D861 located in the adjacent loop, which is known as the D loop. The SUR1 NBD1 homology model indicates that the side chains of K890 and F857 are close together, in a potential cation−π interaction. Removal of the amino group with the K890T mutation would disrupt this interaction, leading to an altered protein conformation and the observed chemical shift changes in HN resonances of many NBD1 residues. For example, large chemical shift changes are seen for residues V850, V851, L853, and D854 of the Walker B motif, even though some of these residues are >10 Å from K890. The Walker B motif is involved in MgATP binding and hydrolysis.24 Chemical shift changes are also observed for residues of other sites involved in ATP binding, such as G713− V715, G718, and S720 in the Walker A motif and Y772, W778, and N781 in the Q loop, which are up to 21 Å from the mutation site. The NMR data indicate that the K890T mutation also affects residues in the α-helical subdomain, including those in and adjacent to the signature sequence (e.g., G827−L830, Q834, and Q836). Some of these residues also contact NBD2. Resonance assignments of the entire signature sequence could not be made because of significant broadening

of resonances from these residues. Finally, the K890T mutation also affects NBD1 residues that contact coupling helix 4. In addition to the aforementioned Y772, W778, and N781, chemical shift changes are also seen for I828 and L830. The number and magnitude of chemical shift changes for residues distant from the mutation site indicate that the K890T mutation causes global conformational changes in NBD1, which may affect binding of NBD1 to MgATP, NBD2, and/or the coupling helices. In contrast to the gross spectral changes in NBD1 from the K890T mutation, mutation of the surfaceexposed H863 to Thr, a mutation that causes diabetes and hence is not expected to decrease the level of channel gating,99 results in limited changes in the NBD1 spectrum (Figure S3). The H863T mutation causes chemical changes in significantly fewer resonances in the NBD1 spectrum compared to the K890T mutation. In addition, the magnitudes of most of the chemical shift changes imparted by the H863T mutation are very small (≤10 Hz). Chemical shift changes of ≥20 Hz are observed for only three resonances in the spectrum of NBD1H863T. In contrast, the K890T mutation causes chemical shift changes of ≥20 Hz in 28 resonances. Thus, differences in spectra of NBD1-WT and NBD1-K890T result from perturbation of the NBD1 structure and may be observed for other mutations that result in impaired trafficking or loss-ofgating function such as those that cause hyperinsulinism. Testing this hypothesis requires structural studies of many NBD mutations that cause hyperinsulinism and diabetes. In addition, the spectrum of NBD1 including mutation of the surface-exposed Trp778, which was acquired to assign the Trp indole resonances, is also very similar to the spectrum of the wild type protein (data not shown). The NMR data described above highlight structural changes in NBD1-WT imparted by the K890T mutation in the MgATP 2407

DOI: 10.1021/acs.biochem.6b00681 Biochemistry 2017, 56, 2400−2416

Article

Biochemistry bound state of the protein. Unfortunately, as seen for SUR2A NBD1,61 samples of SUR1 NBD1 lacking MgATP give NMR spectra that are significantly broadened (Figure S4A), possibly because of aggregation of the apo proteins at the high protein concentrations (≥100 μM) and temperatures (25−30 °C) required for NMR studies of SUR NBDs. Hence, assessing how the K890T mutation affects the structure of apo NBD1 cannot be achieved by NMR spectroscopy. Because NBD1 proteins with and without MgATP yield high-quality fluorescence spectra, including at low temperatures (e.g., 15 °C) where the apo proteins are more stable, we also used Trp fluorescence quenching studies to probe the conformational changes in NBD1 imparted by the K890T mutation. The much lower concentration of NBD1-WT and NBD1-K890T (2 μM) used in the fluorescence studies also ensures that the apo proteins are monomeric, which is confirmed for each sample by size exclusion chromatography used to exchange the NBD1 proteins into the required buffers (Figure 2 and Experimental Procedures). Changes in the fluorescence of Trp residues are widely used to probe conformational changes in proteins.78,100,101 The five Trp residues in SUR1 NBD1 are located throughout the protein (Figure 1) and have varying degrees of solvent exposure, according to the homology model of SUR1 NBD1. Thus, the Trp residues are good reporters for assessing structural changes throughout the protein. Dynamic quenching of Trp fluorescence depends on the exposure of the Trp residue as well as on its local environment.100,101 For example, I− ions will effectively quench an exposed Trp residue close to positively charged groups, such as the side chains of Arg and Lys residues, but will not affect the fluorescence of Trp residues near negatively charged amino acids. Acrylamide will quench surface-exposed Trp residues surrounded by hydrophobic residues. In addition, acrylamide can access the hydrophobic core and hence quench the fluorescence from buried Trp residues. Cs+ will quench an exposed Trp residue close to negatively charged groups. Thus, differences in fluorescence quenching are good indicators of changes in the conformation of NBD1. The fluorescence emission spectrum of SUR1 NBD1-WT shows a λmax of 345 nm, indicating that most of the fluorescence signal arises from exposed Trp residues.78,101,102 Addition of increasing amounts of a quencher (I− or acrylamide) results in successive decreases in the intensity of the fluorescence signal. As for other proteins containing multiple Trp residues, such as P-glycoprotein,78,101 Stern−Volmer plots for SUR1 NBD1-WT and NBD1-K890T are linear (Figure 7). It is likely that some of the Trp residues in the protein are not affected by the quencher, either because they are not surface-exposed or because they are in environments that are not compatible with the specific quencher as explained above. Fluorescence quenching data with I− show differences between NBD1-WT and NBD1-K890T in the absence and presence of MgATP (Figure 7A and Table 1). The larger KSV values for NBD1K890T (apo, 2.27 ± 0.33 M−1; MgATP-bound, 1.70 ± 0.26 M−1) compared with those of NBD1-WT (apo, 1.79 ± 0.18 M−1; MgATP-bound, 1.33 ± 0.07 M−1) indicate greater accessibility of one or more Trp residues located in a positively charged environment in NBD1-K890T in both apo and MgATP-bound states. An examination of the surface potential of the homology model of SUR1 NBD1 suggests that W778 (and possibly W928) are affected by I−, as they are located near positively charged residues, in contrast to other Trp residues

Figure 7. The K890T mutation causes changes in Trp quenching in NBD1. Stern−Volmer plots for fluorescence quenching of the Trp residues in NBD1-WT (black) and NBD1-K890T (red) by (A) I− or (B) acrylamide in fluorescence buffer. Data from fluorescence quenching in the absence and presence of 5 mM MgATP are shown as empty and filled circles, respectively. The fit of the Stern−Volmer plots is shown as a solid line for MgATP-loaded NBD1 proteins and as a dashed line for apo NBD1 proteins. Fluorescence intensities for both acrylamide and I− quenching were corrected for dilution, scattering, and ionic strength. (C) Trp quenching studies of NBD1 with mutation of individual Trp residues to Phe in the presence of MgATP. Trp quenching data are shown for NBD1-W688F (empty gray squares and dotted line), NBD1-W778F (filled gray squares and dotted line), and NBD1-W928F (filled light gray circles and dotted line). Data for additional Trp mutants could not be obtained because of the insolubility of the proteins. The data for NBD1-WT from panel A are shown for comparison. (D) Trp quenching studies of NBD1K890T containing the W778F mutation (filled red squares and dotted line) in the presence of MgATP. The data for NBD1-K890T from panel B and NBD1-W778F from panel C are shown for comparison. (E) Surface representation of the homology model of NBD1-WT with blue and red representing positive and negative electrostatic potential, respectively. The ATP binding site of NBD1 is shown on the left, while the opposite face of NBD1 is shown on the right. Black arrows highlight the side chain of the Trp residues. The calculated accessible surface area of each Trp side chain is given in parentheses.

(W688, W739, and W899) that are near negatively charged or nonpolar residues (Figure 7E). Notably, both the backbone and Trp indole HN NMR resonances for W778 and W928 change with the K890T mutation. In contrast, chemical shift changes are not observed for the other Trp indole resonances (W688′, W739′, and W899′) or for the backbone HN resonances of W739. The overlap in the spectra precludes the assessment of chemical shift changes for the backbone HN resonances of 2408

DOI: 10.1021/acs.biochem.6b00681 Biochemistry 2017, 56, 2400−2416

Article

Biochemistry Table 1. Stern−Volmer Constants for SUR1 NBD1 Proteinsa KI quenching KSV (M−1) NBD1 protein WT K890T W778F W928F W688F W778F/K890T a

MgATP (5 mM) 1.33 1.70 1.11 1.40 1.33 1.47

± ± ± ± ± ±

0.07, 0.26, 0.09, 0.15, 0.17, 0.12,

n n n n n n

= = = = = =

5 4 3 3 2 3

acrylamide quenching KSV (M−1)

EDTA (5 mM) 1.79 2.27 1.35 1.67 1.61 ND

± ± ± ± ±

0.18, 0.33, 0.17, 0.11, 0.17,

n n n n n

= = = = =

MgATP (5 mM) 4 6 3 3 2

4.83 4.96 4.56 4.51 5.45 ND

± ± ± ± ±

0.18, 0.26, 0.28, 0.16, 0.19,

n n n n n

= = = = =

2 2 2 2 3

CsCl quenching KSV (M−1)

EDTA (5 mM) 4.58 4.10 4.25 4.16 5.05 ND

± ± ± ± ±

0.22, 0.04, 0.13, 0.10, 0.35,

n n n n n

= = = = =

MgATP (5 mM) 2 2 2 2 2

0.66 0.66 0.27 0.12 0.63 ND

± ± ± ± ±

0.08, 0.10, 0.06, 0.01, 0.09,

n n n n n

= = = = =

2 2 3 2 2

no MgATP 0.75 0.59 0.63 0.59 0.49 ND

± ± ± ± ±

0.10, n = 2 0.12, n = 2 NDb NDb NDb

ND means not determined. bn = 1.

Figure 8. The K890T mutation reduces the affinity of NBD1 for MgATP. (A) Fluorescence emission spectra of 2 μM NBD1-WT in the absence (●) and presence (○) of 10 μM MANT-ATP. The emission spectra of MANT-ATP () and buffer (---) are also shown. Binding of NBD1-WT (●) and NBD1-K890T (○) to MANT-ATP as monitored by (B) the quenching of the Trp residues at 345 nm and (C) the increase in fluorescence due to the FRET effect at 440 nm. The concentrations of NBD1-WT and NBD1-K890T were 0.25 μM. MANT-ATP titration data were fit assuming a 1:1 complex for the NBD1−nucleotide interaction. Note that experiments performed with 0.05 μM NBD1-WT, rather than 0.25 μM, resulted in the same Kd value.

effective quencher of NBD1. Together, the fluorescence quenching and current NMR studies demonstrate that the K890T mutation alters the conformation of residues near and far from the site of the mutation, in both apo and MgATPbound states. The K890T Mutation Decreases the MgATP Binding Affinity of NBD1. The NMR and fluorescence quenching data indicate that the K890T mutation alters the conformation of residues involved in MgATP binding, such as residues in the Walker A motif, the Walker B motif, and Q loop (also known as the γ-phosphate linker103) (Figure 6). Therefore, we sought to determine whether the K890T mutation alters the affinity of NBD1 for MgATP. We used a fluorescently labeled analogue of ATP, MANT-ATP. MANT-ATP has a methylanthraniloyl group on the ribose of the nucleotide and has been employed to study nucleotide binding to several proteins, including ABC transporters.104−106 Addition of MANT-ATP to NBD1 results in a quenching of the intrinsic Trp fluorescence of the protein (at 345 nm), when the excitation wavelength is 295 nm (Figure 8A). In addition, the fluorescence spectra of samples containing NBD1 and increasing concentrations of MANT-ATP also possess an increase in fluorescence at 440 nm. The decrease in Trp fluorescence (at 345 nm) and the increase in MANT-ATP fluorescence (at 440 nm) are caused by fluorescence resonance energy transfer between a Trp residue in NBD1 and the MANT group of bound MANT-ATP. Thus, the binding affinity of MANT-ATP can be determined from the fluorescence change at either 345 or 440 nm,107 but with different analyses. The decrease in NBD1 fluorescence at 345 nm with increasing amounts of MANT-ATP follows a hyperbolic saturation curve (Figure 8B). Dissociation constants (Kd) obtained from the

W688 and W899. Further support for the contribution of W778 to fluorescence quenching of NBD1-WT and NBD1K890T is seen in the decrease in the KSV values for the two proteins containing the W778F mutation in the presence of MgATP to 1.11 ± 0.09 and 1.47 ± 0.12 M−1, respectively (Figure 7C,D and Table 1). Mutation of W778 to Phe also affects the KSV value for NBD1 in the absence of MgATP, suggesting that W778 contributes to Trp quenching in the apoprotein. In contrast, mutation of W688 or W928 to Phe does not affect the KSV values for NBD1 in the presence or absence of MgATP. The difference in the KSV values for NBD1W778F and NBD1-W778F/K890T indicates that additional Trp residues contribute to quenching of the K890T mutant, such as W928. However, the lack of soluble protein obtained for NBD1-K890T with the W688F or W928F mutation precludes the determination of the contribution of those Trp residues to quenching of NBD1 with the K890T mutation. Soluble protein could also not be obtained for NBD1 proteins with a mutation of W739 or W899 to Phe. In contrast to that observed for I− quenching, KSV values for acrylamide quenching are similar for NBD1-WT and NBD1K890T in the presence of MgATP (Figure 7B and Table 1), indicating that additional Trp residues are not affected by the K890T mutation for the MgATP-loaded proteins. However, KSV values for acrylamide are decreased for NBD1-K890T in the absence of MgATP. Destabilization of the structure of apo NBD1 imparted by the K890T mutation, including but not limited to removal of the K890−F857 cation−π interaction, could cause changes in exposure of Trp residues in hydrophobic environments. The KSV values obtained with Cs+ are very small (e.g., 0.12−0.75 M−1), indicating that Cs+ is not an 2409

DOI: 10.1021/acs.biochem.6b00681 Biochemistry 2017, 56, 2400−2416

Article

Biochemistry

Figure 9. Severe congenital hyperinsulinism-causing mutations cause aggregation of NBD1 in vitro. (A) The size exclusion elution profile of NBD1R842G shows one peak at the void volume, indicating that NBD1-R842G exists as an oligomer of at least 75 kDa. As for NBD1-WT and NBD1K890T (Figure 2), a total of 200 μL of 100 μM protein was loaded onto the column. (B) Overlay of the NMR spectra of NBD1-WT (300 μM) and NBD1-R842G (100 μM). The 15N−1H TROSY-HSQC spectrum of NBD1-WT is in the background and is colored as in Figures 3 and 6A. The spectrum of NBD1-R842G is in the foreground with peaks colored red and blue as for the spectrum of NBD1-K890T in Figure 6A.

fluorescence quenching curves show tight binding of MANTATP to NBD1-WT (0.11 ± 0.03 μM), as is also seen for the interaction of other NBDs with the ATP analogue TNPATP.62,63,94,108 Comparison of the NMR spectra of NBD1 with MANT-ATP and with ATP shows chemical shift changes in several residues (Figure S5), including some in the β-sheet subdomain that are not expected to bind MgATP from structures of other NBDs.91 These chemical shift changes may reflect differences in the binding of ATP and MANT-ATP to NBD1, or differences in conformation of NBD1 with the different nucleotides. Nonetheless, the NMR spectra and hence conformation of NBD1 are similar in the presence of ATP and MANT-ATP, making MANT-ATP a useful probe for nucleotide binding studies of wild type and mutant NBDs. Notably, the K890T mutation results in a decrease in the nucleotide binding affinity of NBD1 (Kd = 0.63 ± 0.11 μM). Decreased MgATP binding affinity has also been observed for other SUR1 NBD mutations that cause congenital hyperinsulinism.109 Changes in the fluorescence emission at 440 nm also demonstrate that the K890T mutation decreases the affinity of the protein for the nucleotide. A plot of the fluorescence at 440 nm as a function of MANT-ATP concentration in the presence of NBD1-WT (Figure 8C) shows a hyperbolic saturation curve at low MANT-ATP concentrations, corresponding to the fluorescence of bound MANT-ATP, and a straight line at high MANT-ATP concentrations that corresponds to the fluorescence of free MANT-ATP. The Kd value determined for the MANT-ATP/NBD1-WT interaction using the fluorescence increase at 440 nm (0.16 ± 0.03 μM) is the same as that determined by monitoring fluorescence quenching at 345 nm. The lower affinity of NBD1-K890T for MANT-ATP does not produce the hyperbolic curve at low MANT-ATP concentrations [Figure 8C (○)], which precludes determination of the Kd value for binding of the nucleotide to NBD1-K890T from the increase in MANT-ATP fluorescence. Increasing the concentration of NBD1-K890T to allow for greater fluorescence enhancement at low nucleotide concentrations cannot be accomplished because of precipitation of the protein in the nucleotide-free state at higher protein concentrations (e.g., 10− 50 μM). Nonetheless, it is clear that the affinity of NBD1K890T for the nucleotide is reduced by at least 5−6-fold compared with that of NBD1-WT.

The binding affinity of NBD1-WT for MANT-ATP decreases 2.5-fold in the absence of Mg2+ (Kd = 0.28 ± 0.01 μM) (Figure S6), consistent with studies that indicate that the nucleotide can bind to NBD1 without Mg2+.32 However, NMR spectra of NBD1 with 5 mM ATP but without Mg2+ yielded spectra that are significantly broadened, identical to NBD1 spectra lacking MgATP (Figure S4). Note that NBD1 samples for these Mg 2+-free studies were exchanged into the fluorescence buffer by gel filtration chromatography. The buffer was treated with Chelex 100 resin to remove any residual metals prior to equilibration of the column. Severe Hyperinsulinism-Causing Mutations Result in Aggregation of NBD1. In contrast to the K890T mutation, NBD1 proteins bearing severe hyperinsulinism-causing mutations (G716V and R842G) produce large aggregates under the same conditions that allow for monomeric NBD1-WT and NBD1-K890T (Figure 9). NBD1-R842G elutes from the gel filtration column at 7.8 mL (Figure 9A), which corresponds to the void volume of the Superdex 75 column. This shows that even at the low concentrations (of ∼10 μM) at which proteins elute from the size exclusion column the majority of the NBD1R842G is aggregated, having a molecular mass of ≥75 kDa. NBD1 with the G716V mutation also shows a similar elution profile on the Superdex 75 column. Thus, these mutant NBD1 proteins do not produce detectable amounts of monomeric protein, possibly resulting from unfolding of the NBD1 protein. Mutation of G716, which is a conserved residue in the Walker A motif, likely inhibits ATP binding and leads to subsequent destabilization of the NBD as seen for SUR2A NBD1.63 Mutation of R842, which is located in helix α5 of the α-helical subdomain, to Gly likely disrupts the secondary structure and destabilizes the domain, as Gly residues are not favored in αhelices.110 Because folding of ABC transporters may involve sequential folding of individual domains during translation,103 the G716V and R842G mutations may also prevent proper folding of SUR1 and, consequently, assembly and trafficking of the KATP channel. Notably, the G716V mutation results in disrupted trafficking of the KATP channel that cannot be rescued with diazoxide.52 The effect of the R842G mutation on KATP channel trafficking or gating has not yet been determined. Consistent with the observed high-molecular weight aggregates, NBD1 proteins bearing the G716V and R842G hyperinsulinism mutations give spectra that contain only a few 2410

DOI: 10.1021/acs.biochem.6b00681 Biochemistry 2017, 56, 2400−2416

Article

Biochemistry resonances centered at ∼8.2 ppm in the 1H dimension (Figure 9B and Figure S7, red). These intense resonances, which are also observed in the spectrum of NBD1-WT, originate from disordered regions of the protein. Resonances with 1H chemical shift values of >8.5 or