Structural and Functional Characterization of the Kindlin-1 Pleckstrin Homology Domain

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin β-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P₃; K_D ∼100 μm) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P₃ as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P₂ on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P₃ is affected by the presence of PO₄ ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.     

Structural and Functional Characterization of the Recombinant Death Domain from Death-Associated Protein Kinase

Death-associated protein kinase (DAPk) is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD) that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2) and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K _d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent ‘D-motif’ located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural bioinformatics assignment of the three-dimensional structure.     

Functional Characterization and Determination of the Physiological Role of a Calcium-Dependent Potassium Channel from Cyanobacteria

Despite the important achievement of the high-resolution structures of several prokaryotic channels, current understanding of their physiological roles in bacteria themselves is still far from complete. We have identified a putative two transmembrane domain-containing channel, SynCaK, in the genome of the freshwater cyanobacterium Synechocystis sp. PCC 6803, a model photosynthetic organism. SynCaK displays significant sequence homology to MthK, a calcium-dependent potassium channel isolated from Methanobacterium thermoautotrophicum. Expression of SynCaK in fusion with enhanced GFP in mammalian Chinese hamster ovary cells’ plasma membrane gave rise to a calcium-activated, potassium-selective activity in patch clamp experiments. In cyanobacteria, Western blotting of isolated membrane fractions located SynCaK mainly to the plasma membrane. To understand its physiological function, a SynCaK-deficient mutant of Synechocystis sp. PCC 6803, ΔSynCaK, has been obtained. Although the potassium content in the mutant organisms was comparable to that observed in the wild type, ΔSynCaK was characterized by a depolarized resting membrane potential, as determined by a potential-sensitive fluorescent probe. Growth of the mutant under various conditions revealed that lack of SynCaK does not impair growth under osmotic or salt stress and that SynCaK is not involved in the regulation of photosynthesis. Instead, its lack conferred an increased resistance to the heavy metal zinc, an environmental pollutant. A similar result was obtained using barium, a general potassium channel inhibitor that also caused depolarization. Our findings thus indicate that SynCaK is a functional channel and identify the physiological consequences of its deletion in cyanobacteria.Detailed structural and mechanistic data now exist for many prokaryotic channels, but their physiological roles remain largely unclear (Martinac et al., 2008). This is especially true for potassium channels. K⁺ is the most abundant cation in organisms, and in general, it plays a crucial role in the survival and development of cells by regulating enzyme activity and tuning electrochemical membrane potential. Potassium channels in prokaryotes have been hypothesized to contribute to the setting of membrane potential rather than to high-affinity potassium uptake normally achieved thanks to specific ATP-dependent potassium transporters (Kuo et al., 2005). K⁺ channel genes are found in almost every prokaryotic genome that has been sequenced, but in most of the few studies where their deletion was obtained, no specific phenotype has been observed, suggesting either functional redundancy or that these channels are only required in case of rather specific environmental stresses. Gain-of-function potassium channel (Kch) mutants of Escherichia coli failed to grow in millimolar-added K⁺ but not Na⁺ (Kuo et al., 2003), and external H⁺ suppressed the gain-of-function phenotype, supporting the hypothesis that KCh might function to regulate membrane potential. However, a clear-cut role of prokaryotic potassium channels by genetic deletion was demonstrated only in a few cases.The model organism Synechocystis sp. PCC 6803 harbors an intracellular membrane system, the thylakoids, where both photosynthesis and respiration take place. In this work, we have identified in Synechocystis sp. PCC 6803 a so-far uncharacterized putative potassium channel, NP_440478, encoded by the open reading frame (ORF) sll0993, with sequence homology to MthK, a Ca²⁺-activated K⁺ channel of the archaeon Methanobacterium thermoautotrophicum. The structure of MthK in an open conformation has been determined (Jiang et al., 2002). The MthK subunit has two transmembrane segments and one pore region, followed by an extension of approximately 200 amino acid residues, which contains a region called the regulator of the conductance of K⁺ (RCK) domain. RCK of MthK binds divalent cations, such as Ca²⁺ or Cd²⁺ (Jiang et al., 2001, 2002). The physiological meaning of the activation of MthK with millimolar Ca²⁺ (Jiang et al., 2002) is, however, unclear, because Ca²⁺ as a second messenger operates at micromolar concentrations in eukaryotes, and the possible signaling roles of Ca²⁺ in prokaryotes are still unclear. Another RCK-containing 160-picoSiemens (pS) K⁺ channel from the archaeon Thermoplasma volcanium, TvoK, was also found to be activated by millimolar Ca²⁺ (Parfenova et al., 2007). Here, we report evidence that, similar to MthK, SynCaK can also be activated by calcium. Furthermore, we show localization of the protein in cyanobacteria and describe a phenotype associated with the lack of the channel in SynCaK-less mutant Synechocystis sp. PCC 6803 cells.     

Biophysical and Biochemical Characterization of the Receptor Binding Domain of SARS-CoV-2 Variants

The Protein Journal - The newly emerging SARS-CoV-2 variants are potential threat and posing new challenges for medical intervention due to high transmissibility and escaping neutralizing antibody...     

Membrane Binding and Self-Association of the Epsin N-Terminal Homology Domain

Epsin possesses a conserved epsin N-terminal homology (ENTH) domain that acts as a phosphatidylinositol 4,5-bisphosphate‐lipid‐targeting and membrane‐curvature‐generating element. Upon binding phosphatidylinositol 4,5‐bisphosphate, the N-terminal helix (H₀) of the ENTH domain becomes structured and aids in the aggregation of ENTH domains, which results in extensive membrane remodeling. In this article, atomistic and coarse-grained (CG) molecular dynamics (MD) simulations are used to investigate the structure and the stability of ENTH domain aggregates on lipid bilayers. EPR experiments are also reported for systems composed of different ENTH-bound membrane morphologies, including membrane vesicles as well as preformed membrane tubules. The EPR data are used to help develop a molecular model of ENTH domain aggregates on preformed lipid tubules that are then studied by CG MD simulation. The combined computational and experimental approach suggests that ENTH domains exist predominantly as monomers on vesiculated structures, while ENTH domains self-associate into dimeric structures and even higher‐order oligomers on the membrane tubes. The results emphasize that the arrangement of ENTH domain aggregates depends strongly on whether the local membrane curvature is isotropic or anisotropic. The molecular mechanism of ENTH‐domain-induced membrane vesiculation and tubulation and the implications of the epsin's role in clathrin-mediated endocytosis resulting from the interplay between ENTH domain membrane binding and ENTH domain self-association are also discussed.     

Functional distinction of human EAG1 and EAG2 potassium channels

Human ether à go-go potassium channel 2 (hEAG2) was cloned and its properties were compared with the previously characterized isoform hEAG1. In the Xenopus oocyte expression system the time course of activation was about four times slower and the voltage required for half-maximal subunit activation was about 10 mV greater for hEAG2 channels. However, its voltage dependence was smaller and, therefore, hEAG2 channels start to open at more negative voltages than hEAG1. Coexpression of both isoforms and kinetic analysis of the resulting currents indicated that they can form heteromeric channel complexes in which the slow activation phenotype of hEAG2 is dominant. Upon expression in mammalian cells, quinidine blocked hEAG1 channels (IC(50) 1.4 microM) more potently than hEAG2 channels (IC(50) 152 microM), thus providing a useful tool for the functional distinction between hEAG1 and hEAG2 potassium channels.     

鼠TNF-α功能位点及其中和性抗体结合位点研究

肿瘤坏死因子TNF-α是一个多功能的细胞因子,许多免疫系统疾病的发生和TNF-α的过量释放有关．对TNF-α的功能研究有助于了解这些疾病的发生机制,而以TNF-α为靶点的抗体可用于治疗多种自身免疫性疾病．通过研究一个抗鼠TNF-α中和性单克隆抗体的结合位点,确定了鼠TNF-α行使其生物学功能的关键部位．首先利用酵母展示技术确定了中和性抗体9C6的结合位点是鼠TNF-α的第29～40氨基酸线性片段．然后在此区域引入点突变,找到鼠TNF-α与9C6抗体结合的关键位点．最终用大肠杆菌表达鼠TNF-α和其突变体蛋白,通过L929细胞杀伤实验,证实与9C6抗体结合的关键位点也是决定鼠TNF-α生物学功能的关键位点．     

Structural and Biophysical Characterization of the Interactions between Calmodulin and the Pleckstrin Homology Domain of Akt

The translocation of Akt, a serine/threonine kinase, to the plasma membrane is a critical step in the Akt activation pathway. It is established that membrane binding of Akt is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃). There is now evidence that Akt activation in many breast cancer cells is also modulated by the calcium-binding protein, calmodulin (CaM). Upon EGF stimulation of breast cancer cells, CaM co-localizes with Akt at the plasma membrane to enhance activation. However, the molecular details of Akt(PHD) interaction with CaM are not known. In this study, we employed NMR, biochemical, and biophysical techniques to characterize CaM binding to Akt(PHD). Our data show that CaM forms a tight complex with the PHD of Akt (dissociation constant = 100 nm). The interaction between CaM and Akt(PHD) is enthalpically driven, and the affinity is greatly dependent on salt concentration, indicating that electrostatic interactions are important for binding. The CaM-binding interface in Akt(PHD) was mapped to two loops adjacent to the PI(3,4,5)P₃ binding site, which represents a rare CaM-binding motif and suggests a synergistic relationship between CaM and PI(3,4,5)P₃ upon Akt activation. Elucidation of the mechanism by which Akt interacts with CaM will help in understanding the activation mechanism, which may provide insights for new potential targets to control the pathophysiological processes of cell survival.     

Structural Determination of Functional Units of the Nucleotide Binding Domain (NBD94) of the Reticulocyte Binding Protein Py235 of Plasmodium yoelii

Background

Invasion of the red blood cells (RBC) by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH) plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94) of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.

Methodology/Principal Findings

In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94_444–547, NBD94_566–663 and NBD94_674–793, respectively. Using fluorescence correlation spectroscopy NBD94_444–547 has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94_444–547 in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 Å in length. The high quality of the constructs, forming the hinge-region, NBD94_566–663 and NBD94_674–793 enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94_566–663 consists of two helices with 97.8 Å and 48.6 Å in length, linked by a loop. By comparison, the low resolution structure of NBD94_674–793 in solution represents a chair–like shape with three architectural segments.

Conclusions

These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target.     

C-Linker of Cyclic Nucleotide–gated Channels Controls Coupling of Ligand Binding to Channel Gating

Cyclic nucleotide–gated channels are composed of a core transmembrane domain, structurally homologous to the voltage-gated K⁺ channels, and a cytoplasmic ligand-binding domain. These two modules are joined by ∼90 conserved amino acids, the C-linker, whose precise role in the mechanism of channel activation by cyclic nucleotides is poorly understood. We examined cyclic nucleotide–gated channels from bovine photoreceptors and Caenorhabditis elegans sensory neurons that show marked differences in cyclic nucleotide efficacy and sensitivity. By constructing chimeras from these two channels, we identified a region of 30 amino acids in the C-linker (the L2 region) as an important determinant of activation properties. An increase in both the efficacy of gating and apparent affinity for cGMP and cAMP can be conferred onto the photoreceptor channel by the replacement of its L2 region with that of the C. elegans channel. Three residues within this region largely account for this effect. Despite the profound effect of the C-linker region on ligand gating, the identity of the C-linker does not affect the spontaneous, ligand-independent open probability. Based on a cyclic allosteric model of activation, we propose that the C-linker couples the opening reaction in the transmembrane core region to the enhancement of the affinity of the open channel for agonist, which underlies ligand gating.     

Structural and Functional Mimicry of the Binding Site of hYAP-WW Domain for Proline-rich Ligands

Based on the 3D structure of the WW domain of human Yes-associated protein (hYAP-WW) in complex with a proline-rich peptide ligand, we have designed and synthesized a cyclic peptide covering a fragment of hYAP-WW that contains its primary contact residues for the interaction with the ligand. This peptide was found to specifically recognize a proline-rich ligand for hYAP-WW. Its conformation was calculated using molecular dynamics simulation, based on long-range NOEs identified by NMR spectroscopy, and indicates an arrangement of primary contact residues similar to hYAP-WW.     

Regulation of KATP Channel Expression and Activity by the SUR1 Nucleotide Binding Fold 1

ATP-sensitive K+ (KATP) channels are oligomeric complexes of pore-forming Kir6 subunits and regulatory Sulfonylurea Receptor (SUR) subunits. SUR, an ATP-Binding Cassette (ABC) transporter, confers Mg-nucleotide stimulation to the channel via nucleotide interactions with its two cytoplasmic domains (Nucleotide Binding Folds 1 and 2; NBF1 and NBF2). Regulation of KATP channel expression is a complex process involving subunit assembly in the ER, SUR glycosylation in the Golgi, and trafficking to the plasma membrane. Dysregulation can occur at different steps of the pathway, as revealed by disease-causing mutations. Here, we have addressed the role of SUR1 NBF1 in gating and expression of reconstituted channels. Deletion of NBF1 severely impairs channel expression and abolishes MgADP stimulation. Total SUR1 protein levels are decreased, suggestive of increased protein degradation, but they are not rescued by treatment with sulfonylureas or the proteasomal inhibitor MG-132. Similar effects of NBF1 deletion are observed in recombinant KATP channels obtained by "splitting" SUR1 into two separate polypeptides (a N-terminal "half" and a C-terminal "half"). Interestingly, the location of the "splitting point" in the vicinity of NBF1 has marked effects on the MgADP stimulation of resulting channels. Finally, ablation of the ER retention motif upstream of NBF1 (in either "split" or full-length SUR1) does not rescue expression of channels lacking NBF1. These results indicate that, in addition to NBF1 being required for MgADP stimulation of the channel, it plays an important role in the regulation of channel expression that is independent of the ER retention checkpoint and the proteasomal degradation pathway.     

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter,ABCA4

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.     

大鼠MUPP1蛋白PDZ8结构域的生化及晶体结构特性

多重PDZ结构域蛋白1型（MUPP1）是一种存在于上皮细胞和神经细胞内含有13个PDZ结构域的重要支架蛋白.在上皮细胞中,MUPP1蛋白在紧密连接结构的形成和上皮细胞的极化过程中发挥重要作用.而在中枢神经系统中,MUPP1基因的1个提前终止突变导致了其最后12个PDZ结构域的缺失,以及严重的先天性脑积水.此外,MUPP1蛋白的表达水平与酒精依赖性和药物戒断的严重性也具有显著的相关性.因此,对MUPP1蛋白所含的PDZ结构域进行纯化和性质鉴定,将有助于深入研究MUPP1蛋白的功能和分子机制.在本文研究中,利用亲和纯化和分子筛技术,对大鼠来源的MUPP1蛋白的第8个PDZ结构域进行了表达和纯化.多角度激光光散射的数据表明: MUPP1-PDZ8结构域在溶液中为单体,分子量为16.4 kD.圆二色谱结果表明,MUPP1-PDZ8结构域具有较好的二级结构折叠,测得其熔解温度为71.6摄氏度,暗示该PDZ结构域在溶液中非常稳定.最后,MUPP1-PDZ8结构域的晶体结构显示,该结构域属于I 型PDZ 结构域,包含3个α螺旋和6个β折叠.其中GLGL模块、β折叠B上的1 351位亮氨酸,以及α螺旋B上的1 405位异亮氨酸/1 398位组氨酸形成的PDZ结合口袋,可以特异性地与其目标蛋白质的羧基末端相结合.综上所述,本文的研究提供了MUPP1-PDZ8结构域的生化特性,以及该结构域与其目标蛋白质相互作用的分子机制,这将为MUPP1蛋白的功能研究提供生物化学与结构生物学的理论基础.     

Biochemical Characterization of a Subtype Pancreatic Cholecystokinin Receptor and of its Agonist Binding Domain

Abstract

This study was undertaken in order to improve photoaffinity labelling efficiency of pancreatic cholecystokinin receptor by the cleavable probe ¹²⁵I-ASD-(Thr²⁸, Ahx³¹)-CCK-25-33 and to further characterize the denaturated receptor and is agonist binding domain. Membrane bound pancreatic cholecystokinin receptor was specifically labelled by ¹²⁵I-ASD-(Thr²⁸, Ahx³¹)-CCK-25-33 as a component of Mr ≈ 85,000-100,000. The efficiency of the photolabelling was 3–4%. Performing photolysis on [¹²⁵I-ASD-(Thr²⁸, Ahx³¹)-CCK-25-33-receptor] complexes solubilized by CHAPS did not affect specificity of the labelling reaction but enhanced its efficiency so that up to 10% of the receptor site population could be cross-linked. Several lectins were tested for their ability to recognize and purify the cholecystokinin receptor denaturated by Nonidet P-40. Wheat germ agglutinin provided the best recovery and purification rate. The receptor was fully adsorbed on immobilized wheat germ agglutinin, while only a fraction was retained on ricin II (28%) and Ulex europaeus (28%), thus suggesting that the receptor is heterogeneously glycosylated. Finally, major labelled receptor fragments were generated by enzymatic digestion. There were: endoproeinase Glu-Mr → C ≈ 34,000; endoproteinase Glu-C/trypsin → Mr ≈ 12,000; chymotrypsinlendoproteinase Glu-C → Mr ≈ 16,000 and 12,000. The fragment of Mr 2 34,000 was deglycosylated to a component of Mr ≈ 22,000 whereas the other fragments were insensitive to deglycosylation Such results strongly suggest that cholecystokinin binding occurs in a non-glycosylated domain of the cholecystokinin receptor protein.     

Purification, Biochemical Characterization, Binding Activity, and Selectivity of a Glutamate Binding Protein from Bovine Brain

A glutamate binding protein was purified from bovine brain to apparent homogeneity. The procedure used for the purification of this protein involved extraction of a crude synaptic membrane fraction with Na-cholate, followed by solubilization of the binding protein from the membranes by Triton X-100, and, finally, affinity batch separation of the protein on L-glutamate-loaded glass fiber. The molecular characteristics of the purified protein were similar to those previously described for the glutamate binding protein from rat brain synaptic membranes and included the following: small Mr (14,000), acidic (pI = 4.7) protein with a single NH2-terminal amino acid (tyrosine), and significant absorption at wave-lengths greater than 300 nm. Complete amino acid analysis of the protein was not achieved, either because of destruction of some amino acids or of incomplete hydrolysis of the protein. The protein bound L-glutamate with high affinity (KD = 0.87 microM), exhibited one class of L-glutamate binding sites, and bound glutamate with a stoichiometry of 0.7 mol ligand/mol protein. The displacement of protein-bound L-glutamic acid by other neuroactive amino acids had characteristics similar to those observed for the displacement of L-glutamate from rat brain synaptic membrane or purified protein binding sites. Finally, the metal ligand formers KCN and NaN3 inhibited the activity of this protein just as they have been shown to do in rat brain synaptic membranes or the purified protein.     

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

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