Neeraj Kumar Mishra

FXYD Proteins Stabilize Na,K-ATPase AMPLIFICATION OF SPECIFIC PHOSPHATIDYLSERINE-PROTEIN INTERACTIONS

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared with the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C12E8), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E1 ↔ E2 conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.

PemK Toxin of Bacillus anthracis Is a Ribonuclease AN INSIGHT INTO ITS ACTIVE SITE, STRUCTURE, AND FUNCTION

Bacillus anthracis genome harbors a toxin-antitoxin (TA) module encoding pemI (antitoxin) and pemK (toxin). This study describes the rPemK as a potent ribonuclease with a preference for pyrimidines (C/U), which is consistent with our previous study that demonstrated it as a translational attenuator. The in silico structural modeling of the PemK in conjunction with the site-directed mutagenesis confirmed the role of His-59 and Glu-78 as an acid-base couple in mediating the ribonuclease activity. The rPemK is shown to form a complex with the rPemI, which is in line with its function as a TA module. This rPemI-rPemK complex becomes catalytically inactive when both the proteins interact in a molar stoichiometry of 1. The rPemI displays vulnerability to proteolysis but attains conformational stability only upon rPemK interaction. The pemI-pemK transcript is shown to be up-regulated upon stress induction with a concomitant increase in the amount of PemK and a decline in the PemI levels, establishing the role of these modules in stress. The artificial perturbation of TA interaction could unleash the toxin, executing bacterial cell death. Toward this end, synthetic peptides are designed to disrupt the TA interaction. The peptides are shown to be effective in abrogating TA interaction in micromolar range in vitro. This approach can be harnessed as a potential antibacterial strategy against anthrax in the future.

Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules

Bromodomain-containing protein dysregulation is linked to cancer, diabetes, and inflammation. Selective inhibition of bromodomain function is a newly proposed therapeutic strategy. We describe a (19)F NMR dual screening method for small molecule discovery using fluorinated tryptophan resonances on two bromodomain-containing proteins. The chemical shift dispersion of (19)F resonances within fluorine-labeled proteins enables the simultaneous analysis of two fluorinated bromodomains by NMR. A library of 229 small molecules was screened against the first bromodomain of Brd4 and the BPTF bromodomain. We report the first small molecule selective for BPTF over Brd4, termed AU1. The Kd = 2.8 μM for AU1, which is active in a cell-based reporter assay. No binding is detected with Brd4. Three new Brd4 inhibitors with submicromolar affinity were also discovered. Brd4 hits were validated in a thermal stability assay and potency determined via fluorescence anisotropy. The speed, ease of interpretation, and low protein concentration needed for protein-observed (19)F NMR experiments in a multiprotein format offers a new method to discover and characterize selective ligands for bromodomain-containing proteins.

Protein-observed 19F-NMR for fragment screening, affinity quantification and druggability assessment

NMR spectroscopy can be used to quantify the binding affinity between proteins and low-complexity molecules, termed fragments; this versatile screening approach allows researchers to assess the druggability of new protein targets. Protein-observed 19F-NMR (PrOF NMR) using 19F-labeled amino acids generates relatively simple spectra that are able to provide dynamic structural information toward understanding protein folding and function. Changes in these spectra upon the addition of fragment molecules can be observed and quantified. This protocol describes the sequence-selective labeling of three proteins (the first bromodomains of Brd4 and BrdT, and the KIX domain of the CREB-binding protein) using commercially available fluorinated aromatic amino acids and fluorinated precursors as example applications of the method developed by our research group. Fragment-screening approaches are discussed, as well as Kd determination, ligand-efficiency calculations and druggability assessment, i.e., the ability to target these proteins using small-molecule ligands. Experiment times on the order of a few minutes and the simplicity of the NMR spectra obtained make this approach well-suited to the investigation of small- to medium-sized proteins, as well as the screening of multiple proteins in the same experiment.

Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase A at low pH.

Osmolytes produced under stress in animal and plant systems have been shown to increase thermal stability of the native state of a number of proteins as well as induce the formation of molten globule (MG) in acid denatured states and compact conformations in natively unfolded proteins. However, it is not clear whether these solutes stabilize native state relative to the MG state under partially denaturing conditions. Yeast hexokinase A exists as a MG state at pH 2.5 that does not show any cooperative transition upon heating. Does the presence of some of these osmolytes at pH 2.5 help in the retention of structure that is typical of native state? To answer this question, the effect of ethylene glycol (EG), glycerol, xylitol, sorbitol, trehalose and glucose at pH 2.5 on the structure and stability of yeast hexokinase A was investigated using spectroscopy and calorimetry. In presence of the above osmolytes, except EG, yeast hexokinase at pH 2.5 retains native secondary structure and hydrophobic core and unfolds with excessive heat absorption upon thermal denaturation. However, the cooperative structure binds to ANS suggesting that it is an intermediate between MG and the native state. Further, we show that at high concentration of polyols at pH 2.5, except EG, which populates a non-native ensemble, ΔH(cal)/ΔH(van) approaches unity indicative of two-state unfolding. The results suggest that osmolytes stabilize cooperative protein structure relative to non-cooperative ensemble. These findings have implications toward the structure formation, folding and stability of proteins produced under stress in cellular systems.

Phospholemman (FXYD1) raises the affinity of the human α1β1 isoform of Na,K-ATPase for Na ions.

The human α(1)/His(10)-β(1) isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-β-maltoside, and purified by metal chelate chromatography. The α(1)β(1) complex spontaneously associates in vitro with the detergent-solubilized purified human FXYD1 (phospholemman) expressed in Escherichia coli. It has been confirmed that FXYD1 spontaneously associates in vitro with the α(1)/His(10)-β(1) complex and stabilizes it in an active mode. The functional properties of the α(1)/His(10)-β(1) and α(1)/His(10)-β(1)/FXYD1 complexes have been investigated by fluorescence methods. The electrochromic dye RH421 which monitors binding to and release of ions from the binding sites has been applied in equilibrium titration experiments to determine ion binding affinities and revealed that FXYD1 induces an ∼30% increase of the Na(+)-binding affinity in both the E(1) and P-E(2) conformations. By contrast, it does not affect the affinities for K(+) and Rb(+) ions. Phosphorylation induced partial reactions of the enzyme have been studied as backdoor phosphorylation by inorganic phosphate and in kinetic experiments with caged ATP in order to evaluate the ATP-binding affinity and the time constant of the conformational transition, Na(3)E(1)-P → P-E(2)Na(3). No significant differences with or without FXYD1 could be detected. Rate constants of the conformational transitions Rb(2)E(1) → E(2)(Rb(2)) and E(2)(Rb(2)) → Na(3)E(1), investigated with fluorescein-labeled Na,K-ATPase, showed only minor or no effects of FXYD1, respectively. The conclusion from all these experiments is that FXYD1 raises the binding affinity of α(1)β(1) for Na ions, presumably at the third Na-selective binding site. In whole cell expression studies FXYD1 reduces the apparent affinity for Na ions. Possible reasons for the difference from this study using the purified recombinant Na,K-ATPase are discussed.

Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase

Background: FXYD1 regulates Na,K-ATPase in heart and other tissues. Results: Purified human isoforms (α1β1 and α2β1) reconstituted with wild-type FXYD1 or phosphomimetic mutants S63E, S68E, and S63E,S68E provide detailed information on kinetic effects. Conclusion: FXYD1 slows conformational transition E2(2K)ATP → E1(3Na)ATP and raises binding affinity to Na+ site III. Significance: A detailed picture emerges of the regulatory mechanism of FXYD1 that complements physiological studies. Phospholemman (FXYD1) is a single-transmembrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle, and brain and phosphorylated by protein kinases A and C at Ser-68 and Ser-63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity, and phosphorylation at Ser-68 or Ser-63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms. As a complementary approach, we utilized purified human Na,K-ATPase (α1β1 and α2β1) reconstituted with FXYD1 or mutants S63E, S68E, and S63E,S68E that mimic phosphorylation at Ser-63 and Ser-68. Compared with control α1β1, FXYD1 reduces Vmax and turnover rate and raises K0.5Na. The phosphomimetic mutants reverse these effects and reduce K0.5Na below control K0.5Na. Effects on α2β1 are similar but smaller. Experiments in proteoliposomes reconstituted with α1β1 show analogous effects of FXYD1 on K0.5Na, which are abolished by phosphomimetic mutants and also by increasing mole fractions of DOPS in the proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 slows the conformational transition E2(2K)ATP → E1(3Na)ATP but does not affect 3NaE1P → E2P3Na. This regulatory effect is explained simply by molecular modeling, which indicates that a cytoplasmic helix (residues 60–70) docks between the αN and αP domains in the E2 conformation, but docking is weaker in E1 (also for phosphomimetic mutants). Taken together with previous work showing that FXYD1 also raises binding affinity for the Na+-selective site III, these results provide a rather comprehensive picture of the regulatory mechanism of FXYD1 that complements the physiological studies.

Specific Acetylation Patterns of H2A.Z Form Transient Interactions with the BPTF Bromodomain

Post-translational lysine acetylation of histone tails affects both chromatin accessibility and recruitment of multifunctional bromodomain-containing proteins for modulating transcription. The bromodomain- and PHD finger-containing transcription factor (BPTF) regulates transcription but has also been implicated in high gene expression levels in a variety of cancers. In this report, the histone variant H2A.Z, which replaces H2A in chromatin, is evaluated for its affinity for BPTF with a specific recognition pattern of acetylated lysine residues of the N-terminal tail region. Although BPTF immunoprecipitates H2A.Z-containing nucleosomes, a direct interaction with its bromodomain has not been reported. Using protein-observed fluorine nuclear magnetic resonance (PrOF NMR) spectroscopy, we identified a diacetylation of H2A.Z on lysine residues 4 and 11, with the highest affinity for BPTF with a Kd of 780 μM. A combination of subsequent 1H NMR Carr-Purcell-Meiboom-Gill experiments and photo-cross-linking further confirmed the specificity of the diacetylation pattern at lysines 4 and 11. Because of an adjacent PHD domain, this transient interaction may contribute to a higher-affinity bivalent interaction. Further evaluation of specificity toward a set of bromodomains, including two BET bromodomains (Brd4 and BrdT) and two Plasmodium falciparum bromodomains, resulted in one midmicromolar affinity binder, PfGCN5 (Kd = 650 μM). With these biochemical experiments, we have identified a direct interaction of histone H2A.Z with bromodomains with a specific acetylation pattern that further supports the role of H2A.Z in epigenetic regulation.

Correction to Letter: Fluorinated Aromatic Amino Acids Are Sensitive (19)F NMR Probes for Bromodomain-Ligand Interactions.

This correction regards a typographical error found in Figure u200bFigure33B of the recent letter in the December issue of ACS Chemical Biology (ACS Chem. Biol., 2014, 9, 2755–2760) by Pomerantz and co-workers. In this report, the authors use chemical shift perturbations of assigned fluorine resonances from aromatic amino acids located on the bromodomain to quantify binding affinity and characterize a binding surface. A typographical error has been found in Figure u200bFigure33B which depicts a 19F NMR spectrum of a 3-fluorotyrosine-labeled Brd4 bromodomain (3FY-Brd4). The assignments in the original version for the most downfield resonance at −128 ppm labeled Y118 and the resonance at −138 ppm labeled as Y119 have been interchanged. The correct assignments are shown in the figure above. All of the assignments and analyses in the Supporting Information remain correct in addition to analyses in the main text. The typographical error does not change any conclusions made in the manuscript. n n n nFigure 3 n nPrOF NMR titration of (+)-JQ1 with Brd4(1). (A) Bottom to top: 5FW-Brd4(1) (25 μM), with 1 equiv (+)-JQ1, with 5FW-BPTF (25 μM), with 5FW-BPTF (25 μM) and 1 equiv (+)-JQ1, with unlabeled BrdT(1) (50 μM) and 1 equiv (+)-JQ1. ...