A spectroscopic study of metal ion and ligand binding to β-lactamase II

β-Lactamase II has two metal-binding sites. The electronic spectra of Cd(II)- and Co(II)-substituted β-lactamase II have been investigated. It is suggested that a thiol ligand is involved in metal binding at the first site. The stoichiometric dissociation constants for Co(II) binding to β-lactamase II were estimated to be 0.13 and 2.66 mM (pH 6.0, 4°C, 1 M NaCl) by equilibrium dialysis. Competition between Zn(II) and Co(II) for the first metal binding site suggests a value of 0.7 μM (pH 6.0, 30°C, 1 M NaCl) for the dissociation constant o Zn(II).The electronic spectra of the Co(II) enzyme lead to the suggestion that the coordination geometries around the metal ions in the first and second sites are related to those of a distorted tetrahedron and octahedron, respectively.     

Crystal structure of the N-terminal region of human Topoisomerase IIβ binding protein 1

Human DNA Topoisomerase IIβ binding protein 1 (TopBP1) is a modulating protein that plays an essential role in the response to DNA damage. The N-terminal region of TopBP1, which contains predicted BRCA1-carboxy terminal (BRCT) domains 1 and 2, binds to Rad9, a component of the cell cycle checkpoint clamp Rad9-Hus1-Rad1 complex. Here, we report the crystal structure of the TopBP1 N-terminal region (residues 1-290) at 2.4 Å resolution. Interestingly, in addition to the predicted tandem BRCT1-2 repeats (residues 103-284), residues 7-98 form a previously unreported BRCT domain (here, BRCT0). In contrast to both BRCT1 and BRCT2, which possess the conventional phosphopeptide binding residues within a surface pocket, the corresponding pocket in BRCT0 is largely hydrophobic. Structural comparisons together with peptide binding studies indicate that the tandem BRCT1-2 domains are the binding region for phosphorylated Ser387 in Rad9.     

A flexible-protein molecular docking study of the binding of ruthenium complex compounds to PIM1, GSK-3β, and CDK2/Cyclin A protein kinases

We employ ensemble docking simulations to characterize the interactions of two enantiomeric forms of a Ru-complex compound (1-R and 1-S) with three protein kinases, namely PIM1, GSK-3β, and CDK2/cyclin A. We show that our ensemble docking computational protocol adequately models the structural features of these interactions and discriminates between competing conformational clusters of ligand-bound protein structures. Using the determined X-ray crystal structure of PIM1 complexed to the compound 1-R as a control, we discuss the importance of including the protein flexibility inherent in the ensemble docking protocol, for the accuracy of the structure prediction of the bound state. A comparison of our ensemble docking results suggests that PIM1 and GSK-3β bind the two enantiomers in similar fashion, through two primary binding modes: conformation I, which is very similar to the conformation presented in the existing PIM1/compound 1-R crystal structure; conformation II, which represents a 180° flip about an axis through the NH group of the pyridocarbazole moiety, relative to conformation I. In contrast, the binding of the enantiomers to CDK2 is found to have a different structural profile including a suggested bound conformation, which lacks the conserved hydrogen bond between the kinase and the ligand (i.e., ATP, staurosporine, Ru-complex compound). The top scoring conformation of the inhibitor bound to CDK2 is not present among the top-scoring conformations of the inhibitor bound to either PIM1 or GSK-3β and vice-versa. Collectively, our results help provide atomic-level insights into inhibitor selectivity among the three kinases.

Agonistic antibody to the α1-adrenergic receptor mobilizes intracellular calcium and induces phosphorylation of a cardiac 15-kDa protein

Hypertension is a major cause for hypertrophic remodelling of the myocardium. Agonistic autoantibodies to extracellular loops of the α₁-adrenergic receptor (α₁-AR) have been identified in patients with arterial hypertension. However, intracellular reactions elicited by these agonistic antibodies remain elusive. An anti-peptide antibody (anti-α₁) was generated against the second extracellular loop of the α₁-AR that bound to its peptide epitope with high affinity (K _D ~50 nM). We studied anti-α₁ effects on intracellular calcium (Ca_i), a key factor in cellular remodelling, and receptor-mediated cardiac protein phosphorylation. Anti-α₁ induced pronounced but transient increases in Ca_i in CHO cells expressing the human α₁-AR (CHO-α₁) and in neonatal cardiomyocytes. Preincubation experiments failed to demonstrate a tonic effect of anti-α₁ on Ca_i. However, preincubation with the antibody attenuated the effect of the α₁-AR antagonist prazosin. In neonatal cardiomyocytes anti-α₁ induced a robust phosphorylation of a 15-kDa protein that is involved in α₁-AR signalling. Our data support the notion that elevation of Ca_i is a general feature of agonistic antibodies’ action and constitute an important pathogenic component of hypertension-associated autoantibodies. Furthermore, we suggest that agonistic antibodies to the α₁-AR contribute to hypertrophic remodelling of cardiac myocytes, and that the cardiac 15-kDa protein is a relevant downstream target of their action.     

β-arrestin-2 regulation of the cAMP response element binding protein

Previous work demonstrated that cystic fibrosis (CF) cells exhibit an increase in cAMP-mediated signaling as a characteristic response to lost CFTR function. Evidence for increased cAMP-mediated signaling in CF included increased phosphorylation of the cAMP response element binding protein (CREB) and elevated β-arrestin-2 (βarr2) expression. However, subsequent studies reveal that CREB activation in CF cells is independent of protein kinase-A (PKA). The goal of this study is to test the hypothesis that elevated βarr2 expression leads to increased CREB activation in a PKA-independent mechanism. βarr2-GFP expressing tracheal epithelial cells (βarr2-GFP) exhibit an increase of pCREB content and subsequent CRE activation compared to GFP expressing control cells. βarr2 activation of the ERK cascade represents a candidate mechanism leading to CREB activation. ERK exhibits increased activation in βarr2-GFP cells compared to cont-GFP cells, and ERK inhibition diminishes CRE activation in both GFP and βarr2-GFP cells. To test directly whether CREB regulation in CF is βarr2-dependent, nasal epithelium excised from wt mice (Cftr +/+; βarr2 +/+), CF mice (Cftr -/-; βarr2 +/+), and DKO mice (Cftr -/-; βarr2 -/-) were analyzed for pCREB protein content. Removal of βarr2 expression from CF mice reduces both pCREB and pERK content to wt levels. These data indicate that CF-related CREB regulation is mediated directly through βarr2 expression via the ERK pathway.     

Computational study of binding of epothilone A to β-tubulin

Understanding the interactions of epothilones with β-tubulin is crucial for computer aided rational design of macrocyclic drugs based on epothilones and epothilone derivatives. Despite numerous structure-activity relationship investigations we still lack substantial knowledge about the binding mode of epothilones and their derivatives to β-tubulin. In this work, we reevaluated the electron crystallography structure of epothilone A/β-tubulin complex (PDB entry 1TVK) and proposed an alternative binding mode of epothilone A to β-tubulin that explains more experimental facts.     

Antiarrhythmic drug amiodarone displays antifungal activity,induces irregular calcium response and intracellular acidification of Aspergillus niger – Amiodarone targets calcium and pH homeostasis of A. niger

The rapidly developing resistance of fungi to antifungal drugs is a serious health problem. Today’s drugs mainly target cell membrane composition and synthesis. Moreover, some of them have serious side effects. New antifungal drugs targeting different molecular pathways are necessary. Amiodarone, an FDA approved antiarrhythmic drug displays antifungal activity. It targets calcium and pH homeostasis. In concentrations above 25 μM, it inhibits the growth of the filamentous fungi Aspergillus niger. It triggers a biphasic calcium response accompanied by a high [Ca²⁺]_c resting level and an intracellular acidification from 7.5 to 6.0, both of which are concentration dependent. Both extracellular calcium and calcium from intracellular organelles are sources of the transient second cytosolic calcium peak, whose amplitude is 0.12 μM for cells treated with 0.1 mM amiodarone. In P-type ATPase deficient A. niger strains pmrAΔ and pmcAΔ, the [Ca²⁺]_c resting level after amiodarone treatment is at least twice as high as that of the wild type, which correlates with fungal viability and hypersensitivity to amiodarone. A combination of amiodarone and amphotericin B is additive in terms of cell viability and cytosolic calcium influx. In contrast, a combination of azole drugs and amiodarone has a synergistic effect on the viability of fungi.     

The relation of protein binding to function: what is the significance of munc18 and synaptotagmin binding to syntaxin 1, and where are the corresponding binding sites?

The Q-SNARE syntaxin 1 is a central component of the synaptic membrane fusion machinery. Syntaxin probably interacts with multiple proteins during synaptic vesicle exocytosis. In vitro, the tightest binding partners for syntaxin 1 are other SNAREs (synaptobrevin/VAMP and SNAP-25) and munc18-1 (also known as rbsec1/nsec1). Recent studies on Drosophila syntaxin led to the surprising finding that a syntaxin mutant which does not bind the munc18-homolog Rop nevertheless functionally substitutes for wild-type syntaxin in membrane fusion (Wu et al., Neuron 23, 593-605, 1999). This observation suggested that syntaxin 1 binding to munc18-1 is not essential for fusion, a puzzling conclusion in view of the tight binding of munc18 and syntaxin homologs in all organisms. To address this issue, we have now reinvestigated the interaction of syntaxin with munc18 and Rop. We find that the syntaxin sequence that was mutated in the Drosophila studies is not essential for munc18/Rop binding, and that the mutant is in fact able to bind to munc18/Rop. Thus the fact that the mutant syntaxin rescues release cannot be used as an argument that munc18 binding is not essential. In addition to munc18 and SNAREs, several other proteins have been suggested to interact with various domains of syntaxin 1, notably the calcium-sensor synaptotagmin and the vesicle protein CSP. Our results confirm that the SNARE motif in syntaxin binds to synaptotagmin, but this interaction does not require the very C-terminus of the motif. Interestingly, binding of synaptotagmin appears to be decreased in the closed conformation of syntaxin. In contrast, no interaction of CSP with syntaxin was detected even under low-stringency conditions. Our data suggest 1., that assays measuring protein/protein interactions that involve syntaxin may be more difficult to evaluate than is often assumed because of the sticky nature of the proteins involved, and 2., that it is currently not possible to draw conclusions about the importance of the various interactions with the available data from Drosophila or vertebrates.     

A quantum-chemical study of the binding ability of βXaaHisGlyHis towards copper(II) ion

The present study analyzed binding of Cu²⁺ to tetrapeptides in water solution at several levels of theoretical approximation. The methods used to study the energetic and structural properties of the complexes in question include semiempirical hamiltonians, density functional theory as well as ab initio approaches including electron correlation effects. In order to shed light on the character of interactions between Cu²⁺ and peptides, which are expected to be mainly electrostatic in nature, decomposition of interaction energy into physically meaningful components was applied.     

Does the S2 rod of myosin II uncoil upon two-headed binding to actin? A leucine-zippered HMM study

Myosin II, like many molecular motors, is a two-headed dimer held together by a coiled-coil rod. The stability of the (S2) rod has implications for head-head interactions, force generation, and possibly regulation. Whether S2 uncoils has been controversial. To test the stability of S2, we constructed a series of "zippered" dimeric smooth muscle myosin II compounds, containing a high-melting temperature 32-amino acid GCN4 leucine zipper in the S2 rod beginning 0, 1, 2, or 15 heptads from the head-rod junction. We then assessed the ability of these and wild-type myosin to bind strongly via two heads to an actin filament by measuring the fluorescence quenching of pyrene-labeled actin induced by myosin binding. Such two-headed binding is expected to exert a large strain that tends to uncoil S2, and hence provide a robust test of S2 stability. We find that wild-type and zippered heavy meromyosin (HMM) are able to bind by both heads to actin under both nucleotide-free and saturating ADP conditions. In addition, we compared the actin affinity and rates for the 0- and 15-zippered HMMs in the phosphorylated "on" state and found them to be very similar. These results strongly suggest that S2 uncoiling is not necessary for two-headed binding of myosin to actin, presumably due to a compliant point in the myosin head(s). We conclude that S2 likely remains intact during the catalytic cycle.     

Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance

Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A_2A receptor (A_2AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A_2AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A_2AR C-terminus from 412 to 316 amino acids (A_2AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A_2AR levels. As cAMP production is downstream of the first activation event—coupling of G protein to its receptor—investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gα_s proteins, we characterize the association of A_2AR and A_2AΔ316R to Gα_s with and without GDP or GTPγs using surface plasmon resonance (SPR). Gα_s affinity for A_2AR was greatest for apo-Gα_s, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A_2AR C-terminus (A_2AΔ316R) decreased the affinity of the unliganded receptor for Gα_s by ～20%, suggesting small changes to binding can greatly impact downstream signaling.     

Development of Photosystem II in dark grown Chlamydomonas reinhardtii. A light-dependent conversion of PS IIβ, QB-nonreducing centers to the PS IIα, QB-reducing form

The green alga Chlamydomonas reinhardtii is a facultative heterotroph and, when cultured in the presence of acetate, will synthesize chlorophyll (Chl) and photosystem (PS) components in the dark. Analysis of the thylakoid membrane composition and function in dark grown C. reinhardtii revealed that photochemically competent PS II complexes were synthesized and assembled in the thylakoid membrane. These PS II centers were impaired in the electron-transport reaction from the primary-quinone electron acceptor, Q_A, to the secondary-quinone electron acceptor, Q_B (Q_B-nonreducing centers). Both complements of the PS II Chl a–b light harvesting antenna (LHC II-inner and LHC II-peripheral) were synthesized and assembled in the thylakoid membrane of dark grown C. reinhardtii cells. However, the LHC II-peripheral was energetically uncoupled from the PS II reaction center. Thus, PS II units in dark grown cells had a -type Chl antenna size with only 130 Chl (a and b) molecules (by definition, PS II units lack LHC II-peripheral). Illumination of dark grown C. reinhardtii caused pronounced changes in the organization and function of PS II. With a half-time of about 30 min, PS II centers were converted froma Q_B-nonreducing form in the dark, to a Q_B-reducing form in the light. Concomitant with this change, PS II units were energetically coupled with the LHC II-peripheral complement in the thylakoid membrane and were converted to a PS II form. The functional antenna of the latter contained more than 250 Chl(a+b) molecules. The results are discussed in terms of a light-dependent activation of the Q_A-Q_B electron-transfer reaction which is followed by association of the PS II unit with a LHC II-peripheral antenna and by inclusion of the mature form of PS II (PS II) in the membrane of the grana partition region.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F₀ non-variable fluorescence yield - F_plf intermediate fluorescence yield plateau leyel - F_max maximum fluorescence yield - F_i initial fluorescence yield increase from F₀ to F_pl (F_pl–F₀) - F_v total variable fluorescence yield (F_m–F0) - DCMU dichlorophenyl-dimethylurea     

Probing the determinants of diacylglycerol binding affinity in the C1B domain of protein kinase Cα

C1 domains are independently folded modules that are responsible for targeting their parent proteins to lipid membranes containing diacylglycerol (DAG), a ubiquitous second messenger. The DAG binding affinities of C1 domains determine the threshold concentration of DAG required for the propagation of signaling response and the selectivity of this response among DAG receptors in the cell. The structural information currently available for C1 domains offers little insight into the molecular basis of their differential DAG binding affinities. In this work, we characterized the C1B domain of protein kinase Cα (C1Bα) and its diagnostic mutant, Y123W, using solution NMR methods and molecular dynamics simulations. The mutation did not perturb the C1Bα structure or the sub-nanosecond dynamics of the protein backbone, but resulted in a > 100-fold increase in DAG binding affinity and a substantial change in microsecond timescale conformational dynamics, as quantified by NMR rotating-frame relaxation-dispersion methods. The differences in the conformational exchange behavior between wild type and Y123W C1Bα were localized to the hinge regions of ligand-binding loops. Molecular dynamics simulations provided insight into the identity of the exchanging conformers and revealed the significance of a particular residue (Gln128) in modulating the geometry of the ligand-binding site. Taken together with the results of binding studies, our findings suggest that the conformational dynamics and preferential partitioning of the tryptophan side chain into the water-lipid interface are important factors that modulate the DAG binding properties of the C1 domains.     

Detergent binding to unmyristylated protein kinase A—Structural implications for the role of Myristate

Myristylation often governs the targeting of protein kinases to the plasma membrane. It is now known that a key member of the src family of protein tyrosine kinases, pp60^v-src, binds to the lipid bilayer of the plasma membrane via a myristylated amino terminal sequence. The mechanism of this interaction is not known; however, myristic acid (Myristic acid may also be referred to as Myristate) and residues 2 through 14 are also absolutely required (Resh and Ling, 1990). This review presents an analysis of crystal structures of detergent-modified recombinant and myristylated mammalian catalytic subunit of protein kinase A. Crystals of unmyristylated recombinant catalytic subunit of protein kinase A are grown in the presence of Mega 8, a glucamide-type of detergent, and only this detergent binds, which results in a resolution extension (Knightonet al., 1991a). Comparisons of these two structures reveal that the detergent association with the recombinant enzyme binds in exactly the same hydrophobic pocket of the protein occupied by myristic acid in the mammalian protein (Karlssonet al., 1993; Zhenget al., 1993a). Removal of the detergent through soaking results in the local unwinding of the first helix, helix A, and disorder of the canonical recognition sequence of the phosphorylation site, Ser 10 (Zhenget al., 1993b). These results suggest that anchoring the myristic acid inside the protein results in formation of a stable structural template, which includes the myristylated amino terminal sequence important for the recognition by protein kinases. This inside out motif might provide a structural paradigm for the recognition of myristylated proteins, including pp60^v-src.     

A proteomics approach to study in vivo protein Nα-modifications

In this article we present a simple method to enrich peptides containing in vivo Nα-modified protein N-termini. We demonstrate that CNBr-activated Sepharose, a commercial amine reactive matrix, can selectively couple peptides via the α-NH₂ group under mild conditions. Following digestion by trypsin, a simple incubation step with the CNBr-activated Sepharose by which the free α-NH₂ containing peptides are coupled with matrix through a covalent bond, allows the separation of N^α-modified peptides from massive free α-NH₂ containing peptides. The removal of contaminant peptides with artificial N^α-modifications, like cyclization of N-terminal S-carbamoylmethylcysteine and glutamine, are also discussed. Application of this method to tryptic digests of HeLa cell proteins resulted by a single LC-MS/MS analysis in the identification of 588 in vivo N^α-modified peptides, of which 507 contain IPI (International Protein Index) annotated protein N-termini and 81 contain IPI unannotated protein N-termini. Most of the identified modifications are acetylations with only a few formylations and propionylations present. Furthermore, Lys-N digestion was also applied and resulted in the identification of 394 in vivo N^α-modified peptides, of which 371 contain IPI annotated protein N-termini and 23 contain IPI unannotated protein N-termini. Combination of the two datasets leads to the identification of 675 N^α-modified IPI annotated protein N-termini and 88 N^α-modified IPI unannotated protein N-termini. Our results suggest that N-terminal acetyltransferases (NATs) may function as N-terminal formyltransferases (NFTs) and N-terminal propionyltransferases (NPTs) in vivo.     

Effects of metal-binding loop mutations on ligand binding to calcium- and integrin-binding protein 1. Evolution of the EF-hand?

Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous, multifunctional regulatory protein consisting of four helix-loop-helix EF-hand motifs. Neither EF-I nor EF-II binds divalent metal ions; however, EF-III is a mixed Mg2+/Ca2+-binding site, and EF-IV is a higher-affinity Ca2+-specific site. Through the generation of several CIB1 mutant proteins, we have investigated the importance of the last (-Z) metal-coordinating position of EF-III (D127) and EF-IV (E172) with respect to the binding of CIB1 to Mg2+, Ca2+, and its biological target, the cytoplasmic domain of the platelet alphaIIb integrin. A D127N mutant had reduced Mg2+ and Ca2+ affinity at EF-III but retained affinity for the alphaIIb domain. A D127E mutant had increased Mg2+ and Ca2+ affinity at EF-III, but unexpectedly, the affinity for the alphaIIb domain was too low for binding to be observed. E172Q and E172D mutants showed no and weak Mg2+ binding at EF-IV, respectively, and each mutant had reduced Ca2+ affinity at EF-IV and showed moderate metal-dependent differences in affinity for the alphaIIb domain. Finally, a D127Q mutant bound Mg2+ and Ca2+ in a manner similar to that of D127N, but like that of D127E, the affinity for the alphaIIb domain was reduced below the detection limit. These data, combined with a NMR-based structural comparison of the Mg2+- and Ca2+-loaded CIB1-alphaIIb peptide complexes, suggest that the D127E and D127Q mutations have a disruptive effect on alphaIIb binding since they expand the metal-binding loop and change the alpha-helix positions in EF-III. Conversely, upon replacement of the ancestral Glu with Asp at the -Z position of EF-III, CIB1 gained affinity for alphaIIb, and the Ca2+ affinity of CIB1 shifted into a range where the protein is able to act as an intracellular Ca2+ sensor.