Removal of 2H-decoupling sidebands in 13CHD2 13C-CEST profiles.

A unique aspect of NMR is its capacity to provide integrated insight into both the structure and intrinsic dynamics of biomolecules. Chemical exchange phenomena that often serve as probes of dynamic processes in biological macromolecules can be quantitatively investigated with chemical exchange saturation transfer (CEST) experiments. 2H-decoupling sidebands, however, always occur in the profiles of 13CHD2 13C-CEST experiments when using the simple CW (continuous wave) method, which may obscure the detection of minor dips of excited states. Traditionally, these sidebands are manually eliminated from the profiles before data analysis by removing experimental points in the range of 2H-decoupling field strength ±50 Hz away from the major dips of the ground state on either side of the dips. Unfortunately, this may also eliminate potential minor dips if they overlap with the decoupling sidebands. Here, we developed methods that use pseudo-continuous waves with variable RF amplitudes distributed onto ramps for 2H decoupling. The new methods were thoroughly validated on Bruker spectrometers at a range of fields (1H frequencies of 600, 700, and 850 MHz, and 1.1 GHz). By using these methods, we successfully removed the sidebands from the NMR profiles of 13CHD2 13C-CEST experiments.

The activation loop of PIP5K functions as a membrane sensor essential for lipid substrate processing.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), a representative member of the phosphatidylinositol phosphate kinase (PIPK) family, is a major enzyme that biosynthesizes the signaling molecule PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) in eukaryotic cells. The stringent specificity toward lipid substrates and the high sensitivity to the membrane environment strongly suggest a membrane-sensing mechanism, but the underlying structural basis is still largely unknown. We present a nuclear magnetic resonance (NMR) study on a peptide commensurate with a PIP5K's activation loop, which has been reported to be a determinant of lipid substrate specificity and subcellular localization of PIP5K. Although the activation loop is severely disordered in the crystal structure of PIP5K, the NMR experiments showed that the largely unstructured peptide folded into an amphipathic helix upon its association with the 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellar surface. Systematic mutagenesis and functional assays further demonstrated the crucial roles of the amphipathic helix and its hydrophobic surface in kinase activity and membrane-sensing function, supporting a working model in which the activation loop is a critical structural module conferring a membrane-sensing mechanism on PIP5K. The activation loop, surprisingly functioning as a membrane sensor, represents a new paradigm of kinase regulation by the activation loop through protein-membrane interaction, which also lays a foundation on the regulation of PIP5K (and other PIPKs) by membrane lipids for future studies.

Structural determinants of host specificity of complement Factor H recruitment by Streptococcus pneumoniae.

Many human pathogens have strict host specificity, which affects not only their epidemiology but also the development of animal models and vaccines. Complement Factor H (FH) is recruited to pneumococcal cell surface in a human-specific manner via the N-terminal domain of the pneumococcal protein virulence factor choline-binding protein A (CbpAN). FH recruitment enables Streptococcus pneumoniae to evade surveillance by human complement system and contributes to pneumococcal host specificity. The molecular determinants of host specificity of complement evasion are unknown. In the present study, we show that a single human FH (hFH) domain is sufficient for tight binding of CbpAN, present the crystal structure of the complex and identify the critical structural determinants for host-specific FH recruitment. The results offer new approaches to the development of better animal models for pneumococcal infection and redesign of the virulence factor for pneumococcal vaccine development and reveal how FH recruitment can serve as a mechanism for both pneumococcal complement evasion and adherence.

Structure of bacterial transcription factor SpoIIID and evidence for a novel mode of DNA binding.

SpoIIID is evolutionarily conserved in endospore-forming bacteria, and it activates or represses many genes during sporulation of Bacillus subtilis. An SpoIIID monomer binds DNA with high affinity and moderate sequence specificity. In addition to a predicted helix-turn-helix motif, SpoIIID has a C-terminal basic region that contributes to DNA binding. The nuclear magnetic resonance (NMR) solution structure of SpoIIID in complex with DNA revealed that SpoIIID does indeed have a helix-turn-helix domain and that it has a novel C-terminal helical extension. Residues in both of these regions interact with DNA, based on the NMR data and on the effects on DNA binding in vitro of SpoIIID with single-alanine substitutions. These data, as well as sequence conservation in SpoIIID binding sites, were used for information-driven docking to model the SpoIIID-DNA complex. The modeling resulted in a single cluster of models in which the recognition helix of the helix-turn-helix domain interacts with the major groove of DNA, as expected. Interestingly, the C-terminal extension, which includes two helices connected by a kink, interacts with the adjacent minor groove of DNA in the models. This predicted novel mode of binding is proposed to explain how a monomer of SpoIIID achieves high-affinity DNA binding. Since SpoIIID is conserved only in endospore-forming bacteria, which include important pathogenic Bacilli and Clostridia, whose ability to sporulate contributes to their environmental persistence, the interaction of the C-terminal extension of SpoIIID with DNA is a potential target for development of sporulation inhibitors.

Role of glutamate 64 in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase.

Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K(m), indicating Glu64 is important for both binding and catalysis in the activation of 5FC. (19)F NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild-type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. (1)H and (15)N NMR analysis revealed trans-hydrogen bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild-type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has a higher activation energy, as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by (1)H and (15)N NMR analysis. To explore the functional role of Glu64 in catalysis, we investigated the deamination of cytosine catalyzed by the E64A mutant by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction intermediate become partially rate-limiting steps. The results of the experimental and computational studies together indicate that Glu64 plays a critical role in both the binding and the chemical transformation in the conversion of the prodrug 5FC to the anticancer drug 5-fluorouracil.

Hydrogen-bond detection, configuration assignment and rotamer correction of side-chain amides in large proteins by NMR spectroscopy through protium/deuterium isotope effects.

The configuration and hydrogen-bonding network of side-chain amides in a 35 kDa protein were determined by measuring differential and trans-hydrogen-bond H/D isotope effects by using the isotopomer-selective (IS)-TROSY technique, which leads to a reliable recognition and correction of erroneous rotamers that are frequently found in protein structures. First, the differential two-bond isotope effects on carbonyl (13)C' shifts, which are defined as Delta(2)Delta(13)C'(ND) = (2)Delta(13)C'(ND(E))-(2)Delta(13)C'(ND(Z)), provide a reliable means for the configuration assignment for side-chain amides, because environmental effects (hydrogen bonds and charges, etc.) are greatly attenuated over the two bonds that separate the carbon and hydrogen atoms, and the isotope effects fall into a narrow range of positive values. Second and more importantly, the significant variations in the differential one-bond isotope effects on (15)N chemical shifts, which are defined as Delta(1)Delta(15)N(D) = (1)Delta(15)N(D(E))-(1)Delta(15)N(D(Z)) can be correlated with hydrogen-bonding interactions, particularly those involving charged acceptors. The differential one-bond isotope effects are additive, with major contributions from intrinsic differential conjugative interactions between the E and Z configurations, H-bonding interactions, and charge effects. Furthermore, the pattern of trans-H-bond H/D isotope effects can be mapped onto more complicated hydrogen-bonding networks that involve bifurcated hydrogen-bonds. Third, the correlations between Delta(1)Delta(15)N(D) and hydrogen-bonding interactions afford an effective means for the correction of erroneous rotamer assignments of side-chain amides. Rotamer correction by differential isotope effects is not only robust, but also simple and can be applied to large proteins.

Using structural analysis to generate parasite-selective monoclonal antibodies.

Diagnosis of eukaryotic parasitic infection using antibody-based tests such as ELISAs (enzyme-linked immunosorbent assays) is often problematic because of the need to differentiate between homologous host and pathogen proteins and to ensure that antibodies raised against a peptide will also bind to the peptide in the context of its three-dimensional protein structure. Filariasis caused by the nematode, Brugia malayi, is an important worldwide tropical disease in which parasites disappear from the bloodstream during daylight hours, thus hampering standard microscopic diagnostic methods. To address this problem, a structural approach was used to develop monoclonal antibodies (mAbs) that detect asparaginyl-tRNA synthetase (AsnRS) secreted from B. malayi. B. malayi and human AsnRS amino acid sequences were aligned to identify regions that are relatively unconserved, and a 1.9 A crystallographic structure of B. malayi AsnRS was used to identify peptidyl regions that are surface accessible and available for antibody binding. Sequery and SSA (Superpositional Structural Analysis) software was used to analyze which of these peptides was most likely to maintain its native conformation as a synthetic peptide, and its predicted helical structure was confirmed by NMR. A 22-residue peptide was synthesized to produce murine mAbs. Four IgG(1) mAbs were identified that recognized the synthetic peptide and the full-length parasite AsnRS, but not human AsnRS. The specificity and affinity of mAbs was confirmed by Western blot, immunohistochemistry, surface plasmon resonance, and enzyme inhibition assays. These results support the success of structural modeling to choose peptides for raising selective antibodies that bind to the native protein.

TROSY of side-chain amides in large proteins.

By using the mixed solvent of 50% H2O/50% D2O and employing deuterium decoupling, TROSY experiments exclusively detect NMR signals from semideuterated isotopomers of carboxamide groups with high sensitivities for proteins with molecular weights up to 80 kDa. This isotopomer-selective strategy extends TROSY experiments from exclusively detecting backbone to both backbone and side-chain amides, particularly in large proteins. Because of differences in both TROSY effect and dynamics between 15N-H(E){D(Z)} and 15N-H(Z){D(E)} isotopomers of the same carboxamide, the 15N transverse magnetization of the latter relaxes significantly faster than that of the former, which provides a direct and reliable stereospecific distinction between the two configurations. The TROSY effects on the 15N-H(E){D(Z)} isotopomers of side-chain amides are as significant as on backbone amides.

Simultaneous NMR assignment of backbone and side chain amides in large proteins with IS-TROSY.

A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H2O/50%D2O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a 13C'-coupled 2D 15N-1H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH2 groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids.

Product release is rate-limiting in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase.

Yeast cytosine deaminase (yCD), a zinc metalloenzyme, catalyzes the hydrolytic deamination of cytosine to uracil. The enzyme is of great biomedical interest because it also catalyzes the deamination of the prodrug 5-fluorocytosine (5FC) to form the anticancer drug 5-fluorouracil (5FU). yCD/5FC is one of the most widely used enzyme/prodrug combinations for gene-directed enzyme prodrug therapy for the treatment of cancers. A pH indicator assay has been developed for the measurement of the steady-state kinetic parameters for the deamination reaction. Transient kinetic studies have shown that the product release is a rate-limiting step in the activation of the prodrug 5FC by yCD. The rate constant of the chemical step for the forward reaction (250 s(-)(1)) is approximately 8 times that of the product release (31 s(-)(1)) and approximately 15 times k(cat) (17 s(-)(1)). The transient kinetic results are consistent with those of the steady-state kinetic analysis in the sense that the k(cat) and K(m) values calculated from the rate constants determined by the transient kinetic analysis are in close agreement with those measured by the steady-state kinetic analysis. NMR experiments have demonstrated that free 5FU is in slow exchange with its complex with yCD but has a low affinity for yCD. The transient kinetic and NMR results together suggest that the release of 5FU is rate-limiting in the activation of the prodrug 5FC by yCD and may involve multiple steps.

Three Dimensional Solution Structure of Neurotoxin CM-11 from the Ophiophagus hannah.

The Ophiophagus hannah (King Cobra) neurotoxin CM-11 is a small protein with 72 amino acid residues. Based on complete assignments of (1)H-NMR resonances and determination of secondary structures of CM-11, 349 distance and 27 dihedral angle constraints including 19 psi's and 8 Xi (1)'s were collected from NOESY and DQF-COSY, and the chemical stereospecific assignment of Beta(1)H was partially achieved. Twelve structures with lower energy were obtained via metric matrix distance geometry and refinement with simulated annealing. These structures have a low RMSD of 0.14 nm for backbone atoms and 0.20 nm for heavy atoms, with no distance constraint violation more than 0.05 nm, and no dihedral angle violation more than 3deg;.

Complete Assignment of (1)H-NMR Resonances of the King Cobra Neurotoxin CM-11.

The king cobra (Ophiophagus Hannah) neurotoxin CM-Il is long-chain peptide with 72 amino acid residues. Its complete assignment of (1)H-NMR resonances was obtained using various 2D-NMR technologies, including DQF-COSY, clean-TOCSY and NOESY.

Determination of the Secondary Structure of the king Cobra Neurotoxin CM-11.

The king cobra neurotoxin CM-11 is a small protein with 72 amino acid residues. After its complete assignments of (1)H-NMR resonance's were obtained using various 2D-NMR technologies, including DQF-COSY, clean-TOCSY and NOESY, the secondary structure was analysed by studying the various NOEs extracted from the NOESY spectra and the distribution of chemical shifts. The secondary structure was finally determined by MCD as follows: a triple-strand antiparallel beta sheet with I20-W26, R37-A43 and V53-S59 as its beta strands, a short alpha helix formed by W30-G35 and four turns formed by P7-K1O, C14-G17, K50-V53 and D61-N64.