Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement.

Hydrogen atoms constitute about half of all atoms in proteins and play a critical role in enzyme mechanisms and macromolecular and solvent structure. Hydrogen atom positions can readily be determined by neutron diffraction, and as such, neutron diffraction is an invaluable tool for elucidating molecular mechanisms. Joint refinement of neutron and X-ray diffraction data can lead to improved models compared with the use of neutron data alone and has now been incorporated into modern, maximum-likelihood based crystallographic refinement programs like CNS. Joint refinement has been applied to neutron and X-ray diffraction data collected on crystals of diisopropyl fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of detoxifying organophosphorus nerve agents. Neutron omit maps reveal a number of important features pertaining to the mechanism of DFPase. Solvent molecule W33, coordinating the catalytic calcium, is a water molecule in a strained coordination environment, and not a hydroxide. The smallest Ca-O-H angle is 53 degrees, well beyond the smallest angles previously observed. Residue Asp-229, is deprotonated, supporting a mechanism involving nucleophilic attack by Asp-229, and excluding water activation by the catalytic calcium. The extended network of hydrogen bonding interactions in the central water filled tunnel of DFPase is revealed, showing that internal solvent molecules form an important, integrated part of the overall structure.

X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase): perdeuteration of proteins for neutron diffraction.

The signal-to-noise ratio is one of the limiting factors in neutron macromolecular crystallography. Protein perdeuteration, which replaces all H atoms with deuterium, is a method of improving the signal-to-noise ratio of neutron crystallography experiments by reducing the incoherent scattering of the hydrogen isotope. Detailed analyses of perdeuterated and hydrogenated structures are necessary in order to evaluate the utility of perdeuterated crystals for neutron diffraction studies. The room-temperature X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase) is reported at 2.1 A resolution. Comparison with an independently refined hydrogenated room-temperature structure of DFPase revealed no major systematic differences, although the crystals of perdeuterated DFPase did not diffract neutrons. The lack of diffraction is examined with respect to data-collection and crystallographic parameters. The diffraction characteristics of successful neutron structure determinations are presented as a guideline for future neutron diffraction studies of macromolecules. X-ray diffraction to beyond 2.0 A resolution appears to be a strong predictor of successful neutron structures.

Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris.

The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D2O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 A resolution on a relatively small (0.43 mm3) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.

Probing the pH-dependent structural features of alpha-KTx12.1, a potassium channel blocker from the scorpion Tityus serrulatus.

Potassium channels are widespread in living cells and are involved in many diseases. The scorpion toxin alpha-KTx(12.1) interacts with various K(+) channels, suggesting its capacity to match diverse channel pores. It is recognized that tissue injuries may affect the pH at toxins site of action, thereby modulating both protein conformation and activity. To better understand its molecular mechanism of action, we studied alpha-KTx(12.1) using pH as a tool to explore its plasticity and NMR in combination with MD calculations to detect it. The toxin solution structure consists of an alpha-helix and a triple-stranded beta-sheet stabilized by four disulfide bridges. The NMR results show, in addition, that His28 possesses an unusually low pK(a) of 5.2. The best set of protein conformers is obtained at pH 4.5, while at pH 7.0, the reduced number of NOEs resulting from a faster hydrogen exchange does not allow to reach a good structural convergence. Nonetheless, MD calculations show that the toxin structure does not vary significantly in that pH range, while conformational changes and modifications of the surface charge distribution occur when His28 is fully protonated. Moreover, essential dynamics analysis reveals variations in the toxin's coherent motions. In conclusion, His28, with its low pK(a) value, provides alpha-KTx(12.1) with the ability to preserve its active conformation over a wide pH interval, thus expanding the range of cellular conditions where the toxin can fully exhibit its activity. Overall, the results further underline the role of histidine as a natural controller of proteins' functionality.

NMR solution structure and dynamics of the peptidyl-prolyl cis-trans isomerase domain of the trigger factor from Mycoplasma genitalium compared to FK506-binding protein.

We have solved the solution structure of the peptidyl-prolyl cis-trans isomerase (PPIase) domain of the trigger factor from Mycoplasma genitalium by homo- and heteronuclear NMR spectroscopy. Our results lead to a well-defined structure with a backbone rmsd of 0.23 A. As predicted, the PPIase domain of the trigger factor adopts the FK506 binding protein (FKBP) fold. Furthermore, our NMR relaxation data indicate that the dynamic behavior of the trigger factor PPIase domain and of FKBP are similar. Structural variations when compared to FKBP exist in the flap region and within the bulges of strand 5 of the beta sheet. Although the active-site crevice is similar to that of FKBP, subtle steric variations in this region can explain why FK506 does not bind to the trigger factor. Sequence variability (27% identity) between trigger factor and FKBP results in significant differences in surface charge distribution and the absence of the first strand of the central beta sheet. Our data indicate, however, that this strand may be partially structured as "nascent" beta strand. This makes the trigger factor PPIase domain the most minimal representative of the FKBP like protein family of PPIases.

Reduced flavin: NMR investigation of N5-H exchange mechanism, estimation of ionisation constants and assessment of properties as biological catalyst.

BACKGROUND: The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases), redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context. RESULTS: N(5)-H exchange reactions of the flavin reduced form and its pH dependence were studied using the 15N-NMR-signals of 15N-enriched, reduced flavin in the pH range from 5 to 12. The chemical shifts of the N(3) and N(5) resonances are not affected to a relevant extent in this pH range. This contrasts with the multiplicity of the N(5)-resonance, which strongly depends on pH. It is a doublet between pH 8.45 and 10.25 that coalesces into a singlet at lower and higher pH values. From the line width of the 15N(5) signal the pH-dependent rate of hydrogen exchange was deduced. The multiplicity of the 15N(5) signal and the proton exchange rates are little dependent on the buffer system used. CONCLUSION: The exchange rates allow an estimation of the pKa value of N(5)-H deprotonation in reduced flavin to be >or= 20. This value imposes specific constraints for mechanisms of flavoprotein catalysis based on this process. On the other hand the pK asymptotically equal to 4 for N(5)-H protonation (to form N(5)+-H2) would be consistent with a role of N(5)-H as a base.

New insights into intracellular lipid binding proteins: The role of buried water.

The crystal structures of most intracellular lipid binding proteins (LBPs) show between 5 and 20 internally bound water molecules, depending on the presence or the absence of ligand inside the protein cavity. The structural and functional significance of these waters has been discussed for several LBPs based on studies that used various biophysical techniques. The present work focuses on two very different LBPs, heart-type fatty acid binding protein (H-FABP) and ileal lipid binding protein (ILBP). Using high-resolution nuclear magnetic resonance spectroscopy, certain resonances belonging to side-chain protons that are located inside the water-filled lipid binding cavity were observed. In the case of H-FABP, the pH- and temperature-dependent behavior of selected side-chain resonances (Ser82 OgH and the imidazole ring protons of His93) indicated an unusually slow exchange with the solvent, implying that the intricate hydrogen-bonding network of amino-acid side-chains and water molecules in the protein interior is very rigid. In addition, holo H-FABP appeared to display a reversible self-aggregation at physiological pH. For ILBP, on the other hand, a more solvent-accessible protein cavity was deduced based on the pH titration behavior of its histidine residues. Comparison with data from other LBPs implies that the evolutionary specialization of LBPs for certain ligand types was not only because of mutations of residues directly involved in ligand binding but also to a refinement of the internal water scaffold.

Correlation of backbone amide and side-chain (13)C resonances in perdeuterated proteins.

Side-chain carbon resonance assignments are difficult to obtain for larger proteins. While standard methods require protons for excitation and detection of magnetization, their presence is often unacceptable and often leads to unacceptable relaxation losses at the directly bound carbon sites. In this paper, pulse sequences are presented which provide connectivities between aliphatic side-chain (13)C and amide (1)H and (15)N chemical shifts in fully deuterated, (13)C/(15)N-enriched proteins. Magnetization either starts off from carbons or from both nitrogens and protons and is passed along the side-chain via (13)C-(13)C isotropic mixing. Direct rather than (13)CO-relayed (15)N-->(13)C(alpha) or (13)C(alpha)-->(15)N transfer steps allow the detection of intraresidual as well as sequential correlations. To avoid ambiguities between these two types in the three-dimensional version of the experiments, a fourth dimension can be introduced to achieve their separation along a (13)C(alpha) frequency axis. The novel methods are demonstrated with the uniformly (2)H/(13)C/(15)N labeled 35-kDa protein diisopropylfluorophosphatase from Loligo vulgaris.

WAVEWAT-improved solvent suppression in NMR spectra employing wavelet transforms.

WAVEWAT is a new processing algorithm to suppress the on-resonance water signal in NMR spectra. It is based on a multiresolution analysis (MRA) of the free induction decay (FID) using a dyadic discrete wavelet transform (DWT). The width of the suppressed signal can be adjusted so that signals close to water are recovered without distortion of the signal shape and intensity. Computational efficiency is comparable to that of convolution filters employing a Fourier transform.

Solution structure of the twelfth cysteine-rich ligand-binding repeat in rat megalin.

Megalin, an approx. 600 kDa transmembrane glycoprotein that acts as multi-ligand transporter, is a member of the low density lipoprotein receptor gene family. Several cysteine-rich repeats, each consisting of about 40 residues, are responsible for the multispecific binding of ligands. The solution structure of the twelfth cysteine-rich ligand-binding repeat with class A motif found in megalin features two short beta-strands and two helical turns, yielding the typical fold with a I-III, II-V and IV-VI disulfide bridge connectivity pattern and a calcium coordination site at the C-terminal end. The resulting differences in electrostatic surface potential compared to other ligand-binding modules of this gene family, however, may be responsible for the functional divergence.

Binding of a designed substrate analogue to diisopropyl fluorophosphatase: implications for the phosphotriesterase mechanism.

A wide range of organophosphorus nerve agents, including Soman, Sarin, and Tabun is efficiently hydrolyzed by the phosphotriesterase enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris. To date, the lack of available inhibitors of DFPase has limited studies on its mechanism. The de novo design, synthesis, and characterization of substrate analogues acting as competitive inhibitors of DFPase are reported. The 1.73 A crystal structure of O,O-dicyclopentylphosphoroamidate (DcPPA) bound to DFPase shows a direct coordination of the phosphoryl oxygen by the catalytic calcium ion. The binding mode of this substrate analogue suggests a crucial role for electrostatics in the orientation of the ligand in the active site. This interpretation is further supported by the crystal structures of double mutants D229N/N120D and D229N/N175D, designed to reorient the electrostatic environment around the catalytic calcium. The structures show no differences in their calcium coordinating environment, although they are enzymatically inactive. Additional double mutants E21Q/N120D and E21Q/N175D are also inactive. On the basis of these crystal structures and kinetic and mutagenesis data as well as isotope labeling we propose a new mechanism for DFPase activity. Calcium coordinating residue D229, in concert with direct substrate activation by the metal ion, renders the phosphorus atom of the substrate susceptible for attack of water, through generation of a phosphoenzyme intermediate. Our proposed mechanism may be applicable to the structurally related enzyme paraoxonase (PON), a component of high-density lipoprotein (HDL).

The electron transfer complex between cytochrome c552 and the CuA domain of the Thermus thermophilus ba3 oxidase. A combined NMR and computational approach.

The structural analysis of the redox complex between the soluble cytochrome c552 and the membrane-integral cytochrome ba3 oxidase of Thermus thermophilus is complicated by the transient nature of this protein-protein interaction. Using NMR-based chemical shift perturbation mapping, however, we identified the contact regions between cytochrome c552 and the CuA domain, the fully functional water-soluble fragment of subunit II of the ba3 oxidase. First we determined the complete backbone resonance assignments of both proteins for each redox state. Subsequently, two-dimensional [15N,1H]TROSY spectra recorded for each redox partner both in free and complexed state indicated those surface residues affected by complex formation between the two proteins. This chemical shift analysis performed for both redox states provided a topological description of the contact surface on each partner molecule. Remarkably, very pronounced indirect effects, which were observed on the back side of the heme cleft only in the reduced state, suggested that alterations of the electron distribution in the porphyrin ring due to formation of the protein-protein complex are apparently sensed even beyond the heme propionate groups. The contact residues of each redox partner, as derived from the chemical shift perturbation mapping, were employed for a protein-protein docking calculation that provided a structure ensemble of 10 closely related conformers representing the complex between cytochrome c552 and the CuA domain. Based on these structures, the electron transfer pathway from the heme of cytochrome c552 to the CuA center of the ba3 oxidase has been predicted.

Mutational and structural studies of the diisopropylfluorophosphatase from Loligo vulgaris shed new light on the catalytic mechanism of the enzyme.

The active site, the substrate binding site, and the metal binding sites of the diisopropylfluorophosphatase (DFPase) from Loligo vulgaris have been modified by means of site-directed mutagenesis to improve our understanding of the reaction mechanism. Enzymatic characterization of mutants located in the major groove of the substrate binding pocket indicates that large hydrophobic side chains at these positions are favorable for substrate turnover. Moreover, the active site residue His287 proved to be beneficial, but not essential, for DFP hydrolysis. In most cases, hydrophobic side chains at position 287 led to significant catalytic activities although reduced relative to the wild-type enzyme. With respect to the Ca-1 binding site, where catalysis occurs, various mutants indicated that the net charge at this calcium-binding site as well as the relative positions of the charged calcium ligands is crucial for catalytic activity. The importance of the electrostatic potential at the active site was furthermore revealed by various mutations of residues lining the interior of the central water-filled tunnel, which traverses the entire protein structure. In this respect, the structural features of residue His181, which is located at the opposite end of the DFPase tunnel relative to the active site, were characterized extensively. It was concluded that a tunnel-spanning hydrogen bond network, which includes a large number of apparently slow exchanging water molecules, relays any modifications in the electrostatics of the system to the active site, thus affecting the catalytic reactivity of the enzyme.

Semiautomatic sequence-specific assignment of proteins based on the tertiary structure--the program st2nmr.

The sequence-specific assignment of resonances is still the most time-consuming procedure that is necessary as the first step in high-resolution NMR studies of proteins. In many cases a reliable three-dimensional (3D) structure of the protein is available, for example, from X-ray spectroscopy or homology modeling. Here we introduce the st2nmr program that uses the 3D structure and Nuclear Overhauser Effect spectroscopy (NOESY) peak list(s) to evaluate and optimize trial sequence-specific assignments of spin systems derived from correlation spectra to residues of the protein. A distance-dependent target function that scores trial assignments based on the presence of expected NOESY crosspeaks is optimized in a Monte Carlo fashion. The performance of the program st2nmr is tested on real NMR data of an alpha-helical (cytochrome c) and beta-sheet (lipocalin) protein using homology models and/or X-ray structures; it succeeded in completely reproducing the correct sequence-specific assignments in most cases using 2D and/or 15N/13C Nuclear Overhauser Effect (NOE) data. Additionally to amino acid residues the program can also handle ligands that are bound to the protein, such as heme, and can be used as a complementary tool to fully automated assignment procedures.

Improved method for unambiguous amino acid side-chain 1H and 13C resonance assignment.

Side-chain proton and carbon-13 resonance assignments of [13C;15N]-enriched proteins usually rely on combinations of several multi-dimensional experiments. Here, we describe a four-dimensional pulse sequence, H(C)C-COSY-TOCSY-(CACO)NH, which provides the information required to assign completely aliphatic side-chain resonance frequencies. As in widely used HCC(CO)NH-TOCSY experiments, problems due to spectral crowding are alleviated by exploiting the dispersion of backbone amide 1H and 15N signals. The modification introduced here allows signals from different side-chains to be distinguished even in the case of overlap in the 1H(N)-15N plane of the spectra. For illustration, the new method is applied to two proteins with molecular masses of 11 and 23 kDa.

Continuum electrostatic analysis of irregular ionization and proton allocation in proteins.

Irregular (nonsigmoidal) ionization behavior of titratable groups in proteins is analyzed theoretically, using a computational algorithm designed to count explicitly for tautomers of titratable groups and different locations of polar hydrogens. On the basis of calculations for different model systems (acid-acid, base-base, acid-base pairs, and cluster of three strongly interacting groups), it is demonstrated that the pK values, extracted from nonsigmoidal titration curves by fitting to a sum of Henderson-Hasselbalch equations, do not describe the ionization equilibrium correctly. The conditions for observation of irregular titration curves are derived analytically for the case of arbitrary couple of interacting ionizable groups. A possible relation between irregularly shaped titration curves and tautomerization is also illustrated. The protonation-deprotonation equilibrium of Asp76 in ribonuclease T1 is shown to be coupled to dipole reorientation of a water molecule bound at the protein-solvent interface. This finding provides a new interpretation of the experimentally observed chemical shift of this residue.

Long-range nature of the interactions between titratable groups in Bacillus agaradhaerens family 11 xylanase: pH titration of B. agaradhaerens xylanase.

Xylanase from Bacillus agaradhaerens belongs to a large group of glycosyl hydrolases which catalyze the degradation of xylan. The protonation behavior of titratable groups of the uniformly (15)N- and (13)C-labeled xylanase was investigated by multinuclear NMR spectroscopy. A total of 224 chemical shift titration curves corresponding to (1)H, (13)C, and (15)N resonances revealed pK(a) values for all aspartic and glutamic acid residues, as well as for the C-terminal carboxylate and histidine residues. Most of the titratable groups exhibit a complex titration behavior, which is most likely due to the mutual interactions with other neighboring groups or due to an unusual local microenvironment. Subsite -1 containing the catalytic dyad shows a long-range interaction over 9 A with Asp21 via two hydrogen bonds with Asn45 as the mediator. This result illuminates the pivotal role of the conserved position 45 among family 11 endoxylanases, determining an alkaline pH optimum by asparagine residues or an acidic pH optimum by an aspartate. The asymmetric interactions of neighboring tryptophan side chains with respect to the catalytic dyad can be comprehended as a result of hydrogen bonding and aromatic stacking. Most of the chemical shift-pH profiles of the backbone amides exhibit biphasic behavior with two distinct inflection points, which correspond to the pK(a) values of the nearby acidic side chains. However, the alternation of both positive and negative slopes of individual amide titration curves is interpreted as a consequence of a simultaneous reorganization of side chain conformational space at pH approximately 6 and/or an overall change in the hydrogen network in the substrate binding cleft.

Cofactor-apoprotein hydrogen bonding in oxidized and fully reduced flavodoxin monitored by trans-hydrogen-bond scalar couplings.

Hydrogen bonding plays a key role in the tight binding of the FMN cofactor and the regulation of its redox properties in flavodoxins. Hydrogen bonding interactions can be directly observed in solution by multidimensional heteronuclear NMR spectroscopy through the scalar couplings between donor and acceptor nuclei. Here we report on the detection of intermolecular trans-hydrogen-bond couplings ((h)J) between the flavin ring system and the backbone of Desulfovibrio vulgaris flavodoxin in the oxidized and the two-electron reduced states. For this purpose, experiments are adapted from pulse sequences previously applied to determining (h)J coupling constants in nucleic acid-base pairs and proteins. The resulting (h2)J(N,N), (h4)J(N,N), (h3)J(C,N), and (h1)J(H,N) couplings involve the (15)N(1), (13)C(2), and (15)N(3) nuclei of the pyrimidine moiety of FMN, whereas no such interactions are detectable for (13)C(4) and (15)N(5). Several long-range (15)N-(15)N, (13)C-(15)N, and (1)H-(15)N J-coupling constants within the flavin are obtained as "by-products". The magnitudes of both (h)J and regular J couplings are found to be dependent on the redox state. In general, good correlations between (h)J coupling constants and donor-group (1)H chemical shifts and also crystallographic donor-acceptor distances are observed.

Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins.

Methods are described to correlate aromatic 1H(delta)2/13C(delta)2 or 1H(epsilon)1/15N(epsilon)1 with aliphatic 13C(beta) chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [15N, 1H]- and/or [13C, 1H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with beta-carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [15N, 1H]-TROSY-H(N)Car experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropyl-fluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine delta-CH and tryptophan epsilon-NH groups.

Statistical analysis of crystallographic data obtained from squid ganglion DFPase at 0.85 A resolution.

The X-ray crystal structure of squid-type diisopropylfluorophosphatase (DFPase) has been refined to a resolution of 0.85 A and a crystallographic R value of 9.4%. Crystal annealing improved both the mosaicity and resolution of the crystals considerably. The overall structure of this protein represents a six-bladed beta-propeller with two calcium ions bound in a central water-filled tunnel. 496 water, two glycerol and two MES buffer molecules and 18 PEG fragments of different lengths could be refined in the solvent region. 45 of the 314 residues have been refined with alternative orientations. H atoms have been omitted from disordered residues. For the residues of the inner beta-strands, H atoms are visible in a normal F(o) - F(c) difference map of a hydrogen-deficient structure model. The 208 most reliable residues, without disorder or reduced occupancy in their side chains, were finally refined without restraints. A subsequent full-matrix refinement cycle for the positional parameters yielded estimated standard deviations (e.s.d.s) by matrix inversion. The thus calculated bond lengths and bond angles and their e.s.d.s were used to obtain averaged bond lengths and bond angles, which were compared with the restraints applied in the preceding refinement cycles. The lengths and angles of the hydrogen bonds inside the antiparallel beta-sheets of the DFPase structure were compared with data averaged over 11 high-resolution protein structures. Torsion angles were averaged according to angle types used as restraints in X-PLOR and CNS and subsequently compared with values obtained from 46 high-resolution structures. Side-chain torsion angles were also classified into rotamer types according to the Penultimate Rotamer Library. Moreover, precise dimensions for both Ca(2+)-coordination polyhedra could be obtained and the coordination of one Ca(2+) ion by an imidazole N atom was confirmed. This statistical analysis thus provides a first step towards a set of restraints that are founded completely on macromolecular data; however, 10-20 additional protein data sets of comparable accuracy and size will be required to obtain a larger statistical base, especially for side-chain analysis.