Heinz Rüterjans

Crystal structure of diisopropylfluorophosphatase from Loligo vulgaris.

BACKGROUND Phosphotriesterases (PTE) are enzymes capable of detoxifying organophosphate-based chemical warfare agents by hydrolysis. One subclass of these enzymes comprises the family of diisopropylfluorophosphatases (DFPases). The DFPase reported here was originally isolated from squid head ganglion of Loligo vulgaris and can be characterized as squid-type DFPase. It is capable of hydrolyzing the organophosphates diisopropylfluorophosphate, soman, sarin, tabun, and cyclosarin. RESULTS Crystals were grown of both the native and the selenomethionine-labeled enzyme. The X-ray crystal structure of the DFPase from Loligo vulgaris has been solved by MAD phasing and refined to a crystallographic R value of 17.6% at a final resolution of 1.8 A. Using site-directed mutagenesis, we have structurally and functionally characterized essential residues in the active site of the enzyme. CONCLUSIONS The crystal structure of the DFPase from Loligo vulgaris is the first example of a structural characterization of a squid-type DFPase and the second crystal structure of a PTE determined to date. Therefore, it may serve as a structural model for squid-type DFPases in general. The overall structure of this protein represents a six-fold beta propeller with two calcium ions bound in a central water-filled tunnel. The consensus motif found in the blades of this beta propeller has not yet been observed in other beta propeller structures. Based on the results obtained from mutants of active-site residues, a mechanistic model for the DFP hydrolysis has been developed.

Solution structure of the LexA repressor DNA binding domain determined by 1H NMR spectroscopy.

The structure of the 84 residue DNA binding domain of the Escherichia coli LexA repressor has been determined from NMR data using distance geometry and restrained molecular dynamics. The assignment of the 1H NMR spectrum of the molecule, derived from 2‐ and 3‐D homonuclear experiments, is also reported. A total of 613 non‐redundant distance restraints were used to give a final family of 28 structures. The structured region of the molecule consisted of residues 4‐69 and yielded a r.m.s. deviation from an average of 0.9 A for backbone and 1.6 A for all heavy atoms. The structure contains three regular alpha‐helices at residues 6‐21 (I), 28‐35 (II) and 41‐52 (III), and an antiparallel beta‐sheet at residues 56‐58 and 66‐68. Helices II and III form a variant helix‐turn‐helix DNA binding motif, with an unusual one residue insert at residue 38. The topology of the LexA DNA binding domain is found to be the same as for the DNA binding domains of the catabolic activator protein, human histone 5, the HNF‐3/fork head protein and the Kluyveromyces lactis heat shock transcription factor.

Backbone dynamics of ribonuclease T1 and its complex with 2′GMP studied by two-dimensional heteronuclear NMR spectroscopy

SummaryThe backbone dynamics of free ribonuclease T1 and its complex with the competitive inhibitor 2′GMP have been studied by 15N longitudinal and transverse relaxation experiments, combined with {1H, 15H} NOE measurements. The intensity decay of individual amide cross peaks in a series of (1H, 15N)-HSQC spectra with appropriate relaxation periods (Kay, L.E. et al. (1989) Biochemistry, 28, 8972–8979; Kay, L.E. et al. (1992) J. Magn. Reson., 97, 359–375) was fitted to a single exponential by using a simplex algorithm in order to obtain 15N T1 and T2 relaxation times. These experimentally obtained values were analysed in terms of the ‘model-free’ approach introduced by Lipari and Szabo (Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559; 4559–4570). The microdyramical parameters accessible by this approach clearly indicate a correlation between the structural flexibility and the tertiary structure of ribonuclease T1, as well as restricted mobility of certain regions of the protein backbone upon binding of the inhibitor. The results obtained by NMR are compared to X-ray crystallographic data and to observations made in molecular dynamics simulations.

Self-consistent 3J coupling analysis for the joint calibration of Karplus coefficients and evaluation of torsion angles

The concept of self-consistent J coupling evaluation exploits redundant structure information inherent in large sets of 3J coupling constants. Application to the protein Desulfovibrio vulgaris flavodoxin demonstrates the simultaneous refinement of torsion-angle values and related Karplus coefficients. The experimental basis includes quantitative coupling constants related to the polypeptide backbone φ torsion originating from a variety of heteronuclear 2D and 3D NMR correlation experiments, totalling 124 3J(HN,Hα), 129 3J(HN,C′), 121 3J(HN,Cβ), 128 3J(C′i−1,Hαi), 121 3J(C′i−1,C′i), and 122 3J(C′i−1,Cβi). Without prior knowledge from either X-ray crystallography or NMR data, such as NOE distance constraints, accurate φ dihedral angles are specified for 122 non-glycine and non-proline residues out of a total of 147 amino acids. Different models of molecular internal mobility are considered. The Karplus coefficients obtained are applicable to the conformational analysis of φ torsions in other polypeptides.

3D Triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins

SummaryTwo new 3D 1H-15N-13C triple-resonance experiments are presented which provide sequential cross peaks between the amide proton of one residue and the amide nitrogen of the preceding and succeeding residues or the amide proton of one residue and the amide proton of the preceding and succeeding residues, respectively. These experiments, which we term 3D-HN(CA)NNH and 3D-H(NCA)NNH, utilize an optimized magnetization transfer via the 2JNCα coupling to establish the sequential assignment of backbone NH and 15N resonances. In contrast to NH-NH connectivities observable in homonuclear NOESY spectra, the assignments from the 3D-H(NCA)NNH experiment are conformation independent to a first-order approximation. Thus the assignments obtained from these experiments can be used as either confirmation of assignments obtained from a conventional homonuclear approach or as an initial step in the analysis of backbone resonances according to Ikura et al. (1990) [Biochemistry, 29, 4659–4667]. Both techniques were applied to uniformly 15N- and 13C-labelled ribonuclease T1.

A new triple-resonance experiment for the sequential assignment of backbone resonances in proteins

SummaryA new protocol is described for obtaining intraresidual and sequential correlations between carbonyl carbons and amide 1H and 15N resonances of amino acids. Frequency labeling of 13CO spins occurs during a period required for the 13Cα-15N polarization transfer, leading to an optimized transfer efficiency. In a four-dimensional version of the experiment, 13Cα chemical shifts are used to improve the dispersion of signals. The resonance frequencies of all backbone nuclei can be detected in a 3D variant in which cross peaks are split along two frequency axes. This pulse scheme is the equivalent of a five-dimensional experiment. The novel pulse sequences are applied to flavodoxin from Desulfovibrio vulgaris.

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°, 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.

Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase

A membrane-bound c-type cytochrome, c552, acts as the electron mediator between the cytochrome bc1 complex and cytochrome c oxidase in the branched respiratory chain of the bacterium Paracoccus denitrificans. Unlike in mitochondria where a soluble cytochrome c interacts with both complexes, the bacterial c552, the product of the cycM gene, shows a tripartite structure, with an N-terminal membrane anchor separated from a typical class I cytochrome domain by a highly charged region. Two derivative fragments, lacking either only the membrane spanning region or both N-terminal domains, were constructed on the genetic level, and expressed in Escherichia coli cotransformed with the ccm gene cluster encoding host-specific cytochrome c maturation factors. High levels of cytochromes c were expressed and located in the periplasm as holo-proteins; both these purified c552 fragments are functional in electron transport to oxidase, as ascertained by kinetic measurements, and will prove useful for future structural studies of complex formation by NMR and X-ray diffraction.

Solution structure and backbone dynamics of human epidermal-type fatty acid-binding protein (E-FABP).

Human epidermal-type fatty acid-binding protein (E-FABP) belongs to a family of intracellular 14-15 kDa lipid-binding proteins, whose functions have been associated with fatty acid signalling, cell growth, regulation and differentiation. As a contribution to understanding the structure-function relationship, we report in the present study features of its solution structure and backbone dynamics determined by NMR spectroscopy. Applying multi-dimensional high-resolution NMR techniques on unlabelled and 15N-enriched recombinant human E-FABP, the 1H and 15N resonance assignments were completed. On the basis of 2008 distance restraints, the three-dimensional solution structure of human E-FABP was subsequently obtained (backbone atom root-mean-square deviation of 0.92+/-0.11 A; where 1 A=0.1 nm), consisting mainly of 10 anti-parallel beta-strands that form a beta-barrel structure. 15N relaxation experiments (T1, T2 and heteronuclear nuclear Overhauser effects) at 500, 600 and 800 MHz provided information on the internal dynamics of the protein backbone. Nearly all non-terminal backbone amide groups showed order parameters S(2)>0.8, with an average value of 0.88+/-0.04, suggesting a uniformly low backbone mobility in the nanosecond-to-picosecond time range. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the beta-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics elaborated in the present study differ markedly from those of the phylogenetically closely related heart-type FABP and the more distantly related ileal lipid-binding protein, implying a strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family.

Determination of HN,H? and HN,C? coupling constants in 13C, 15N-labeled proteins

SummarySensitive three-dimensional NMR experiments, based on the E.COSY principle, are presented for the measurement of the 3J(HN,Hα) and 3J(HN,C′) coupling constants in uniformly 13C- and 15N-labeled proteins. They employ gradient coherence selection in combination with the sensitivity enhancement method in HSQC-type spectra (Cavanagh et al., 1991; Palmer et al., 1991). In most cases, the two measured coupling constants unambiguously define the ϕ-angle for protein structure determination. The method is applied to uniformly 13C, 15N-labeled ribonuclease T1.