The single cysteine residue of the Sud protein is required for its function as a polysulfide-sulfur transferase in Wolinella succinogenes.

The periplasmic Sud protein which is induced in Wolinella succinogenes growing by polysulfide respiration, has been previously proposed to serve as a polysulfide binding protein and to transfer polysulfide-sulfur to the active site of polysulfide reductase [Klimmek, O, Kreis, V., Klein, C., Simon, J., Wittershagen, A. & Kr?ger, A. (1998) Eur. J. Biochem. 253, 263-269.]. The results presented in this communication suggest that polysulfide-sulfur is covalently bound to the single cysteine residue (Cys109) of the Sud monomer, and that Cys109 is required for tight binding of polysulfide-sulfur and for sulfur transfer. A modified Sud protein [(C109S)Sud-His6] in which the cysteine residue was replaced by serine, did not catalyze sulfur transfer from polysulfide to cyanide and did not stimulate electron transport to polysulfide, in contrast to Sud-His6. The polysulfide-sulfur bound to (C109S)Sud-His6 was fully removed upon dialysis against sulfide. After this treatment, Sud-His6 retained one sulfur atom per monomer; thiocyanate was formed upon addition of cyanide to the preparation. After incubation of Sud-His6 with polysulfide, a proportion of the Sud-His6 monomers carried one or two sulfur atoms, as shown by matrix-assisted laser desorption ionization mass spectrometry. The sulfur atoms were absent from monomers derived from Sud-His6 treated with cyanide and from (C109S)Sud-His6 incubated with polysulfide.     

Structure of the PIN/LC8 dimer with a bound peptide.

The structure of the protein known both as neuronal nitric oxide synthase inhibitory protein, PIN (protein inhibitor of nNOS), and also as the 8 kDa dynein light chain (LC8) has been solved by X-ray diffraction. Two PIN/LC8 monomers related by a two-fold axis form a rectangular dimer. Two pairs of alpha-helices cover opposite faces, and each pair of helices packs against a beta-sheet with five antiparallel beta-strands. Each five-stranded beta-sheet contains four strands from one monomer and a fifth strand from the other monomer. A 13-residue peptide from nNOS is bound to the dimer in a deep hydrophobic groove as a sixth antiparallel beta-strand. The structure provides key insights into dimerization of and peptide binding by the multifunctional PIN/LC8 protein.     

Human interleukin 4. The solution structure of a four-helix bundle protein.

Heteronuclear 13C and 15N three-dimensional nuclear magnetic resonance (n.m.r.) techniques have been used to determine the solution structure of human interleukin 4, a four-helix bundle protein. A dynamical simulated annealing protocol was used to calculate an ensemble of structures from an n.m.r. data set of 1735 distance restraints, 101 phi angle restraints and 27 pairs of hydrogen bond restraints. The protein structure has a left-handed up-up-down-down topology for the four helices with the two long overhand loops in the structure being connected by a short section of irregular antiparallel beta-sheet. Analysis of the side-chains in the protein shows a clustering of hydrophobic residues, particularly leucines, in the core of the bundle with the side-chains of charged residues being located on the protein surface. The solution structure has been compared with a recent structure prediction for human interleukin 4 and with crystal structures of other helix bundle proteins.     

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor.

Stromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium-resolution structure of a 20-kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed beta-sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine-containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166-residue protein fragment complexed with a 4-residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 A. The structure has good stereochemical properties, including its backbone torsion angles. The beta-sheet and alpha-helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X-ray crystallographic model of Lovejoy B et al., 1994b, Biochemistry 33:8207-8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 A when three loop segments are disregarded. The hydroxamate-substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal S'1 subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic S'2 surface subsite, and a third hydrophobic side chain (P'3) lies at the surface.     

Three-dimensional solution structure of beta cryptogein, a beta elicitin secreted by a phytopathogenic fungus Phytophthora cryptogea.

Cryptogein belongs to a new family of 10-kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of beta cryptogein from Phytophthora cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE-derived distance restraints and 40 dihedral angle restraints obtained from 3JHNH alpha couplings. The RMS deviation from the mean structure is 0.87 +/- 0.14 A for backbone atoms and 1.34 +/- 0.14 A for all the non-hydrogen atoms of residues 2 to 98. The structure of beta cryptogein reveals a novel protein fold, with five helices and a double-stranded beta-sheet facing an omega-loop. One edge of the beta-sheet and the adjacent face of the omega-loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X-ray structure, with slight differences lightly due to the crystal packing.     

Crystal structure of cholesterol oxidase from Brevibacterium sterolicum refined at 1.8 A resolution

Cholesterol oxidase (3 beta-hydroxysteroid oxidase, EC 1.1.3.6) is an FAD-dependent enzyme that carries out the oxidation and isomerization of steroids with a trans A : B ring junction. The crystal structure of the enzyme from Brevibacterium sterolicum has been determined using the method of isomorphous replacement and refined to 1.8 A resolution. The refined model includes 492 amino acid residues, the FAD prosthetic group and 453 solvent molecules. The crystallographic R-factor is 15.3% for all reflections between 10.0 A and 1.8 A resolution. The structure is made up of two domains: an FAD-binding domain and a steroid-binding domain. The FAD-binding domain consists of three non-continuous segments of sequence, including both the N terminus and the C terminus, and is made up of a six-stranded beta-sheet sandwiched between a four-stranded beta-sheet and three alpha-helices. The overall topology of this domain is very similar to other FAD-binding proteins. The steroid-binding domain consists of two non-continuous segments of sequence and contains a six-stranded antiparallel beta-sheet forming the "roof" of the active-site cavity. This large beta-sheet structure and the connections between the strands are topologically similar to the substrate-binding domain of the FAD-binding protein para-hydroxybenzoate hydroxylase. The active site lies at the interface of the two domains, in a large cavity filled with a well-ordered lattice of 13 solvent molecules. The flavin ring system of FAD lies on the "floor" of the cavity with N-5 of the ring system exposed. The ring system is twisted from a planar conformation by an angle of approximately 17 degrees, allowing hydrogen-bond interactions between the protein and the pyrimidine ring of FAD. The amino acid residues that line the active site are predominantly hydrophobic along the side of the cavity nearest the benzene ring of the flavin ring system, and are more hydrophilic on the opposite side near the pyrimidine ring. The cavity is buried inside the protein molecule, but three hydrophobic loops at the surface of the molecule show relatively high temperature factors, suggesting a flexible region that may form a possible path by which the substrate could enter the cavity. The active-site cavity contains one charged residue, Glu361, for which the side-chain electron density suggests a high degree of mobility for the side-chain. This residue is appropriately positioned to act as the proton acceptor in the proposed mechanism for the isomerization step.     

Solution structure of Escherichia coli Par10: The prototypic member of the Parvulin family of peptidyl-prolyl cis/trans isomerases

E. coli Par10 is a peptidyl-prolyl cis/trans isomerase (PPIase) from Escherichia coli catalyzing the isomerization of Xaa-Pro bonds in oligopeptides with a broad substrate specificity. The structure of E. coli Par10 has been determined by multidimensional solution-state NMR spectroscopy based on 1207 conformational constraints (1067 NOE-derived distances, 42 vicinal coupling-constant restraints, 30 hydrogen-bond restraints, and 68 phi/psi restraints derived from the Chemical Shift Index). Simulated-annealing calculations with the program ARIA and subsequent refinement with XPLOR yielded a set of 18 convergent structures with an average backbone RMSD from mean atomic coordinates of 0.50 A within the well-defined secondary structure elements. E. coli Par10 is the smallest known PPIase so far, with a high catalytic efficiency comparable to that of FKBPs and cyclophilins. The secondary structure of E. coli Par10 consists of four helical regions and a four-stranded antiparallel beta-sheet. The N terminus forms a beta-strand, followed by a large stretch comprising three alpha-helices. A loop region containing a short beta-strand separates these helices from a fourth alpha-helix. The C terminus consists of two more beta-strands completing the four-stranded anti-parallel beta-sheet with strand order 2143. Interestingly, the third beta-strand includes a Gly-Pro cis peptide bond. The curved beta-strand forms a hydrophobic binding pocket together with alpha-helix 4, which also contains a number of highly conserved residues. The three-dimensional structure of Par10 closely resembles that of the human proteins hPin1 and hPar14 and the plant protein Pin1At, belonging to the same family of highly homologous proteins.     

Solution structure of NPr, a bacterial signal-transducing protein that controls the phosphorylation state of the potassium transporter-regulating protein IIANtr

A nitrogen-related signal transduction pathway, consisting of the three phosphotransfer proteins EI(Ntr), NPr, and IIA(Ntr), was discovered recently to regulate the uptake of K(+) in Escherichia coli. In particular, dephosphorylated IIA(Ntr) inhibits the activity of the K(+) transporter TrkA. Since the phosphorylation state of IIA(Ntr) is partially determined by its reversible phosphorylation by NPr, we have determined the three-dimensional structure of NPr by solution NMR spectroscopy. In total, we obtained 973 NOE-derived distance restraints, 112 chemical shift-derived backbone angle restraints, and 35 hydrogen-bond restraints derived from temperature coefficients (wave). We propose that temperature wave is useful for identifying exposed beta-strands and assists in establishing protein folds based on chemical shifts. The deduced structure of NPr contains three alpha-helices and four beta-strands with the three helices all packed on the same face of the beta-sheet. The active site residue His16 of NPr for phosphoryl transfer was found to be neutral and in the N epsilon 2-H tautomeric state. There appears to be increased motion in the active site region of NPr compared to HPr, a homologous protein involved in the uptake and regulation of carbohydrate utilization.     

Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.     

Solution structure of DinI provides insight into its mode of RecA inactivation

The Escherichia coli RecA protein triggers both DNA repair and mutagenesis in a process known as the SOS response. The 81-residue E. coli protein DinI inhibits activity of RecA in vivo. The solution structure of DinI has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of residual dipolar couplings, obtained in bicelle and phage media, supplemented with J couplings and a moderate number of NOE restraints. DinI has an alpha/beta fold comprised of a three-stranded beta-sheet and two alpha-helices. The beta-sheet topology is unusual: the central strand is flanked by a parallel and an antiparallel strand and the sheet is remarkably flat. The structure of DinI shows that six negatively charged Glu and Asp residues on DinI's kinked C-terminal alpha-helix form an extended, negatively charged ridge. We propose that this ridge mimics the electrostatic character of the DNA phospodiester backbone, thereby enabling DinI to compete with single-stranded DNA for RecA binding. Biochemical data confirm that DinI is able to displace ssDNA from RecA.     

Solution structure of a recombinant mouse major urinary protein.

Major urinary proteins (MUPs) form an ensemble of protein isoforms which are expressed and secreted by sexually mature male mice only. They belong to the lipocalin superfamily and share with other members of this family the capacity to bind hydrophobic molecules, some of which are odorants. MUPs, either associated with or free of their natural ligands, play an important role in the reproductive cycle of these rodents by acting as pheromones. In fact, they are able to interact with receptors in the vomeronasal organ of the female mice, inducing hormonal and physiological responses by an as yet unknown mechanism. In order to investigate the structural and dynamical features of these proteins in solution, one of the various wild-type isoforms (rMUP: 162 residues) was cloned and subsequently isotopically labeled. The complete 1H, 13C and 15N resonance assignment of that isoform, achieved by using a variety of multidimensional heteronuclear NMR experiments, has been reported recently. Here, we describe the refined high-resolution three-dimensional solution structure of rMUP in the native state, obtained by a combination of distance geometry and energy minimization calculations based on 2362 NOE-derived distance restraints. A comparison with the crystal structure of the wild-type MUPs reveals, aside from minor differences, a close resemblance in both secondary structure and overall topology. The secondary structure of the protein consists of eight antiparallel beta-strands forming a single beta-sheet and an alpha-helix in the C-terminal region. In addition, there are several helical and hairpin turns distributed throughout the protein sequence, mostly connecting the beta-strands. The tertiary fold of the beta-sheet creates a beta-barrel, common to all members of the lipocalin superfamily. The shape of the beta-barrel resembles a calyx, lined inside by mostly hydrophobic residues that are instrumental for the binding and transport of small nonpolar ligand molecules.     

GENFOLD: a genetic algorithm for folding protein structures using NMR restraints.

We report the development and validation of the program GENFOLD, a genetic algorithm that calculates protein structures using restraints obtained from NMR, such as distances derived from nuclear Overhauser effects, and dihedral angles derived from coupling constants. The program has been tested on three proteins: the POU domain (a small three-helix DNA-binding protein), bovine pancreatic trypsin inhibitor (BPTI), and the starch-binding domain from Aspergillus niger glucoamylase I, a 108-residue beta-sheet protein. Structures were calculated for each protein using published NMR restraints. In addition, structures were calculated for BPTI using artificial restraints generated from a high-resolution crystal structure. In all cases the fittest calculated structures were close to the target structure, and could be refined to structures indistinguishable from the target structures by means of a low-temperature simulated annealing refinement. The effectiveness of the program is similar to that of distance geometry and simulated annealing methods, and it is capable of using a very wide range of restraints as input. It can thus be readily extended to the calculation of structures of large proteins, for which few NOE restraints may be available.     

Refined structure of villin 14T and a detailed comparison with other actin-severing domains.

Villin 14T is the amino terminal actin monomer binding domain from the actin-severing and bundling protein villin. Its structure has been determined in solution using heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy (Markus MA, Nakayama T, Matsudaira P, Wagner G. 1994. Solution structure of villin 14T, a domain conserved among actin-severing proteins. Protein Science 3:70-81). An additional nuclear Overhauser effect (NOE) spectroscopy data set, acquired using improved gradient techniques, and further detailed analysis of existing data sets, produced an additional 601 NOE restraints for structure calculations. The overall fold does not change significantly with the additional NOE restraints but the definition of the structure is improved, as judged by smaller deviations among an ensemble of calculated structures that adequately satisfy the NMR restraints. Some of the side chains, especially those in the hydrophobic core of the domain, are much more defined. This improvement in the detail of the solution structure of villin 14T makes it interesting to compare the structure with the crystal structure of gelsolin segment 1, which shares 58% sequence identity with villin 14T, in an effort to gain insight into villin 14T's weaker affinity for actin monomers. Villin 14T has smaller side chains at several positions that make hydrophobic contacts with actin in the context of gelsolin segment 1. The structure is also compared with the structure of the related actin-severing domain, severin domain 2.     

Solution structure of PAFP-S: a new knottin-type antifungal peptide from the seeds of Phytolacca americana

The three-dimensional solution structure of PAFP-S, an antifungal peptide extracted from the seeds of Phytolacca americana, was determined using 1H NMR spectroscopy. This cationic peptide contains 38 amino acid residues. Its structure was determined from 302 distance restraints and 36 dihedral restraints derived from NOEs and coupling constants. The peptide has six cysteines involved in three disulfide bonds. The previously unassigned parings have now been determined from NMR data. The solution structure of PAFP-S is presented as a set of 20 structures using ab initio dynamic simulated annealing, with an average RMS deviation of 1.68 A for the backbone heavy atoms and 2.19 A for all heavy atoms, respectively. For the well-defined triple-stranded beta-sheet involving residues 8-10, 23-27, and 32-36, the corresponding values were 0.39 and 1.25 A. The global fold involves a cystine-knotted three-stranded antiparallel beta-sheet (residues 8-10, 23-27, 32-36), a flexible loop (residues 14-19), and four beta-reverse turns (residues 4-8, 11-14, 19-22, 28-32). This structure features all the characteristics of the knottin fold. It is the first structural model of an antifungal peptide that adopts a knottin-type structure. PAFP-S has an extended hydrophobic surface comprised of residues Tyr23, Phe25, Ile27, Tyr32, and Val34. The side chains of these residues are well-defined in the NMR structure. Several hydrophilic and positively charged residues (Arg9, Arg38, and Lys36) surround the hydrophobic surface, giving PAFP-S an amphiphilic character which would be the main structural basis of its biological function.     

The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 A, 1.8 A and 2.4 A resolution

Escherichia coli nitroreductase is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Its ability to convert relatively non-toxic prodrugs such as CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide) into highly cytotoxic derivatives has led to interest in its potential for cancer gene therapy. We have determined the structure of the enzyme bound to a substrate analogue, nicotinic acid, from three crystal forms at resolutions of 1.7 A, 1.8 A and 2.4 A, representing ten non-crystallographically related monomers. The enzyme is dimeric, and has a large hydrophobic core; each half of the molecule consists of a five-stranded beta-sheet surrounded by alpha-helices. Helices F and F protrude from the core region of each monomer. There is an extensive dimer interface, and the 15 C-terminal residues extend around the opposing monomer, contributing the fifth beta-strand. The active sites lie on opposite sides of the molecule, in solvent-exposed clefts at the dimer interface. The FMN forms hydrogen bonds to one monomer and hydrophobic contacts to both; its si face is buried. The nicotinic acid stacks between the re face of the FMN and Phe124 in helix F, with only one hydrogen bond to the protein. If the nicotinamide ring of the coenzyme NAD(P)H were in the same position as that of the nicotinic acid ligand, its C4 atom would be optimally positioned for direct hydride transfer to flavin N5. Comparison of the structure with unliganded flavin reductase and NTR suggests reduced mobility of helices E and F upon ligand binding. Analysis of the structure explains the broad substrate specificity of the enzyme, and provides the basis for rational design of novel prodrugs and for site-directed mutagenesis for improved enzyme activity.     

Study of the correlation of secondary structure of beta-amyloid peptide (Abeta40) with the hydrophobic exposure under different conditions

Abeta is the core protein of extracellular plaque of Alzheimer's disease, and its neurotoxicity is relative to its conformation. In the current work, the effects of various factors, such as pH, ionic strength and lipid membranes, on the secondary structure of Abeta were studied by circular dichroism. In addition, we detected the exposure of hydrophobic sites of Abeta under different conditions using ANS fluorescence. The results showed that the hydrophobic exposure of the protein was correlated with the content of 3betasheet conformation in the phospholipid-containing environment. The beta-sheet content and hydrophobic exposure of Abeta both increased when reacted with pure PC vesicles, while no beta-sheet content and very low hydrophobic exposure were detected after reaction with 30% cholesterol containing PC vesicles. Since beta-sheet conformation is considered as the toxic conformation of Afbeta such correlation may be important for the pathology of AD.     

Solution structure of villin 14T, a domain conserved among actin-severing proteins.

The solution structure of the N-terminal domain of the actin-severing protein villin has been determined by multidimensional heteronuclear resonance spectroscopy. Villin is a member of a family of actin-severing proteins that regulate the organization of actin in the eukaryotic cytoskeleton. Members of this family are built from 3 or 6 homologous repeats of a structural domain of approximately 130 amino acids that is unrelated to any previously known structure. The N-terminal domain of villin (14T) contains a central beta-sheet with 4 antiparallel strands and a fifth parallel strand at one edge. This sheet is sandwiched between 2 helices on one side and a 2-stranded parallel beta-sheet with another helix on the other side. The strongly conserved sequence characteristic of the protein family corresponds to internal hydrophobic residues. Calcium titration experiments suggest that there are 2 binding sites for Ca2+, a stronger site near the N-terminal end of the longest helix, with a Kd of 1.8 +/- 0.4 mM, and a weaker site near the C-terminal end of the same helix, with a Kd of 11 +/- 2 mM. Mutational and biochemical studies of this domain in several members of the family suggest that the actin monomer binding site is near the parallel strand at the edge of the central beta-sheet.     

Biochemical analysis of the modular enzyme inosine 5'-monophosphate dehydrogenase.

Two prominent domains have been identified in the X-ray crystal structure of inosine-5'-monophosphate dehydrogenase (IMPDH), a core domain consisting of an alpha/beta barrel which contains the active site and an inserted subdomain whose structure is less well defined. The core domain encompassing amino acids 1-108 and 244-514 of wild-type human IMPDH (II) connected by the tetrapeptide linker Ile-Arg-Thr-Gly was expressed. The subdomain including amino acids 99-244 of human wild-type IMPDH (II) was expressed as a His-tagged fusion protein, where the His-tag was removable by enterokinase cleavage. These two proteins as well as wild-type human IMPDH (II), all proteins expressed in Escherichia coli, have been purified to apparent homogeneity. Both the wild-type and core domain proteins are tetrameric and have very similar enzymatic activities. In contrast, the subdomain migrates as a monomer or dimer on a gel filtration column and lacks enzymatic activity. Circular dichroism spectropolarimetry indicates that the core domain retains secondary structure very similar to full-length IMPDH, with 30% alpha-helix and 30% beta-sheet vs 33% alpha-helix and 29% beta-sheet for wild-type protein. Again, the subdomain protein is distinguished from both wild-type and core domain proteins by its content of secondary structure, with only 15% each of alpha-helix and beta-sheet. These studies demonstrate that the core domain of IMPDH expressed separately is both structurally intact and enzymatically active. The availability of the modules of IMPDH will aid in dissecting the architecture of this enzyme of the de novo purine nucleotide biosynthetic pathway, which is an important target for immunosuppressive and antiviral drugs.