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The C-terminal (haemopexin-like) domain structure of human gelatinase A (MMP2): structural implications for its function.

In common with most other matrix metalloproteinases, gelatinase A has a non-catalytic C-terminal domain that displays sequence homology to haemopexin. Crystals of this domain were used by molecular replacement to solve its molecular structure at 2.6 A resolution, which was refined to an R value of 17.9%. This structure has a disc-like shape, with the chain folded into a beta-propeller structure that has pseudo four-fold symmetry. Although the topology and the side-chain arrangement are very similar to the equivalent domain of fibroblast collagenase, significant differences in surface charge and contouring are observable on 1 side of the gelatinase A disc. This difference might be a factor in allowing the gelatinase A C-terminal domain to bind to natural inhibitor TIMP-2.

Amino Acid Sequence↗

Crystal structures of archaerhodopsin-1 and -2: Common structural motif in archaeal light-driven proton pumps.

Archaerhodopsin-1 and -2 (aR-1 and aR-2) are light-driven proton pumps found in Halorubrum sp. aus-1 and -2, which share 55-58% sequence identity with bacteriorhodopsin (bR), a proton pump found in Halobacterium salinarum. In this study, aR-1 and aR-2 were crystallized into 3D crystals belonging to P4(3)2(1)2 (a = b = 128.1 A, c = 117.6 A) and C222(1) (a = 122.9 A, b = 139.5 A, c = 108.1 A), respectively. In both the crystals, the asymmetric unit contains two protein molecules with slightly different conformations. Each subunit is composed of seven helical segments as seen in bR but, unlike bR, aR-1 as well as aR-2 has a unique omega loop near the N terminus. It is found that the proton pathway in the extracellular half (i.e. the proton release channel) is more opened in aR-2 than in aR-1 or bR. This structural difference accounts for a large variation in the pKa of the acid purple-to-blue transition among the three proton pumps. All the aromatic residues surrounding the retinal polyene chain are conserved among the three proton pumps, confirming a previous argument that these residues are required for the stereo-specificity of the retinal isomerization. In the cytoplasmic half, the region surrounded by helices B, C and G is highly conserved, while the structural conservation is very low for residues extruded from helices E and F. Structural conservation of the hydrophobic residues located on the proton uptake pathway suggests that their precise arrangement is necessary to prevent a backward flow of proton in the presence of a large pH gradient and membrane potential. An empty cavity is commonly seen in the vicinity of Leu93 contacting the retinal C13 methyl. Existence of such a cavity is required to allow a large rotation of the side-chain of Leu93 at the early stage of the photocycle, which has been shown to accompany water translocation across the Schiff base.

Amino Acid Sequence↗

Tertiary structural models for human interleukin-6 and evaluation by a sequence-structure compatibility method and NMR experimental information.

Tertiary structure models of Interleukin-6 were constructed using a routine prediction method based on the X-ray crystal structures of granulocyte colony-stimulating factor (GCSF) and leukemia inhibitory factor (LIF). The models were evaluated with the aid of the sequence-structure compatibility (3D-1D) method program compass and NMR experimental information. The model constructed from GCSF gained higher scores on compass examination than did that from LIF, and the NOE data [Nishimura et al. (1996) Biochemistry 35, 273-281] also turned to be more consistent with the former model.

Amino Acid Sequence↗

Structure-function relationship of three neurotoxins from the venom of Naja kaouthia: a comparison between the NMR-derived structure of NT2 with its homologues, NT1 and NT3.

Three homologous short-chain neurotoxins, named NT1, NT2 and NT3, were purified from the venom of Naja kaouthia. NT1 has an identical amino acid sequence to cobrotoxin from Naja naja atra [Biochemistry 32 (1993) 2131]. NT3 shares the same sequence with cobrotoxin b [J. Biochem. (Tokyo) 122 (1997) 1252], whereas NT2 is a novel 61-residue neurotoxin. Tests of their physiological functions indicate that NT1 shows a greater inhibition of muscle contraction induced by electrical stimulation of the nerve than do NT2 and NT3. Homonuclear proton two-dimensional NMR methods were utilized to study the solution tertiary structure of NT2. A homology model-building method was employed to predict the structure of NT3. Comparison of the structures of these three toxins shows that the surface conformation of NT1 facilitates the substituted base residues, Arg28, Arg30, and Arg36, to occupy the favorable spatial location in the central region of loop II, and the cation groups of all three arginines face out of the molecular surface of NT1. This may contribute greatly to the higher binding of NT1 with AchR compared to NT2 and NT3.

Amino Acid Sequence↗

Solution structure of bromelain inhibitor IV from pineapple stem: structural similarity with Bowman-Birk trypsin/chymotrypsin inhibitor from soybean.

Bromelain inhibitor VI from pineapple stem (BI-VI) is a unique double-chain inhibitor with an 11-residue light chain and a 41-residue heavy chain by disulfide bonds and inhibits the cysteine proteinase bromelain competitively. The structure of BI-VI in aqueous solution was determined using nuclear magnetic resonance spectroscopy and simulated annealing-based calculations. Its three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded antiparallel beta-sheet. Unexpectedly, BI-VI was found to share a similar folding and disulfide bond connectivities not with cystatin superfamily inhibitors which inhibit the same cysteine proteinases but with the Bowman-Birk trypsin/chymotrypsin inhibitor from soybean (BBI-I). BBI-I is a 71-residue inhibitor which has two independent inhibitory sites toward the serine proteinases trypsin and chymotrypsin. These structural similarities with BBI-I suggest that they have evolved from a common ancestor and differentiated in function during a course of molecular evolution.

Amino Acid Sequence↗

Structural studies on peanut lectin complexed with disaccharides involving different linkages: further insights into the structure and interactions of the lectin.

Crystal structures of peanut lectin complexed with Galbeta1-3Gal, methyl-T-antigen, Galbeta1-6GalNAc, Galalpha1-3Gal and Galalpha1-6Glc and that of a crystal grown in the presence of Galalpha1-3Galbeta1-4Gal have been determined using data collected at 100 K. The use of water bridges as a strategy for generating carbohydrate specificity was previously deduced from the complexes of the lectin with lactose (Galbeta1-4Glc) and T-antigen (Galbeta1-3GalNAc). This has been confirmed by the analysis of the complexes with Galbeta1-3Gal and methyl-T-antigen (Galbeta1-3GalNAc-alpha-OMe). A detailed analysis of lectin-sugar interactions in the complexes shows that they are more extensive when the beta-anomer is involved in the linkage. As expected, the second sugar residue is ill-defined when the linkage is 1-->6. There are more than two dozen water molecules which occur in the hydration shells of all structures determined at resolutions better than 2.5 A. Most of them are involved in stabilizing the structure, particularly loops. Water molecules involved in lectin-sugar interactions are also substantially conserved. The lectin molecule is fairly rigid and does not appear to be affected by changes in temperature.

Disaccharides↗

Fluctuations in follicular structures of rat thyroid glands during 24 hours: fine structural and morphometric studies.

The thyroid follicles of adult male Wistar rats were examined at six evenly spaced times over 24 hr with a morphometric technique. Follicular structures were subjected to distinct variations during 24 hr, with respect to volume and numerical densities of follicles in the thyroid gland, and diameters, volumes, cell numbers, and luminal surface areas of individual follicles. Variations in follicular structures were divided into two phases: a large follicular phase at 1200, 1600, and 2000 hr and a small follicular phase at the other times. Although volume densities of follicles in the gland varied with a small amplitude, diameters, volumes, and cell numbers of individual follicles exhibited distinct fluctuations during 24 hr. Numerical densities of follicles in the gland changed distinctly during the small follicular phase as well. Degenerating follicular cells appeared in the follicular lumen especially at 1600 hr. No mitotic follicular cells were found throughout the experiment. Furthermore, one to three follicular cells of two adjacent follicles were often in contact with each other at 0400, 0800, and 1200 hr, and these follicles were lined by the common basement membranes. These results suggest that the variations in follicular structures during the small follicular phase occur in the form of follicle separation and fusion. Moreover, the morphological and morphometric variations in follicles reflect those in subcellular structures of follicular cells previously reported by us.

Animals↗

Protein conformational transitions coupled to binding in molecular recognition of unstructured proteins: hierarchy of structural loss from all-atom Monte Carlo simulations of p27Kip1 unfolding-unbinding and structural determinants of the binding mechanism.

Conformational transitions coupled to binding are studied for the p27(Kip1) protein which undergoes a functional disorder-to-order folding transition during tertiary complex formation with the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) binary complex. Temperature-induced Monte Carlo simulations of p27(Kip1) unfolding-unbinding carried out from the crystal structure of the tertiary complex have revealed a systematic trend in the hierarchy of structural loss for p27(Kip1) and a considerable difference in mobility of p27(Kip1) secondary structure elements. The most persistent interactions of p27(Kip1) at the intermolecular interface during unfolding-unbinding simulations are formed by beta-hairpin and beta-strand that on average maintain their structural integrity considerably longer than other p27(Kip1) elements. We have found that the ensemble of unfolded p27(Kip1) conformations is characterized by transitions between mostly unbound, collapsed conformations and entropically favorable p27(Kip1) conformations, which are weakly bound to the cyclin A side of the binary complex. The results of this study are consistent with the experimental evidence pointing to this region of the intermolecular interface as a potential initiation docking site during binding reaction and may reconcile conflicting experimental hypotheses on the recognition of substrate recruitment motifs.

Cell Cycle Proteins↗

Packing motifs and magneto-structural correlations in crystal structures of metallo-tetrakis(1,2,5-thiadiazole)porphyrazine series, MTTDPz (M=H2, Fe, Co, Ni, Cu, Zn).

Single crystals of tetrakis(thiadiazole)porphyrazine and the corresponding metal(II) derivatives, MTTDPz (M=H2, Fe, Co, Ni, Cu, and Zn) were grown by sublimation under reduced pressure with continuous N2 gas flow. Their structures, obtained by X-ray crystallographic analysis, depend significantly on the central metal ion, and the M=Ni and Cu derivatives exhibit polymorphism. They can be classified into three forms, alpha, beta, and gamma. The alpha form (M=H2, Ni, and Cu) is composed of two-dimensional hexagonal close packing formed by side-by-side contacts between thiadiazole rings, whereas the beta form (M=Fe, Co, and Zn) crystallizes into a one-dimensional coordination polymer. The gamma form (M=Ni and Cu) consists of a ladder structure caused by pi stacking, similar to the beta form of phthalocyanine, and by side-by-side contacts between thiadiazole rings. Although the crystal structures of the MTTDPz series exhibited multi-dimensional network structures, magnetic measurements revealed relatively weak exchange interactions, probably reflecting the long distances between the metal ions.

Journal Article↗

A physiological and structural study of neuron types in the cochlear nucleus. II. Neuron types and their structural correlation with response properties.

The present study examined the morphological cell types of neurons labeled with intracellular horseradish peroxidase injections, many of them following electrophysiological recordings in the cochlear nucleus of gerbils and chinchillas. Most of the subdivisions and neuronal types previously described in the cat were identified in the present material, including spherical and globular bushy cells, stellate, bushy multipolar, elongate, octopus, and giant cells in the ventral cochlear nucleus, and a cartwheel cell in the dorsal cochlear nucleus. In many cases these structurally distinct neurons were correlated with their characteristic responses to stimulation by sound or intracellular injection of depolarizing current. The dendritic terminals of the elongate, antenniform, and clavate cells of the posteroventral cochlear nucleus link each of these cell types with neighboring structures in distinct patterns, which may provide a basis for differences in synaptic organization. These cell types differ from each other and from the stellate cells of the anteroventral cochlear nucleus. Despite their heterogeneous morphology, most of these neurons had a regular discharge in response to stimulation (choppers). Irregularly firing neurons (primary-like) had very different structures, e.g., the spherical and globular bushy cells and the bushy multipolar neuron. They, too, represent a heterogeneous population. An onset neuron was identified as an octopus cell. This paper compares the morphological observations with the electrophysiological properties of different cell types reported in a companion paper (Feng et al. [1994] J. Comp. Neurol.). Together, these findings imply that response properties may be partially independent of neuronal structure. Morphologically distinct neurons can generate similar temporal patterns in response to simple acoustic stimuli. Nevertheless, the synaptic organization of these different neuron types, including their connections, would be expected to affect or alter the cells' responses to appropriate stimuli. The possibility is raised that membrane properties and synaptic organization complement and interact with each other.

Animals↗

Characterization of "H-2K,D-like structures" on MM2 x mouse L cell hybrids. II. An "H-2Kd-like" structure on C3H-derived tumor.

Anti-MM2 serum, which had been prepared by immunizing C3H/He mice with syngeneic MM2 mouse mammary ascites tumor, immunoprecipitated "H-2K,D-like" molecules on DBA/2 and C3H.H-2 degrees lymph node cells as well as on somatic cell hybrids between MM2 tumor and mouse L cells. Preclearing of lysates from C3H.H-2 degrees lymph node cells with anti-H-2.31 serum removed all "H-2K,D-like" molecules reactive with anti-MM2 serum, indicating that the molecules detected by anti-MM2 serum are H-2Kd antigens. The anti-H-2.31 serum detected an "H-2K,D-like structure" on the hybrid cells and absorption of the anti-H-2.31 serum with the hybrid cells deprived the serum of anti-H-2Kd reactivity. The hybrid cells could induce antibodies against the H-2Kd antigen in C3H/He mice. These results indicate that on the hybrid cells, whose parental cells were both derived from C3H mice, there is an "H-2K,D-like structure" that has the H-2Kd private specificity. Absorption of anti-MM2 serum with EL4 cells did not affect the capacity of the serum to detect the H-2Kd antigen on C3H.H-2 degrees lymph node cells, indicating that the "H-2Kd-like structure" is distinct from "H-2K,D-like structure A" which was previously reported. Nine isozymes were examined and MM2 cells, mouse L cells, and the hybrids were found to have the same isozyme markers as those of the C3H/He mouse.

Animals↗

Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome.

The Mu in vitro strand transfer reaction proceeds via two stable higher order nucleoprotein complexes, the Type 1 and Type 2 transpososomes. The Mu A protein is responsible for the structural and functional integrity of the Type 1 transpososome. We have investigated the quaternary structure of the Mu A protein within this complex by chemical cross-linking experiments and found that the basic structural unit is an A tetramer. Three Mu A binding sites in the transpososome are protected by DNase I footprinting: the outermost A binding sites L1 and R1, as well as R2. Genetic evidence is also presented which corroborates this result. Efficient formation of Type 1 complexes occurs in mini-Mus with the L3 or R3 sites deleted or when the L2 site has been substituted; but no reaction occurs in the absence of R2. The protection at the L1 and R1 sites extends 12-13 bp beyond the Mu-host junctions as seen by DNase I and methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] foot-printing, indicating Mu A contacts with the flanking host sequences in the transpososome but not on linear DNA; furthermore, hydroxyl radical footprinting shows an unprecedentedly large enhancement on the continuous strand, 2 bp beyond the nick site outside the Mu right end, which suggests that an altered DNA structure is induced upon Type 1 complex formation.

Bacterial Proteins↗

Theoretical study of the gas-phase structures of sodiated and cesiated leucine and isoleucine: zwitterionic structure disfavored in kinetic method experiments.

The most stable charge-solvated (CS) and zwitterionic (ZW) structures of sodiated and cesiated leucine and isoleucine were studied by density functional theory methods. According to the Boltzmann distribution in gas phase, both forms of LeuNa+ and IleNa+ exist, but in LeuCs+ and IleCs+, the ZW forms are dominant. Results for the sodiated compounds are consistent with the relationship found between decrease in relative stability of CS versus ZW form and aliphatic amino acid side chain length. The observed degeneracy in energy for IleNa+ conformers is at odds with kinetic method results. Additional calculations showed that kinetic method structural determinations for IleNa+ do not reflect relative order of populations in the lowest energy conformers. Since complexation of cationized amino acids into ion-bound dimers disfavors ZW structure by approximately 8 kJ mol(-1), it is suggested that for energy close conformers of sodium-cationized amino acids, the kinetic method may not be reliable for structural determinations.

Cesium↗

Crystal structures of CheY from Thermotoga maritima do not support conventional explanations for the structural basis of enhanced thermostability.

The crystal structure of CheY protein from Thermotoga maritima has been determined in four crystal forms with and without Mg++ bound, at up to 1.9 A resolution. Structural comparisons with CheY from Escherichia coli shows substantial similarity in their folds, with some concerted changes propagating away from the active site that suggest how phosphorylated CheY, a signal transduction protein in bacterial chemotaxis, is recognized by its targets. A highly conserved segment of the protein (the "y-turn loop," residues 55-61), previously suggested to be a rigid recognition determinant, is for the first time seen in two alternative conformations in the different crystal structures. Although CheY from Thermotoga has much higher thermal stability than its mesophilic counterparts, comparison of structural features previously proposed to enhance thermostability such as hydrogen bonds, ion pairs, compactness, and hydrophobic surface burial would not suggest it to be so.

Amino Acid Sequence↗

1.70 A X-ray structure of human apo kallikrein 1: structural changes upon peptide inhibitor/substrate binding.

Human kallikreins are serine proteases that comprise a recently identified large and closely related 15-member family. The kallikreins include both regulatory- and degradative-type proteases, impacting a variety of physiological processes including regulation of blood pressure, neuronal health, and the inflammatory response. While the function of the majority of the kallikreins remains to be elucidated, two members are useful biomarkers for prostate cancer and several others are potentially useful biomarkers for breast cancer, Alzheimer's, and Parkinson's disease. Human tissue kallikrein (human K1) is the best functionally characterized member of this family, and is known to play an important role in blood pressure regulation. As part of this function, human K1 exhibits unique dual-substrate specificity in hydrolyzing low molecular weight kininogen between both Arg-Ser and Met-Lys sequences. We report the X-ray crystal structure of mature, active recombinant human apo K1 at 1.70 A resolution. The active site exhibits structural features intermediate between that of apo and pro forms of known kallikrein structures. The S2 to S2' pockets demonstrate a variety of conformational changes in comparison to the porcine homolog of K1 in complex with peptide inhibitors, including the displacement of an extensive solvent network. These results indicate that the binding of a peptide substrate contributes to a structural rearrangement of the active-site Ser 195 resulting in a catalytically competent juxtaposition with the active-site His 57. The solvent networks within the S1 and S1' pockets suggest how the Arg-Ser and Met-Lys dual substrate specificity of human K1 is accommodated.

Animals↗

Influence of Anionic Layer Structure of Fe-Oxyhydroxides on the Structure of Cd Surface Complexes.

The nature of crystallographic reactive sites on the lepidocrocite (gammaFeOOH) surface has been determined by atomic force microscopy (AFM) and extended X-ray absorption fine structure (EXAFS) spectroscopy and compared to the surface bonding properties of goethite. To this end, the specific surface areas of lepidocrocite particles, and of their crystal faces, were calculated from the size and shape of individual particles determined by AFM, and the structure of Cd surface complexes was determined from Cd-Fe EXAFS distances. The combined results show that Cd forms solely mononuclear surface complexes, even at 100% surface coverage, and that hydrated Cd octahedra sorb on basal {010} and lateral {hk0}, {h0l} faces of lepidocrocite platelets by sharing edges with surface Fe octahedra. The absence, or scarcity, of corner-sharing linkage between Fe and Cd octahedra on the surface of lepidocrocite is in contrast to goethite (alphaFeOOH), where this type of complex is predominant. The explanation for the observed difference of Cd sorption mechanism on these two polymorphs lies not in the shape and relative surface area of their crystallographic faces, but in their different bulk structures and, specifically, in the stacking mode of anion layers (O(2-), OH(-)) which is hexagonal in alphaFeOOH and cubic in gammaFeOOH. This study demonstrates that the stacking mode of anions in the sorbent solid is a key factor in determining the structure of surface complexes on mineral surfaces. Copyright 2000 Academic Press.

Journal Article↗

Structural and mechanistic analysis of two refined crystal structures of the pyridoxal phosphate-dependent enzyme dialkylglycine decarboxylase.

Two refined structures, differing in alkali metal ion content, of the bifunctional, pyridoxal phosphate-dependent enzyme dialkylglycine decarboxylase (DGD) are presented in detail. The enzyme is an alpha 4 tetramer, built up as a dimer of dimers, with a subunit molecular mass of 46.5 kDa. The fold of DGD is similar to those of aspartate aminotransferase, omega-amino acid aminotransferase and tyrosine phenol-lyase. The structure has two binding sites for alkali metal ions. DGD with potassium in site 1 (near the active site) and sodium in site 2 (at the surface of the molecule) has been refined against 2.6A resolution data (R-factor = 17.6%), and DGD with sodium at both sites has been refined against 2.1 A resolution data (R-factor = 17.8%). The proximity of site 1 to the active site accounts for the dependence of enzyme activity on potassium ions, and the observed active site structural changes caused by ion exchange at this site explain the inhibition of activity by sodium. DGD catalyzes both the decarboxylation of dialkylglycine species and the transamination of L-amino acids in its normal catalytic cycle. The active site structure of DGD is moderately homologous to that of aspartate aminotransferase, which catalyzes only transamination; both the differences and similarities provide mechanistic guidelines for the DGD-catalyzed reactions. Models of the L-isovaline and L-alanine external aldimine intermediates suggest mechanisms by which the decarboxylation and transamination reactions could be accomplished within the single active site. Decarboxylation is proposed to be at least partially catalyzed by stereoelectronic activation of the C alpha-carboxylate bond achieved by orienting this bond perpendicular to the plane of the pyridinium ring in the dialkylglycine external aldimine intermediate. Transamination is proposed to be catalyzed by a similar effect on the C alpha-H bond of the L-amino acid external aldimine intermediate, combined with general base catalysis provided by Lys272, in analogy to the mechanism of aspartate aminotransferase.

Amino Acid Sequence↗

Towards structure-based drug design: crystal structure of a multisubstrate adduct complex of glycinamide ribonucleotide transformylase at 1.96 A resolution.

An inhibitor complex structure of glycinamide ribonucleotide transformylase (GAR-Tfase; EC 2.1.2.2) from Escherichia coli has been determined with a multisubstrate adduct BW1476U89 to an R-value of 19.1% at 1.96 A resolution. The structure was determined by a combination of molecular and single isomorphous replacement using data from two different monoclinic crystal lattices and collecting data from crystals soaked in 20% (w/v) methyl-pentanediol as cryoprotectant for shock-freezing at -150 degrees C. The multisubstrate adduct is bound in an extended crevice at the interface between the two functional domains of the enzyme. This inhibitor is positioned in the binding site by three sets of tight interactions with its phosphate, glutamate and pyrimidone ring moieties, while its interventing linker atoms are more flexible and adopt two distinct sets of conformations. The highly conserved Arg103, His108 and Gln170 residues that are key in ligand binding and catalysis (His108), have compensatory conformational variation that gives some clues as to their role in substrate specificity and in the formyl transfer. The molecular design of 1476U89 as a multisubstrate adduct inhibitor (Ki approximately 100 pM at pH 8.5), is confirmed as it closely mimics the shape, molecular interaction and combined binding constants of the natural 10-formyltetrahydrofolate (10-CHO-H4F; Km approximately 77.4 microM at pH 8.5) and glycinamide-ribonucleotide (GAR; Km approximately 8.1 microM at pH 8.5) substrates. The stereochemistry of this ligand complex suggests that His108 may act as an electrophile stabilizing the oxyanion of the tetrahedral intermediate that is formed as a result of the direct attack on the 10-CHO-H4F by the amino group of GAR. Structural comparison of the folate binding modes among GAR-Tfase, dihydrofolate reductase and thymidylate synthase reveals that folate derivates bound to GAR-Tfase differentially adopt the trans conformation for the dihedral angle between atoms C-6 and C-9 providing a handle for targeting specific folate-dependent enzymes. The structural information derived from two different discrete conformations of the ligand in the binding site also suggests several leads for the de novo design of inhibitors of GAR-Tfase that may develop into useful chemotherapeutic agents.

Acyltransferases↗