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At least 811 records · Page 45Linked to original sources
PubMed2003

Structures of phosphate and trivanadate complexes of Bacillus stearothermophilus phosphatase PhoE: structural and functional analysis in the cofactor-dependent phosphoglycerate mutase superfamily.

Bacillus stearothermophilus phosphatase PhoE is a member of the cofactor-dependent phosphoglycerate mutase superfamily possessing broad specificity phosphatase activity. Its previous structural determination in complex with glycerol revealed probable bases for its efficient hydrolysis of both large, hydrophobic, and smaller, hydrophilic substrates. Here we report two further structures of PhoE complexes, to higher resolution of diffraction, which yield a better and thorough understanding of its catalytic mechanism. The environment of the phosphate ion in the catalytic site of the first complex strongly suggests an acid-base catalytic function for Glu83. It also reveals how the C-terminal tail ordering is linked to enzyme activation on phosphate binding by a different mechanism to that seen in Escherichia coli phosphoglycerate mutase. The second complex structure with an unusual doubly covalently bound trivanadate shows how covalent modification of the phosphorylable His10 is accompanied by small structural changes, presumably to catalytic advantage. When compared with structures of related proteins in the cofactor-dependent phosphoglycerate mutase superfamily, an additional phosphate ligand, Gln22, is observed in PhoE. Functional constraints lead to the corresponding residue being conserved as Gly in fructose-2,6-bisphosphatases and Thr/Ser/Cys in phosphoglycerate mutases. A number of sequence annotation errors in databases are highlighted by this analysis. B. stearothermophilus PhoE is evolutionarily related to a group of enzymes primarily present in Gram-positive bacilli. Even within this group substrate specificity is clearly variable highlighting the difficulties of computational functional annotation in the cofactor-dependent phosphoglycerate mutase superfamily.

Amino Acid Sequence↗
PubMed2001

A computer system to perform structure comparison using TOPS representations of protein structure.

We describe the design and implementation of a fast topology-based method for protein structure comparison. The approach uses the TOPS topological representation of protein structure, aligning two structures using a common discovered pattern and generating measure of distance derived from an insert score. Heavy use is made of a constraint-based pattern-matching algorithm for TOPS diagrams that we have designed and described elsewhere (Bioinformatics 15(4) (1999) 317). The comparison system is maintained at the European Bioinformatics Institute and is available over the Web at tops.ebi.ac.uk/tops. Users submit a structure description in Protein Data Bank (PDB) format and can compare it with structures in the entire PDB or a representative subset of protein domains, receiving the results by email.

Algorithms↗
PubMed1998

Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes.

New levels in the understanding of DNA replication have been achieved from recent crystal structure determinations of several DNA polymerases and their substrate complexes. The structure of an alpha family DNA polymerase from bacteriophage RB69 shows some similarities, but also considerable differences in structure and organization from the pol I family DNA polymerases. Also, the functions of three polymerase domains and their conserved residues have been clarified by studying structures of pol I family DNA polymerases complexed to their substrates. These structures also confirm that an identical two-metal ion catalytic mechanism proposed previously is used by both the nonhomologous pol I and pol beta family DNA polymerases.

Amino Acid Sequence↗
PubMed2000

The crystal structure of the conserved GTPase of SRP54 from the archaeon Acidianus ambivalens and its comparison with related structures suggests a model for the SRP-SRP receptor complex.

BACKGROUND: Protein targeting to the endoplasmic reticulum in eukaryotes and to the cell membrane in prokaryotes is mediated by the signal recognition particle (SRP) and its receptor (SR). Both contain conserved GTPase domains in the signal-peptide-binding proteins (SRP54 and Ffh) and the SR proteins (SRalpha and FtsY). These GTPases are involved in the regulation of protein targeting. Most studies so far have focussed on the SRP machinery of mammals and bacteria, leaving the SRP system of archaea less well understood. RESULTS: We report the crystal structure of the conserved GTPase (NG-Ffh) from the thermophilic archaeon Acidianus ambivalens at 2.0 A resolution and of the Thr112-->Ala mutant, which is inactive in GTP hydrolysis. This is the first structure of an SRP component from an archaeon and allows for a detailed comparison with related structures from Escherichia coli and thermophilic bacteria. In particular, differences in the conserved consensus regions for nucleotide binding and the subdomain interfaces are observed, which provide information about the regulation of the GTPase. These interactions allow us to propose a common signalling mechanism for the SRP-SR system. CONCLUSIONS: The overall structure of SRP-GTPases is well conserved between bacteria and archaea, which indicates strong similarities in the regulation of the SRP-targeting pathway. Surprisingly, structure comparisons identified a homodimeric ATP-binding protein as the closest relative. A heterodimer model for the SRP-SR interaction is presented.

Archaea↗
PubMed1994

The 2.8 A crystal structure of peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: a five-layered alpha beta alpha beta alpha structure constructed from two core domains of identical topology.

BACKGROUND: The peroxisomal enzyme 3-ketoacyl-coenzyme A thiolase of the yeast Saccharomyces cerevisiae is a homodimer with 417 residues per subunit. It is synthesized in the cytosol and subsequently imported into the peroxisome where it catalyzes the last step of the beta-oxidation pathway. We have determined the structure of this thiolase in order to study the reaction mechanism, quaternary associations and intracellular targeting of thiolases generally, and to understand the structural basis of genetic disorders associated with human thiolases. RESULTS: Here we report the crystal structure of unliganded yeast thiolase refined at 2.8 A resolution. The enzyme comprises three domains; two compact core domains having the same fold and a loop domain. Each of the two core domains is folded into a mixed five-stranded beta-sheet covered on each side by helices and the two are assembled into a five-layered alpha beta alpha beta alpha structure. The central layer is formed by two helices, which point with their amino termini towards the active site. The loop domain, which is to some extent stabilized by interactions with the other subunit, runs over the surface of the two core domains, encircling the active site of its own subunit. CONCLUSIONS: The crystal structure of thiolase shows that the active site is a shallow pocket, shaped by highly conserved residues. Two conserved cysteines and a histidine at the floor of this pocket probably play key roles in the reaction mechanism. The two active sites are on the same face of the dimer, far from the amino and carboxyl termini of both subunits and the disordered amino-terminal import signal sequence.

Acetyl-CoA C-Acyltransferase↗
PubMed1998

Structure of Escherichia coli ribokinase in complex with ribose and dinucleotide determined to 1.8 A resolution: insights into a new family of kinase structures.

BACKGROUND: D-ribose must be phosphorylated at O5' before it can be used in either anabolism or catabolism. This reaction is catalysed by ribokinase and requires the presence of ATP and magnesium. Ribokinase is a member of a family of carbohydrate kinases of previously unknown structure. RESULTS: The crystal structure of ribokinase from Escherichia coli in complex with ribose and dinucleotide was determined at 1.84 A resolution by multiple isomorphous replacement. There is one 33 kDa monomer of ribokinase in the asymmetric unit but the protein forms a dimer around a crystallographic twofold axis. Each subunit consists of a central alpha/beta unit, with a new type of nucleotide-binding fold, and a distinct beta sheet that forms a lid over the ribose-binding site. Contact between subunits involves orthogonal packing of beta sheets, in a novel dimer interaction that we call a beta clasp. CONCLUSIONS: Inspection of the complex indicates that ribokinase utilises both a catalytic base for activation of the ribose in nucleophilic attack and an anion hole that stabilises the transition state during phosphoryl transfer. The structure suggests an ordered reaction mechanism, similar to those proposed for other carbohydrate kinases that probably involves conformational changes. We propose that the beta-clasp structure acts as a lid, closing and opening upon binding and release of ribose. From these observations, an understanding of the structure and catalytic mechanism of related sugar kinases can be obtained.

Adenylyl Imidodiphosphate↗
PubMed1998

Crystal structure of the phosphatidylethanolamine-binding protein from bovine brain: a novel structural class of phospholipid-binding proteins.

BACKGROUND: Phosphatidylethanolamine-binding protein (PEBP) is a basic protein found in numerous tissues from a wide range of species. The screening of gene and protein data banks defines a family of PEBP-related proteins that are present in a variety of organisms, including Drosophila and inferior eukaryotes. PEBP binds to phosphatidylethanolamine and nucleotides in vitro, but its biological function in vivo is not yet known. The expression of PEBP and related proteins seems to be correlated with development and cell morphogenesis, however. To obtain new insights into the PEBP family and its potential functions, we initiated a crystallographic study of bovine brain PEPB. RESULTS: The X-ray crystal structure of bovine brain PEBP has been solved using multiple isomorphous replacement methods, and refined to 1.84 A resolution. The structure displays a beta fold and exhibits one nonprolyl cis peptide bond. Analysis of cavities within the structure and sequence alignments were used to identify a putative ligand-binding site. This binding site is defined by residues of the C-terminal helix and the residues His85, Asp69, Gly109 and Tyr119. This site also corresponds to the binding site of phosphorylethanolamine, the polar head group of phosphatidylethanolamine. CONCLUSIONS: This study shows that PEBP is not related to the G-protein family nor to known lipid-binding proteins, and therefore defines a novel structural family of phospholipid-binding proteins. The PEBP structure contains no internal hydrophobic pocket, as described for lipocalins or small phospholipid-transfer proteins. Nevertheless, in PEBP, a small cavity close to the protein surface has a high affinity for anions, such as phosphate and acetate, and also phosphorylethanolamine. We suggest that this cavity corresponds to the binding site of the polar head group of phosphatidylethanolamine.

Amino Acid Sequence↗
PubMed1999

The crystal structure of pyroglutamyl peptidase I from Bacillus amyloliquefaciens reveals a new structure for a cysteine protease.

BACKGROUND: The N-terminal pyroglutamyl (pGlu) residue of peptide hormones, such as thyrotropin-releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH), confers resistance to proteolysis by conventional aminopeptidases. Specialized pyroglutamyl peptidases (PGPs) are able to cleave an N-terminal pyroglutamyl residue and thus control hormonal signals. Until now, no direct or homology-based three-dimensional structure was available for any PGP. RESULTS: The crystal structure of pyroglutamyl peptidase I (PGP-I) from Bacillus amyloliquefaciens has been determined to 1.6 A resolution. The crystallographic asymmetric unit of PGP-I is a tetramer of four identical monomers related by noncrystallographic 222 symmetry. The protein folds into an alpha/beta globular domain with a hydrophobic core consisting of a twisted beta sheet surrounded by five alpha helices. The structure allows the function of most of the conserved residues in the PGP-I family to be identified. The catalytic triad comprises Cys144, His168 and Glu81. CONCLUSIONS: The catalytic site does not have a conventional oxyanion hole, although Cys144, the sidechain of Arg91 and the dipole of an alpha helix could all stabilize a negative charge. The catalytic site has an S1 pocket lined with conserved hydrophobic residues to accommodate the pyroglutamyl residue. Aside from the S1 pocket, there is no clearly defined mainchain substrate-binding region, consistent with the lack of substrate specificity. Although the overall structure of PGP-I resembles some other alpha/beta twisted open-sheet structures, such as purine nucleoside phosphorylase and cutinase, there are important differences in the location and organization of the active-site residues. Thus, PGP-I belongs to a new family of cysteine proteases.

Amino Acid Sequence↗
PubMed2003

Overview of structural genomics: from structure to function.

The unprecedented increase in the number of new protein sequences arising from genomics and proteomics highlights directly the need for methods to rapidly and reliably determine the molecular and cellular functions of these proteins. One such approach, structural genomics, aims to delineate the total repertoire of protein folds, thereby providing three-dimensional portraits for all proteins in a living organism and to infer molecular functions of the proteins. The goal of obtaining protein structures on a genomic scale has motivated the development of high-throughput technologies for macromolecular structure determination, which have begun to produce structures at a greater rate than previously possible. These new structures have revealed many unexpected functional and evolution relationships that were hidden at the sequence level.

Amino Acid Sequence↗
PubMed1992

The three-dimensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution.

The crystal structure of a mu class glutathione S-transferase (EC 2.5.1.18) from rat liver (isoenzyme 3-3) in complex with the physiological substrate glutathione (GSH) has been solved at 2.2-A resolution by multiple isomorphous replacement methods. The enzyme crystallized in the monoclinic space group C2 with unit cell dimensions of a = 87.98 A, b = 69.41 A, c = 81.34 A, and beta = 106.07 degrees. Oligonucleotide-directed site-specific mutagenesis played an important role in the solution of the structure in that the cysteine mutants C86S, C114S, and C173S were used to help locate the positions of mercuric ion sites in nonisomorphous derivatives with ethylmercuric phosphate and to align the sequence with the model derived from MIR phases. A complete model for the protein was not obtained until part of the solvent structure was interpreted. The dimer in the asymmetric unit refined to a crystallographic R = 0.171 for 19,298 data and I > or = 1.5 sigma (I). The final model consists of 4150 atoms, including all non-hydrogen atoms of 434 amino acid residues, two GSH molecules, and oxygen atoms of 474 water molecules. The dimeric enzyme is globular in shape with dimensions of 53 x 62 x 56 A. Crystal contacts are primarily responsible for conformational differences between the two subunits which are related by a noncrystallographic 2-fold axis. The structure of the type 3 subunit can be divided into two domains separated by a short linker, a smaller alpha/beta domain (domain I, residues 1-82), and a larger alpha domain (domain II, residues 90-217). Domain I contains four beta-strands which form a central mixed beta-sheet and three alpha-helices which are arranged in a beta alpha beta alpha beta beta alpha motif. Domain II is composed of five alpha-helices. Domain I can be considered the glutathione binding domain, while domain II seems to be primarily responsible for xenobiotic substrate binding. The active site is located in a deep (19-A) cavity which is composed of three relatively mobile structural elements: the long loop (residues 33-42) of domain I, the alpha 4/alpha 5 helix-turn-helix segment, and the C-terminal tail. GSH is bound at the active site in an extended conformation at one end of the beta-sheet of domain I with its backbone facing the cavity and the sulfur pointing toward the subunit to which it is bound.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Crystal structure of the complex of carboxypeptidase A with a strongly bound phosphonate in a new crystalline form: comparison with structures of other complexes.

O-[[(1R)-[[N-(Phenylmethoxycarbonyl)-L-alanyl]amino]ethyl] hydroxyphosphinyl]-L-3-phenyllacetate [ZAAP(O)F], an analogue of (benzyloxycarbonyl)-Ala-Ala-Phe or (benzyloxycarbonyl)-Ala-Ala-phenyllactate, binds to carboxypeptidase A with great affinity (Ki = 3 pM). Similar phosphonates have been shown to be transition-state analogues of the CPA-catalyzed hydrolysis [Hanson, J. E., Kaplan, A. P., & Bartlett, P. A. (1989) Biochemistry 28, 6294-6305]. In the present study, the structure of the complex of this phosphonate with carboxypeptidase A has been determined by X-ray crystallography to a resolution of 2.0 A. The complex crystallizes in the space group P2(1)2(1)2(1) with cell dimensions a = 61.9 A, b = 67.2 A, and c = 76.2 A. The structure of the complex was solved by molecular replacement. Refinement of the structure against 20,776 unique reflections between 10.0 and 2.0 A yields a crystallographic residual of 0.193, including 140 water molecules. The two phosphinyl oxygens of the inhibitor bind to the active-site zinc at 2.2 A on the electrophilic (Arg-127) side and 3.1 A on the nucleophilic (Glu-270) side. Various features of the binding mode of this phosphonate inhibitor are consistent with the hypothesis that carboxypeptidase A catalyzed hydrolysis proceeds through a general-base mechanism in which the carbonyl carbon of the substrate is attacked by Zn-hydroxyl (or Zn-water). An unexpected feature of the bound inhibitor, the cis carbamoyl ester bond at the benzyloxycarbonyl linkage to alanine, allows the benzyloxycarbonyl phenyl ring of the inhibitor to interact favorably with Tyr-198. This complex structure is compared with previous structures of carboxypeptidase A, including the complexes with the potato inhibitor, a hydrated keto methylene substrate analogue, and a phosphonamidate inhibitor. Comparisons are also made with the complexes of thermolysin with some phosphonamidate inhibitors.

Alanine↗
PubMed2003

Crystal structures of ferrous and CO-, CN(-)-, and NO-bound forms of rat heme oxygenase-1 (HO-1) in complex with heme: structural implications for discrimination between CO and O2 in HO-1.

Heme oxygenase (HO) catalyzes heme degradation by utilizing O(2) and reducing equivalents to produce biliverdin IX alpha, iron, and CO. To avoid product inhibition, the heme[bond]HO complex (heme[bond]HO) is structured to markedly increase its affinity for O(2) while suppressing its affinity for CO. We determined the crystal structures of rat ferrous heme[bond]HO and heme[bond]HO bound to CO, CN(-), and NO at 2.3, 1.8, 2.0, and 1.7 A resolution, respectively. The heme pocket of ferrous heme-HO has the same conformation as that of the previously determined ferric form, but no ligand is visible on the distal side of the ferrous heme. Fe[bond]CO and Fe[bond]CN(-) are tilted, whereas the Fe[bond]NO is bent. The structure of heme[bond]HO bound to NO is identical to that bound to N(3)(-), which is also bent as in the case of O(2). Notably, in the CO- and CN(-)-bound forms, the heme and its ligands shift toward the alpha-meso carbon, and the distal F-helix shifts in the opposite direction. These shifts allow CO or CN(-) to bind in a tilted fashion without a collision between the distal ligand and Gly139 O and cause disruption of one salt bridge between the heme and basic residue. The structural identity of the ferrous and ferric states of heme[bond]HO indicates that these shifts are not produced on reduction of heme iron. Neither such conformational changes nor a heme shift occurs on NO or N(3)(-) binding. Heme[bond]HO therefore recognizes CO and O(2) by their binding geometries. The marked reduction in the ratio of affinities of CO to O(2) for heme[bond]HO achieved by an increase in O(2) affinity [Migita, C. T., Matera, K. M., Ikeda-Saito, M., Olson, J. S., Fujii, H., Yoshimura, T., Zhou, H., and Yoshida, T. (1998) J. Biol. Chem. 273, 945-949] is explained by hydrogen bonding and polar interactions that are favorable for O(2) binding, as well as by characteristic structural changes in the CO-bound form.

Animals↗
PubMed2003

Three-dimensional structure in lipid micelles of the pediocin-like antimicrobial peptide sakacin P and a sakacin P variant that is structurally stabilized by an inserted C-terminal disulfide bridge.

The three-dimensional structures in dodecylphosphocholine (DPC) micelles and in trifluoroethanol (TFE) of the pediocin-like antimicrobial peptide sakacin P and an engineered variant of sakacin P (termed sakP[N24C+44C]) have been determined by use of nuclear magnetic resonance spectroscopy. SakP[N24C+44C] has an inserted non-native activity- and structure-stabilizing C-terminal disulfide bridge that ties the C-terminus to the middle part of the peptide. In the presence of DPC, the cationic N-terminal region (residues 1-17) of both peptides has an S-shaped conformation that is reminiscent of a three-stranded antiparallel beta-sheet and that is more pronounced when the peptide was dissolved in TFE instead of DPC. The four positively charged residues located in the N-terminal part are found pointing to the same direction. For both peptides, the N-terminal region is followed by a well-defined central amphiphilic alpha-helix (residues 18-33), and this in turn is followed by the C-terminal tail (residues 34-43 for sakacin P and 34-44 for sakP[N24C+44C]) that lacks any apparent common secondary structural motif. In the presence of DPC, the C-terminal tails in both peptides fold back onto the central alpha-helix, thereby creating a hairpin-like structure in the C-terminal halves. The lack of long-range NOEs between the beta-sheet Nu-terminal region and the hairpin-like C-terminal half indicates that there is a flexible hinge between these regions. We discuss which implications such a structural arrangement has on the interaction with the target cell membrane.

Amino Acid Sequence↗
PubMed2005

Crystal structure of a hyperthermophilic archaeal acylphosphatase from Pyrococcus horikoshii--structural insights into enzymatic catalysis, thermostability, and dimerization.

Acylphosphatases catalyze the hydrolysis of the carboxyl-phosphate bond in acyl phosphates. Although acylphosphatase-like sequences are found in all three domains of life, no structure of acylphosphatase has been reported for bacteria and archaea so far. Here, we report the characterization of enzymatic activities and crystal structure of an archaeal acylphosphatase. A putative acylphosphatase gene (PhAcP) was cloned from the genomic DNA of Pyrococcus horikoshii and was expressed in Escherichia coli. Enzymatic parameters of the recombinant PhAcP were measured using benzoyl phosphate as the substrate. Our data suggest that, while PhAcP is less efficient than other mammalian homologues at 25 degrees C, the thermophilic enzyme is fully active at the optimal growth temperature (98 degrees C) of P. horikoshii. PhAcP is extremely stable; its apparent melting temperature was 111.5 degrees C and free energy of unfolding at 25 degrees C was 54 kJ mol(-)(1). The 1.5 A crystal structure of PhAcP adopts an alpha/beta sandwich fold that is common to other acylphosphatases. PhAcP forms a dimer in the crystal structure via antiparallel association of strand 4. Structural comparison to mesophilic acylphosphatases reveals significant differences in the conformation of the L5 loop connecting strands 4 and 5. The extreme thermostability of PhAcP can be attributed to an extensive ion-pair network consisting of 13 charge residues on the beta sheet of the protein. The reduced catalytic efficiency of PhAcP at 25 degrees C may be due to a less flexible active-site residue, Arg20, which forms a salt bridge to the C-terminal carboxyl group. New insights into catalysis were gained by docking acetyl phosphate to the active site of PhAcP.

Acid Anhydride Hydrolases↗
PubMed2006

Structural studies of Apo NosL, an accessory protein of the nitrous oxide reductase system: insights from structural homology with MerB, a mercury resistance protein.

The formation of the unique catalytic tetranuclear copper cluster (Cu(Z)) of nitrous oxide reductase, N(2)OR, requires the coexpression of a multiprotein assembly apparatus encoded by the nosDFYL operon. NosL, one of the proteins encoded by this transcript, is a 20 kDa lipoprotein of the periplasm that has been shown to bind copper(I), although its function has yet to be detemined. Cu(I) EXAFS data collected on the holo protein demonstrated that features of the copper binding site are consistent with a role for this protein as a metallochaperone, a class of metal ion transporters involved in metal resistance, homeostasis, and metallocluster biosynthesis. To test this hypothesis and to gain insight into other potential functional roles for this protein in the N(2)OR system, the three-dimensional solution structure of apo NosL has been solved by solution NMR methods. The structure of apo NosL consists of two relatively independent homologous domains that adopt an unusual betabetaalphabeta topology. The fold of apo NosL displays structural homology to only one other protein, MerB, an organomercury lyase involved in bacterial mercury resistance (Di Lello et al. (2004) Biochemistry 43, 8322-32). The structural similarity between apo NosL and MerB, together with the absolute conservation of Met109 in all NosL sequences, indicates that this residue may be involved in copper ligation, and that the metal binding site is likely to be solvent-accessible and contiguous with a large binding cleft. The structural observations suggest that NosL is exceptionally adapted for a role in copper and/or sulfur delivery and possibly for metallochaperone function.

Achromobacter cycloclastes↗
PubMed1996

Effect of adenine methylation on the structure and dynamics of TpA steps in DNA: NMR structure determination of [d(CGAGGTTTAAACCTCG)]2 and its A9-methylated derivative at 750 MHz.

At TpA steps in DNA, the adenine base experiences exceptionally large amplitude (20 degrees-50 degrees) and slow (10 ms-1 microsecond) motion [Kennedy et al. (1993) Biochemistry 32, 8022-8035] which has been correlated with transitions between multiple conformational states [Lefevre et al. (1985) FEBS Lett. 190, 37-40]. The base dynamics can be detected in one-dimensional 1H NMR spectra as excess line width of the aromatic proton resonances. The magnitude of the excess line width is temperature dependent and reaches a maximum at some temperature. In order to better understand the origin of the dynamics, we have studied the effect of N6-methylation of the TpA adenine on both the line widths and its local structure. Here, solution-state 500 and 750 MHz 1H NMR data collected on [d(CGAGGTTTAAACCTCG)]2 show that the excess line width of the TpA adenine-H2 is diminished when the TpA adenine is N6-methylated and that the line width no longer experiences a maximum as the temperature is varied. The resonances sharpen upon methylation because the amplitude of base motion is restricted due to steric effects and due to other structural changes at the TpA site. Additionally, both the TpA adenine-H8 and the exchangeable imino resonance of thymine at the TpA step were also found to have excess line width that is diminished upon N6-methylation. In order to elucidate the structural features responsible for TpA base dynamics, solution-state NMR structures of [d(CGAGGTTTAAACCTCG)]2 and its A9 N6-methylated derivative were determined at 750 MHz. Comparison of the structures shows that poor base stacking at the TpA step may contribute to, or be the origin of, its base dynamics.

Adenine↗
PubMed1996

Three-dimensional structure of the reduced C77S mutant of the Chromatium vinosum high-potential iron-sulfur protein through nuclear magnetic resonance: comparison with the solution structure of the wild-type protein.

The full 1H NMR assignment of the reduced C77S mutant of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) was achieved by taking advantage of the assignment available for the wild-type protein. A total of 1565 nuclear Overhauser effect (NOE) spectroscopy cross peaks were integrated and converted into distance constraints, of which 497 were found to be irrelevant. An additional 24 dipolar constraints were obtained from one-dimensional NOE difference spectra by saturating hyperfine-shifted beta CH2 cysteine/serine protons. Forty-six 3JNH-H alpha coupling constants and eight hydrogen bonds provided further constraints. Through a distance geometry approach, a family of 15 structures was calculated, which was subsequently subjected to restrained energy minimization. The root mean square deviations of the minimized structures were 0.62 +/- 0.09 and 1.09 +/- 0.11 A for backbone and heavy atoms, respectively. The resulting solution structures are very similar to those of the reduced wild-type protein (WT). An analysis of the NOEs experienced by the protons of Ser-77 in both the reduced and oxidized forms reveals that they are very similar to those experienced by Cys-77 in WT. On the basis of the hyperfine shifts observed for the Ser-77 protons and of the present structural analysis, it is concluded that the serine O gamma atom is coordinated to the polymetallic center, thus confirming the strict analogy of the electronic structures of the polymetallic center in both proteins. Capillary electrophoresis experiments demonstrate coordination of Ser-77 as an anion. Serine versus cysteine coordination in iron-sulfur proteins is briefly discussed.

Amino Acid Sequence↗
PubMed2000

Transient structure of the amyloid precursor protein cytoplasmic tail indicates preordering of structure for binding to cytosolic factors.

The cytoplasmic tail of the amyloid precursor protein (APP) appears to play two important roles in the cell through participation in intracellular signaling and proteolytic processing of APP. Hence, knowledge of the structure of the 47 residue cytoplasmic tail of APP is important for understanding the molecular interactions involved in normal cell function as well as in the pathogenesis of Alzheimer's disease. Multidimensional solution NMR spectroscopy has been applied to examine the structural features of a 49-residue peptide (APP-C) containing two N-terminal residues (GS) and the APP cytoplasmic tail, over the pH range of 4.2-7.1. Although the peptide does not adopt a stable folded structure, regions of unstable structure exist over the pH range examined and have been characterized by a combination of H(alpha) chemical shifts, NOE analysis, and (3)J(HNH)(alpha) coupling constants and by identification of transient hydrogen bonds between amide protons and titrating carboxylate groups. These studies extend the work of others [Kroenke et al. (1997) Biochemistry 36, 8145-8152] by identifying an additional nascent helix and a hydrophobic cluster within the N-terminal 20 amino acid residues and by further characterizing the TPEE turn as a helix capping box. The transient structure of APP-C provides insight into the importance of preordering of this cytoplasmic tail in governing specificity and affinity for cytosolic binding partners.

Adaptor Proteins, Signal Transducing↗