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PubMed2001

Crystal structure of human AUH protein, a single-stranded RNA binding homolog of enoyl-CoA hydratase.

BACKGROUND: The AU binding homolog of enoyl-CoA hydratase (AUH) is a bifunctional protein that has two distinct activities: AUH binds to RNA and weakly catalyzes the hydration of 2-trans-enoyl-coenzyme A (enoyl-CoA). AUH has no sequence similarity with other known RNA binding proteins, but it has considerable sequence similarity with enoyl-CoA hydratase. A segment of AUH, named the R peptide, binds to RNA. However, the mechanism of the RNA binding activity of AUH remains to be elucidated. RESULTS: We determined the crystal structure of human AUH at 2.2 A resolution. AUH adopts the typical fold of the enoyl-CoA hydratase/isomerase superfamily and forms a hexamer as a dimer of trimers. Interestingly, the surface of the AUH hexamer is positively charged, in striking contrast to the negatively charged surfaces of the other members of the superfamily. Furthermore, wide clefts are uniquely formed between the two trimers of AUH and are highly positively charged with the Lys residues in alpha helix H1, which is located on the edge of the cleft and contains the majority of the R peptide. A mutational analysis showed that the lysine residues in alpha helix H1 are essential to the RNA binding activity of AUH. CONCLUSIONS: Alpha helix H1 exposes a row of Lys residues on the solvent-accessible surface. These characteristic Lys residues are named the "lysine comb." The distances between these Lys residues are similar to those between the RNA phosphate groups, suggesting that the lysine comb may continuously bind to a single-stranded RNA. The clefts between the trimers may provide spaces sufficient to accommodate the RNA bases.

Amino Acid Sequence↗

PubMed1996

Refined solution structure of type III antifreeze protein: hydrophobic groups may be involved in the energetics of the protein-ice interaction.

BACKGROUND: Antifreeze proteins are found in certain fish inhabiting polar sea water. These proteins depress the freezing points of blood and body fluids below that of the surrounding sea water by binding to and inhibiting the growth of seed ice crystals. The proteins are believed to bind irreversibly to growing ice crystals in such a way as to change the curvature of the ice-water interface, leading to freezing point depression, but the mechanism of high-affinity ice binding is not yet fully understood. RESULTS: The solution structure of the type III antifreeze protein was determined by multidimensional NMR spectroscopy. Twenty-two structures converged and display a root mean square difference from the mean of 0.26 A for backbone atoms and 0.62 A for all non-hydrogen atoms. The protein exhibits a compact fold with a relatively large hydrophobic core, several short and irregular beta sheets and one helical turn. The ice-binding site, which encompasses parts of the C-terminal sheet and a loop, is planar and relatively nonpolar. The site is further characterized by the low solvent accessibilities and the specific spatial arrangement of the polar side-chain atoms of the putative ice-binding residues Gln9, Asn14, Thr15, Thr18 and Gln44. CONCLUSIONS: In agreement with the adsorption-inhibition mechanism of action, interatomic distances between active polar protein residues match the spacing of water molecules in the prism planes (¿10&1macr;0¿) of the hexagonal ice crystal. The particular side-chain conformations, however, limit the number and strength of possible proten-ice hydrogen bonds. This suggests that other entropic and enthalpic contributions, such as those arising from hydrophobic groups, could play a role in the high-affinity protein-ice adsorption.

Animals↗

PubMed1997

The crystal structure of a triacylglycerol lipase from Pseudomonas cepacia reveals a highly open conformation in the absence of a bound inhibitor.

BACKGROUND: . Lipases, a family of enzymes which catalyze the hydrolysis of triglycerides, are widely distributed in many organisms. True lipases are distinguished from esterases by the characteristic interfacial activation they exhibit at an oil-water interface. Lipases are one of the most frequently used biocatalysts for organic reactions performed under mild conditions. Their biotechnological applications include food and oil processing and the preparation of chiral intermediates for the synthesis of enantiomerically pure pharmaceuticals. Recent structural studies on several lipases have provided some clues towards understanding the mechanisms of hydrolytic activity, interfacial activation, and stereoselectivity. This study was undertaken in order to provide structural information on bacterial lipases, which is relatively limited in comparison to that on the enzymes from other sources. RESULTS: . We have determined the crystal structure of a triacylglycerol lipase from Pseudomonas cepacia (PcL) in the absence of a bound inhibitor using X-ray crystallography. The structure shows the lipase to contain an alpha/beta-hydrolase fold and a catalytic triad comprising of residues Ser87, His286 and Asp264. The enzyme shares several structural features with homologous lipases from Pseudomonas glumae (PgL) and Chromobacterium viscosum (CvL), including a calcium-binding site. The present structure of PcL reveals a highly open conformation with a solvent-accessible active site. This is in contrast to the structures of PgL and PcL in which the active site is buried under a closed or partially opened 'lid', respectively. CONCLUSIONS: . PcL exhibits some structural features found in other lipases. The presence of the Ser-His-Asp catalytic triad, an oxyanion hole, and the opening of a helical lid suggest that this enzyme shares the same mechanisms of catalysis and interfacial activation as other lipases. The highly open conformation observed in this study is likely to reflect the activated form of the lipase at an oil-water interface. The structure suggests that the interfacial activation of bacterial lipases involves the reorganization of secondary structures and a large movement of the lid to expose the active site. This is similar to the mechanism described for other well characterized fungal and mammalian lipases.

Amino Acid Sequence↗

PubMed1998

Structure of translation initiation factor 5A from Pyrobaculum aerophilum at 1.75 A resolution.

BACKGROUND: Translation initiation factor 5A (IF-5A) is reported to be involved in the first step of peptide bond formation in translation, to be involved in cell-cycle regulation and to be a cofactor for the Rev and Rex transactivator proteins of human immunodeficiency virus-1 and T-cell leukemia virus I, respectively. IF-5A contains an unusual amino acid, hypusine (N-epsilon-(4-aminobutyl-2-hydroxy)lysine), that is required for its function. The first step in the post-translational modification of lysine to hypusine is catalyzed by the enzyme deoxyhypusine synthase, the structure of which has been published recently. RESULTS: IF-5A from the archebacterium Pyrobaculum aerophilum has been heterologously expressed in Escherichia coli with selenomethionine substitution. The crystal structure of IF-5A has been determined by multiwavelength anomalous diffraction and refined to 1.75 A. Unmodified P. aerophilum IF-5A is found to be a beta structure with two domains and three separate hydrophobic cores. CONCLUSIONS: The lysine (Lys42) that is post-translationally modified by deoxyhypusine synthase is found at one end of the IF-5A molecule in an turn between beta strands beta4 and beta5; this lysine residue is freely solvent accessible. The C-terminal domain is found to be homologous to the cold-shock protein CspA of E. coli, which has a well characterized RNA-binding fold, suggesting that IF-5A is involved in RNA binding.

Amino Acid Sequence↗

PubMed1999

Electrospray ionization mass spectrometry and hydrogen/deuterium exchange for probing the interaction of calmodulin with calcium.

The extent of H/D exchange of the protein calmodulin in solution was monitored by mass spectrometry following electrospray ionization (ESI) of the protein. In the absence of Ca2+, approximately 115 protons are exchanged for deuteriums after 60 min. As the calmodulin is titrated with Ca2+, the extent of exchange decreases significantly (i.e., by 24 protons), indicating Ca(2+)-induced folding of the protein to a tighter, less solvent-accessible form. The extent of H/D exchange ceases to decrease when the amount of added Ca2+ is sufficient to convert greater than 80% of the calmodulin to a form bound by four calcium ions. Lysozyme, a protein of similar molecular weight, does not show a significant decrease in the extent of H/D exchange as it binds to Ca2+, indicating that the changes in H/D exchange for calmodulin reflect tertiary structural change that occur upon binding with Ca2+.

Algorithms↗

PubMed2001

Thermodynamic analysis of the interaction between YY1 and the AAV P5 promoter initiator element.

BACKGROUND: We previously determined the co-crystal structure of the zinc finger region of transcription factor YY1 (YY1Delta) bound to the initiator element (Inr) of the adenoassociated virus (AAV) P5 gene promoter [Houbaviy, H.B. et al. (1996) Proc. Natl. Acad. Sci. USA 93, 13577-13582]. Our structure explained both binding specificity and the ability of YY1 to support specific, unidirectional transcription initiation. RESULTS: To further understand Inr recognition by YY1, we analyzed the YY1Delta-Inr interaction by isothermal titration calorimetry (ITC) and used limited proteolysis, DNase I footprinting and missing nucleoside experiments to show that YY1Delta and full-length YY1 (YY1WT) have indistinguishable DNA binding properties. CONCLUSIONS: YY1 binding occurs at an equilibrium dissociation constant (K(d)) of about 1 microM, and exhibits a large negative heat capacity change (DeltaC(p)). We analyzed the thermodynamic behavior of YY1Delta in terms of buried solvent-accessible surface area resulting from interaction of two rigid bodies, which could not explain our measured value of DeltaC(p). We must, therefore, postulate conformational changes in YY1 and/or the Inr or question the validity of current DeltaC(p) analysis methods for protein-DNA interactions.

DNA-Binding Proteins↗

PubMed2000

Stereolithographic models of biopolymers.

Stereolithography (STL) has been used to make plastic models of the solvent accessible surfaces of biopolymers. Models have been made of proteins and proteins bound to DNA and RNA. The STL process uses a laser to photopolymerize a liquid resin. Using the ACES (accurate, clear, epoxy, solid) building technique, parts are made with minimum postcure shrinkage. Protein Data Bank files are converted to STL files that represent the surface topology of the biopolymer as a series of triangles and an index that describes their orientation. The models are useful in teaching biomolecular structure and the principle of docking. They are especially useful to the visually impaired.

Acetylcholinesterase↗

PubMedDate not supplied

Efficient combinatorial filtering for desired molecular properties of reaction products.

Two combinatorial filtering methods for efficiently selecting reaction products with desired properties are presented. The first, "direct reactants" method is applicable only to those molecular properties that are strictly additive or approximately additive, with relatively small interference between neighboring fragments. This method uses only the molecular properties of reactants. The second, "basis products" method can be used to filter not only the strictly additive properties but also the approximately additive molecular properties where a certain degree of mutual influence occurs between neighboring fragments. This method requires the molecular properties of the "basis products," which are the products formed by combining all the reactants for a given reaction component with the simplest set of complementary reactant partners. There is a one-to-one correspondence between the reactants and the "basis products." The latter is a product representation of the former. High efficiency of both methods is enhanced further by a tree-sorting and hierarchical selection algorithm, which is performed on the reaction components in a limited space determined systematically from the filtering criteria. The methods are illustrated with product logPs, van der Waals volumes, solvent accessible surface areas, and other product properties. Good results are obtained when filtering for a number of important molecular properties in a virtual library of 1.5 billion.

Algorithms↗

PubMed2001

The conserved residues of the ligand-binding domains of steroid receptors are located in the core of the molecules.

The relationship between conserved residues and biochemical functions of steroid receptors was investigated. Pairwise three-dimensional (3D) alignment of the ligand-binding domains of the human estrogen (1A52) and progesterone (1A28) receptors revealed two conserved domains; Asn313-Ser456 and Gln471-Lys531 (numbering reflects the sequence in the human estrogen receptor). Alignment of the protein sequences of 39 steroid receptors revealed 36 highly conserved residues (i.e., the residues commonly found in more than 80% of sequences aligned). They were distributed throughout the sequences but formed a contiguous 3D structure. Most of these highly conserved residues were buried in the ligand-binding domain, but several residues were exposed on the surface. The well-known functions commonly associated with the ligand-binding domain of steroid receptors are ligand binding, HSP90 binding, transcriptional activation and dimerization. The relationship between the residues and these functions were checked. To determine the residues involved in dimerization, the differences between the solvent accessibilities of the monomeric and dimeric forms were calculated. These results revealed 32 residues of 1A52 and 15 residues of 1A28 potentially involved in dimerization. Their distribution areas do not overlap greatly. Comparing these putative dimerization sites with highly conserved residues, many of the exposed conserved residues were observed on the side of the domain opposite are the dimerization sites. Some highly conserved residues are located in a steroid-binding site and in transcriptional activation domain. However, few of them were observed in the HSP90 binding site. These results indicate that the core structure made by most of the highly conserved residues among the ligand-binding domains of steroid receptors is important. These conserved residues may be essential for conformational change in the ligand-binding domain from its inactive to active form.

Amino Acid Motifs↗

PubMed2001

Prediction of membrane protein orientation in lipid bilayers: a theoretical approach.

Over the past few years, several three-dimensional (3-D) structures of membrane proteins have been described with increasing accuracy, but their relationship with membranes are still not well understood. Recently, we have developed an empirical method, Integral Membrane Protein and Lipid Association (IMPALA), to predict the insertion of molecules (lipids, drugs) into lipid bilayers (Proteins 30 (1998) 357). The IMPALA uses a Monte Carlo minimisation procedure to calculate the depth and the angle of insertion of membrane-interacting molecules taking into account the restraints dictated by a lipid bilayer. In this paper, we use IMPALA to test the insertion of 23 integral membranous proteins (IMPs) and 2 soluble proteins into membranes. Four IMP are studied in detail: OmpA, maltoporin, MsCl channel and bacteriorhodopsin. The 3-D structures of the proteins are kept constant and the insertion into membrane is monitored by minimising the value of the restraint representing the sum of two terms, one for lipid perturbation and the other for hydrophobicity. The two soluble proteins are rejected from the membrane whereas, under the same conditions, all the membrane proteins remain inside, if the solvent accessible surface of the amino acids located inside the pore of porins is ignored. The results give the tilt angle of the IMP helices or strands with respect to the membrane surface and the depth of the protein mass centre insertion. We conclude that the restraint terms of IMPALA could be used to study the insertion of model structures or complexes of proteins within membranes.

Bacterial Proteins↗

PubMed1998

Development of filter functions for protein-ligand docking.

Current docking methods can generate bound conformations of a ligand close to the experimentally observed structure of a protein-ligand complex. However, the scoring functions used to evaluate the potential solutions are not yet reliable enough at giving the highest ranks to the best structure predictions. One approach to this problem is the use of filter functions that are applied to all docked conformations to remove structures with certain energetically unfavorable properties. We present a computationally efficient scheme for such a postprocessing of docking results. For each of the conformations generated for a given protein-ligand complex, four properties are calculated: the fraction of the ligand volume buried inside the binding pocket, the size of lipophilic cavities along the protein-ligand interface, the solvent-accessible surface (SAS) of nonpolar parts of the ligand, and the number of close contacts between nonhydrogen-bonded polar atoms of the ligand and the protein. These four terms were used to filter out the majority of the calculated solutions and to rescore the remaining ones. On a test set of 32 protein-ligand complexes, this protocol significantly improves the accuracy of the structure predictions.

Crystallography, X-Ray↗

PubMed1996

Scrapie prions: a three-dimensional model of an infectious fragment.

BACKGROUND: A conformational change seems to represent the major difference between the scrapie prion protein (PrPSc) and its normal cellular isoform (PrPC). We recently proposed a set of four helix bundle models for the three-dimensional structure of PrPC that are consistent with a variety of spectroscopic and genetic data. RESULTS: We report a plausible model for the three-dimensional structure of a biologically important fragment of PrPSc. The model of residues 108-218 was constructed by an approach that combines computational techniques and experimental data. The proposed structures of this fragment of PrPSc display a four-stranded beta-sheet covered on one face by two alpha-helices. Residues implicated in the prion species barrier are found to cluster on the solvent-accessible surface of the beta-sheet of one of the models. This interface could provide a structural template that would assist the conversion of PrPC to PrPSc and hence direct prion propagation. CONCLUSIONS: Molecular models of the PrP isoforms should prove very useful in developing structural hypotheses about the process by which PrPC is transformed into PrPSc, the mechanisms by which PrP gene mutations give rise to the inherited human prion diseases, and the species barrier that seems to protect humans from animal prions. It seems likely that PrPC represents a kinetically trapped intermediate in PrP folding.

Animals↗

PubMed1998

Residual structure in the Alzheimer's disease peptide: probing the origin of a central hydrophobic cluster.

BACKGROUND: . Structure-function studies on the Alzheimer's disease peptide sh w that a central hydrophobic cluster - Abeta(17-21), LVFFA - is a prominent structural feature linked to plaque competence. The origin and stability of this cluster was probed in a 17-residue fragment which includes flanking residues that potentially help stabilize the cluster. RESULTS: After residue substitution, the measurement of pKas, amide exchange rates and other NMR data show that any coulombic interactions between His14 and Glu22 are not required for the stability of the central hydrophobic cluster. In contrast, a single substitution within the cluster disrupts its integrity and causes the largest pKa shift for flanking residues, while increasing the solvent accessibility of the backbone. CONCLUSIONS: The integrity of the structurally dominant cluster relies primarily upon local hydrophobic interactions, rather than on interactions between the sidechains of charged flanking residues. Moreover, the conformational disposition of the cluster affects the pKas of flanking residues, underscoring its structural dominance.

Alzheimer Disease↗

PubMed1998

Steady-state and time-resolved fluorescence of Esperase: comparison with the X-ray structure in the region of the two tryptophans.

Fluorescence emission properties of the alkaline protease Esperase have been investigated using steady-state and time-resolved fluorescence spectroscopy. The local polarity and solvent accessibility of the tryptophyl chromophores is characterized. Quenching studies demonstrated that Trp 6 and Trp 113 are 'buried' to acrylamide, iodide ions and caesium ions. An abnormally low tryptophan quantum yield was calculated showing that the emission of the two indole rings is significantly quenched by nearby side chains or peptide bonds. The fluorescence decay of PMS-Esperase was well fitted by two exponentials with lifetimes of 2.7 and 0.35 ns. X-ray data for Esperase (S. Klupsch, Ph.D. Thesis, University of Hamburg, Hamburg, Germany) in the region of the two tryptophans were used to explain the observed emission properties. Gln 182 and Asn 204 as well as Asn 117 and Met 119 are the most likely quenchers, respectively, of the Trp 6 and Trp 113 fluorescence. The two tryptophans in Esperase are 'buried' in hydrophobic regions and are excellent intrinsic probes to study folding-unfolding reactions. Experiments in the presence and absence of added calcium ions demonstrated the stabilizing role of the Ca(2+)-binding sites.

Computer Graphics↗

PubMed2000

Phosphatidylinositol-specific phospholipase C-gamma1 undergoes pH-induced activation and conformational change.

Phospholipase C-gamma1 displayed sigmoidal kinetics with a S(0.5) value of 0.17 mole fraction PIP(2) when assayed at pH 6.8 using detergent:lipid mixed micelles. The pH optimum for hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C-gamma1 was dependent on the mole fraction of substrate in the micelle. The pH optimum was 5.5 when the enzyme was assayed below the S(0.5). The pH optima shifted to a pH range of 6.0-6.3 when the enzyme was assayed above the S(0.5). The kinetic parameters for phospholipase C-gamma1 assayed at various pH values from pH 7.0 to 5.0 yielded similar n values (n=4), but the constant, K', decreased from 1x10(-2) (mole fraction)(2) at pH 7.0 to 1x10(-5) (mole fraction)(2) at pH 5.0. Maximum enzyme specificity occurred at pH values below pH 6.0 as determined by the plot of logk(cat)/S(0.5) versus pH. Intrinsic fluorescence spectroscopy revealed that at a pH value above 7.0 or below 6.3, tryptophan quenching occurred. Fluorescence quenching experiments performed with acrylamide determined phospholipase C-gamma1 incubated at pH 5.0 had a larger collisional quenching constant than enzyme incubated at pH 7.0. Lowering the pH to 5.0 apparently resulted in interior tryptophans becoming more solvent accessible. These data suggest that pH may activate phospholipase C-gamma1 by disrupting ionizable groups leading to a conformational change.

Acrylamide↗

PubMed2002

A model for the structure of the Escherichia coli SOS-regulated UmuD2 protein.

The ubiquitous Y-family of DNA polymerases, exemplified by the Escherichia coli UmuC protein (the catalytic subunit of DNA Pol V), possess the remarkable ability to replicate imperfect DNA templates that cannot be replicated by other types of DNA polymerases. Since this ability comes at the cost of a reduced fidelity, it is important that organisms manage these unique polymerases to coordinate their actions with those of the replication machinery. In E. coli, it is becoming evident that a sophisticated series of protein-protein interactions involving the two forms of the umuD gene product, UmuD and UmuD' and components of the replicative DNA polymerase serve to manage the actions of the umuC-encoded DNA polymerase. The purpose of this study was to better understand how structural differences between UmuD2 and UmuD2' help to determine which biological role the umuDC gene products will play; the UmuD2C complex functions as a DNA damage checkpoint effector, while the UmuD2'C complex participates in translesion DNA synthesis, which serves as the mechanistic basis for most chemical and UV light mutagenesis. Based on the results of a combination of disulfide cross-linking experiments, measurements of solvent accessibility and electron paramagnetic spin resonance (EPR) studies, we have developed a refined model for the structure of the UmuD2 homodimer. In the model that we are proposing, the N-terminal arms of UmuD (residues 1-39) form an extended interface in the UmuD2 homodimer by folding down over the globular domains of their intradimer partners. As a result, significant portions of the surface of each globular domain are buried in the UmuD2 homodimer. Based on the structure of the UmuD2' homodimer, both in the crystal and in solution, these same surfaces are exposed. Implications of these structural differences between the UmuD2 and the UmuD2' homodimers with respect to their roles in managing the actions of the umuC-encoded DNA polymerase are discussed.

Binding Sites↗

PubMed2003

Support vector machines for predicting rRNA-, RNA-, and DNA-binding proteins from amino acid sequence.

Classification of gene function remains one of the most important and demanding tasks in the post-genome era. Most of the current predictive computer methods rely on comparing features that are essentially linear to the protein sequence. However, features of a protein nonlinear to the sequence may also be predictive to its function. Machine learning methods, for instance the Support Vector Machines (SVMs), are particularly suitable for exploiting such features. In this work we introduce SVM and the pseudo-amino acid composition, a collection of nonlinear features extractable from protein sequence, to the field of protein function prediction. We have developed prototype SVMs for binary classification of rRNA-, RNA-, and DNA-binding proteins. Using a protein's amino acid composition and limited range correlation of hydrophobicity and solvent accessible surface area as input, each of the SVMs predicts whether the protein belongs to one of the three classes. In self-consistency and cross-validation tests, which measures the success of learning and prediction, respectively, the rRNA-binding SVM has consistently achieved >95% accuracy. The RNA- and DNA-binding SVMs demonstrate more diverse accuracy, ranging from approximately 76% to approximately 97%. Analysis of the test results suggests the directions of improving the SVMs.

Computational Biology↗

PubMed2002

RNA footprinting analysis using ion pair reverse phase liquid chromatography.

Hydroxyl radical footprinting is a powerful technique often employed in characterization of the tertiary interactions between proteins and nucleic acids. Following the generation of a nucleic acid "ladder" either by chemical or enzymatic reactions, the radiolabeled products are traditionally separated by denaturing gel electrophoresis and further quantified by phosphorimaging techniques. Here we report the use of ion pair reverse phase liquid chromatography to analyze the products of an RNA footprinting reaction using fluorescently labeled RNA molecules. This technique offers several advantages over existing procedures, including rapid analysis, automation, and direct quantification of the cleavage products without the need to employ radiolabeling. To illustrate the resolving power of this technique, we have analyzed the products of base hydrolysis, generated from a fluorescently labeled RNA molecule and have subsequently used this method to define the solvent accessibility of the substrate strand as it docks with the hairpin ribozyme.

Base Sequence↗