Mass spectrometry as a tool for studying the action of human aspartic proteases on peptides and proteins.
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Amino Acid Sequence↗
Biomedical subjects
J Lubkowski
Publications and source records attributed to J Lubkowski.
The structural basis of phage display elucidated by the crystal structure of the N-terminal domains of g3p.
The structure of the two N-terminal domains of the gene 3 protein of filamentous phages (residues 1-217) has been solved by multiwavelength anomalous diffraction and refined at 1.46 A resolution. Each domain consists of either five or eight beta-strands and a single alpha-helix. Despite missing sequence homology, their cores superimposed with a root-mean-square deviation of 2 A. The domains are engaged in extensive interactions, resulting in a horseshoe shape with aliphatic amino acids and threonines lining the inside, delineating the likely binding site for the F-pilus. The glycine-rich linker connecting the domains is invisible in the otherwise highly ordered structure and may confer flexibility between the domains required during the infection process.
Amino Acid Sequence↗
Crystal structures of the inactive D30N mutant of feline immunodeficiency virus protease complexed with a substrate and an inhibitor.
Crystal structures of complexes of a D30N mutant of feline immunodeficiency virus protease (FIV PR) complexed with a statine-based inhibitor (LP-149), as well as with a substrate based on a modification of this inhibitor (LP-149S), have been solved and refined at resolutions of 2.0 and 1.85 A, respectively. Both the inhibitor and the substrate are bound in the active site of the mutant protease in a similar mode, which also resembles the mode of binding of LP-149 to the native protease. The carbonyl oxygen of the scissile bond in the substrate is not hydrated and is located within the distance of a hydrogen bond to an amido nitrogen atom from one of the two asparagines in the active site of the enzyme. The nitrogen atom of the scissile bond is 3.25 A from the conserved water molecule (Wat301). A model of a tetrahedral intermediate bound to the active site of the native enzyme was built by considering the interactions observed in all three crystal structures of FIV PR. Molecular dynamics simulations of this model bound to native wild-type FIV PR were carried out, to investigate the final stages of the catalytic mechanism of aspartic proteases.
Asparagine↗
Time-resolved fluorescence of hemoglobin species.
We used time-resolved fluorescence in the pico- to nanosecond time range to monitor the presence of tetramers, dimers and monomers in carbonmonoxyhemoglobin (COHb) solutions and to investigate how their distributions change under different experimental conditions. Comparison of fluorescence lifetime computed from the atomic coordinates of COHb (Vasquez et al., 1996) with those experimentally measured allowed identification of molecular species present in the hemoglobin solution. It was possible to observe modification of the distribution of tetramers, dimers, monomers and species with disordered hemes produced by different experimental conditions. Protein concentration affected the detectable lifetimes, indicating increasing amounts of dimers and monomers at low protein concentrations, while the amount of inverted hemes was not modified. Titration with up to 1 M NaCl modified only the extent of dissociation of hemoglobin into dimers, without affecting heme inversion and monomer formation. Hyperbaric pressure increased the amounts of dimers and monomers. This is the first time that monomeric subunits of hemoglobin have been detected at neutral pH in the normal system.
Carboxyhemoglobin↗
Intrinsic fluorescence of hemoglobins and myoglobins.
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Animals↗
The structure of MCP-1 in two crystal forms provides a rare example of variable quaternary interactions.
The X-ray crystal structure of recombinant human monocyte chemoattractant protein (MCP-1) has been solved in two crystal forms. One crystal form (P), refined to 1.85 A resolution, contains a dimer in the asymmetric unit, while the other (I) contains a monomer and was refined at 2.4 A. Although both crystal forms grow together in the same droplet, the respective quaternary structures of the protein differ dramatically. In addition, both X-ray structures differ to a similar extent from the solution structure of MCP-1. Such extent of variability of quaternary structures is unprecedented. In the crystal structures, the well-ordered N termini of MCP-1 form 3(10)-helices. Comparison of the three MCP-1 structures revealed a direct correlation between the main-chain conformation of the first two cysteine residues and the quaternary arrangements. These data can be used to explain the structural basis for the assignment of residues responsible for biological activity.
Biopolymers↗
The oligomerization domain of p53: crystal structure of the trigonal form.
The structure of the oligomerization domain of the p53 tumor suppressor protein was determined in the trigonal crystal form, using a refined NMR structure as a model. A synthetic peptide comprising residues 319-360 of human p53 crystallized in the space group P3(1)21. There is one biologically relevant tetrameric domain in the crystallographic asymmetric unit. The structure was refined jointly with NMR data, only the third such case (the previous examples being IL-1beta (Shaanan, B., Gronenborn, A.M., Cohen, G.H., Gilliland, G.L., Veerapandian, B., Davies, D.R. and Clore, G.M. (1992) Science 257, 961-964 [1]) and BPTI (Schiffer, C., Huber, R., Wuthrich, K. and Van Gunsteren, W.F. (1994) J. Mol. Biol. 241, 588-599 [21)), to 2.5 A resolution with an R factor of 0.207. The distribution of tumor-derived mutations in the oligomerization region together with structural and biological data suggest a strategy for the design of antitumor therapeutics.
Biopolymers↗
Crystal structure and amino acid sequence of Wolinella succinogenes L-asparaginase.
The amino acid sequence and tertiary structure of Wolinella succinogenes L-asparaginase were determined, and were compared with the structures of other type-II bacterial L-asparaginases. Each chain of this homotetrameric enzyme consists of 330 residues. The amino acid sequence is 40-50% identical to the sequences of related proteins from other bacterial sources, and all residues previously shown to be crucial for the catalytic action of these enzymes are identical. Differences between the amino acid sequence of W. succinogenes L-asparaginase and that of related enzymes are discussed in terms of the possible influence on the substrate specificity. The overall fold of the protein subunit is almost identical to that observed for other L-asparaginases. Two fragments in each subunit, a very highly flexible loop (approximately 20 amino acids) that forms part of the active site, and the N-terminus (two amino acids), are not defined in the structure. The orientation of Thr14, a residue probably involved in the catalytic activity, indicates the absence of ligand in the active-site pocket. The rigid part of the active site, which includes the asparaginase triad Thr93-Lys 166-Asp94, is structurally very highly conserved with equivalent regions found in other type-II bacterial L-asparaginases.
Amino Acid Sequence↗
A covalently bound catalytic intermediate in Escherichia coli asparaginase: crystal structure of a Thr-89-Val mutant.
Escherichia coli asparaginase II catalyzes the hydrolysis of L-asparagine to L-aspartate via a threonine-bound acyl-enzyme intermediate. A nearly inactive mutant in which one of the active site threonines, Thr-89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 A resolution, shows high overall similarity to the wild-type enzyme, but an aspartyl moiety is covalently bound to Thr-12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.
Asparaginase↗
Fluorescence and Monte Carlo conformational studies of the (1-15) galanin amide fragment.
Galanin (GAL) is a 29 amino acid C-terminally aminated linear neuropeptide showing diverse biological activities. The N-terminal (1-15)GAL-NH2 fragment was shown to have a very high affinity to the galanin receptor. In this work we describe the results of a combined fluorescence and Monte Carlo studies, the latter carried out using the ECEPP/3 force field with and without including hydration, on the (1-15)GAL-NH2 fragment. Using the time-domain technique we measured fluorescence decay times of the tyrosine residue in position 9. Based on the Forster energy transfer theory we calculated the distance and distance distribution between the Trp2 (acceptor) and Tyr9 (donor) aromatic side chains. The distance obtained was about 10.5 angstrom and half-width, hw, of the distance distribution was 5.6 angstrom. This results were found to be in good agreement with the chromophore distances calculated for the low-energy solution confirmations obtained in Monte Carlo simulations. All the low-energy conformations obtained in the absence of water were almost all-helical with the exception of a few C-terminal residues. In contrast, none of the low-energy solution conformations contained any significant amount of secondary structure. These findings are in agreement with the results of earlier CD and NMR conformational studies of galanin in water and non-aqueous solvents. On the other hand, the conformations obtained in the presence of water turned out to be largely compact in the N-terminal hydrophobic part. This explains the relatively short distance between chromophores and narrow distance distribution obtained in fluorescence measurements.
Amides↗
Heme-protein interactions in horse heart myoglobin at neutral pH and exposed to acid investigated by time-resolved fluorescence in the pico- to nanosecond time range.
We measured the steady state and time-resolved emission intensity decay of horse heart myoglobin at various pH values from neutral to pH 4.42. The steady state intensity was reversibly increased with the decreasing pH, almost doubling at pH 4.5. Frequency domain data for emission decay were analyzed separately for each pH and simultaneously by global analyses. The results indicated the presence of four lifetime components, conserved throughout the pH titrations at 40, 116, 1363, and 4822 ps, respectively. The titration affected only their fractional intensities. Assignments of the lifetimes were based on the Förster theory of radiationless dipole-dipole interaction and the atomic coordinates of the system. We assigned the two shorter lifetimes to Trp-14 and Trp-7, respectively, in the presence of normal hemes. The 1363-ps lifetime was assigned to Trp-7 with inverted hemes (i.e. rotated 180 degrees around the alpha-gamma-meso axis of the porphyrin ring). The 4822-ns lifetime was assigned to reversibly heme-dissociated myoglobin. Lorentzian lifetime distributions were narrow for the lifetimes at 40, 116, and 4822 ps, indicating a homogeneous protein structure. Instead the lifetime at 1363 ns had a broad, pH-independent distribution consistent with small angle wobblings of inverted hemes inside the heme pocket. These analyses revealed the presence of three species originating from heme-protein interactions: the native form of crystalline myoglobin, the conformation with disorder hemes, and the reversibly dissociated heme-free myoglobin. There was increased heme inversion and heme dissociability at lower pH, consistent with the titration of the proximal and distal histidines inside the heme pocket.
Animals↗
Structure of an inhibitor complex of the proteinase from feline immunodeficiency virus.
The crystal structure of a recombinant form of the proteinase encoded by the feline immunodeficiency virus (FIV PR) has been solved at 2 A resolution and refined to an R-factor of 0.148. The refined structure includes a peptidomimetic, statine-based inhibitor, LP-149, which is an even more potent inhibitor of HIV PR. Kinetic parameters were obtained for the cleavage of five substrates by FIV PR, and inhibition constants were measured for four inhibitors. The structure of FIV PR resembles other related retroviral enzymes although few inhibitors of HIV PR are capable of inhibiting FIV PR. The structure of FIV PR will enhance our knowledge of this class of enzymes, and will direct testing of new proteinase inhibitors in a feline animal model.
Amino Acid Sequence↗
Refined crystal structure of Acinetobacter glutaminasificans glutaminase-asparaginase.
The crystal structure of glutaminase-asparaginase from Acinetobacter glutaminasificans has been reinterpreted and refined to an R factor of 0.171 at 2.9 A resolution, using the same X-ray diffraction data that were used to build a preliminary model of this enzyme [Ammon, Weber, Wlodawer, Harrison, Gilliland, Murphy, Sjölin & Roberts (1988). J. Biol. Chem. 263, 150-156]. The current model, which does not include solvent, is based in part on the related structure of Escherichia coli asparaginase and is significantly different from the structure of the enzyme from A. glutaminasificans described previously. The reason for the discrepancies has been traced to insufficient phasing power of the original heavy-atom derivative data, which could not be compensated for fully by electron-density modification techniques. The corrected structure of A. glutaminasificans glutaminase-asparaginase is presented and compared with the preliminary model and with the structure of E. coli asparaginase.
Journal Article↗
Structural characterization of Pseudomonas 7A glutaminase-asparaginase.
The amino acid sequence and a 2-A-resolution crystallographic structure of Pseudomonas 7A glutaminase-asparaginase (PGA) have been determined. PGA, which belongs to the family of tetrameric bacterial amidohydrolases, deamidates glutamine and asparagine. The amino acid sequence of PGA has a high degree of similarity to the sequences of other members of the family. PGA has the same fold as other bacterial amidohydrolases, with the exception of the position of a 20-residue loop that forms part of the active site. In the PGA structure presented here, the active site loop is observed clearly in only one monomer, in an open position, with a conformation different from that observed for other amidohydrolases. In the other three monomers the loop is disordered and cannot be traced. This phenomenon is probably a direct consequence of a very low occupancy of product(s) of the enzymatic reaction bound in the active sites of PGA in these crystals. The active sites are composed of a rigid part and the flexible loop. The rigid part consists of the residues directly involved in the catalytic reaction as well as residues that assist in orienting the substrate. Two residues that are important for activity residue on the flexible loop. We suggest that the flexible loops actively participate in the transport of substrate and product molecules through the amidohydrolase active sites and participate in orienting the substrate molecules properly in relation to the catalytic residues.
Amidohydrolases↗