Crystal structure of a bacterial albumin-binding domain at 1.4 A resolution

The albumin-binding domain, or GA module, of the peptostreptococcal albumin-binding protein expressed in pathogenic strains of Finegoldia magna is believed to be responsible for the virulence and increased growth rate of these strains. Here we present the 1.4A crystal structure of this domain, and compare it with the crystal structure of the GA-albumin complex. An analysis of protein-protein interactions in the two crystals, and the presence of multimeric GA species in solution, indicate the GA module is "sticky", and is capable of forming contacts with a range of protein surfaces. This might lead to interactions with different host proteins.     

Verdoheme formation in Proteus mirabilis catalase

Background

Heme oxidative degradation has been extensively investigated in peroxidases but not in catalases. The verdoheme formation, a product of heme oxidation which inactivates the enzyme, was studied in Proteus mirabilis catalase.

Methods

The verdoheme was generated by adding peracetic acid and analyzed by mass spectrometry and spectrophotometry.

Results

Kinetics follow-up of different catalase reactional intermediates shows that i) the formation of compound I always precedes that of verdoheme, ii) compound III is never observed, iii) the rate of compound II decomposition is not compatible with that of verdoheme formation, and iv) dithiothreitol prevents the verdoheme formation but not that of compound II, whereas NADPH prevents both of them. The formation of verdoheme is strongly inhibited by EDTA but not increased by Fe³⁺ or Cu²⁺ salts. The generation of verdoheme is facilitated by the presence of protein radicals as observed in the F194Y mutated catalase. The inability of the inactive variant (H54F) to form verdoheme, indicates that the heme oxidation is fully associated to the enzyme catalysis.

Conclusion

These data, taken together, strongly suggest that the verdoheme formation pathway originates from compound I rather than from compound II.

General significance

The autocatalytic verdoheme formation is likely to occur in vivo.     

Crystal structure of plant asparaginase

In plants, specialized enzymes are required to catalyze the release of ammonia from asparagine, which is the main nitrogen-relocation molecule in these organisms. In addition, K+-independent plant asparaginases are also active in splitting the aberrant isoaspartyl peptide bonds, which makes these proteins important for seed viability and germination. Here, we present the crystal structure of potassium-independent L-asparaginase from yellow lupine (LlA) and confirm the classification of this group of enzymes in the family of Ntn-hydrolases. The alpha- and beta-subunits that form the mature (alphabeta)2 enzyme arise from autoproteolytic cleavage of two copies of a precursor protein. In common with other Ntn-hydrolases, the (alphabeta) heterodimer has a sandwich-like fold with two beta-sheets flanked by two layers of alpha-helices (alphabetabetaalpha). The nucleophilic Thr193 residue, which is liberated in the autocatalytic event at the N terminus of subunit beta, is part of an active site that is similar to that observed in a homologous bacterial enzyme. An unusual sodium-binding loop of the bacterial protein, necessary for proper positioning of all components of the active site, shows strictly conserved conformation and metal coordination in the plant enzyme. A chloride anion complexed in the LlA structure marks the position of the alpha-carboxylate group of the L-aspartyl substrate/product moiety. Detailed analysis of the active site suggests why the plant enzyme hydrolyzes asparagine and its beta-peptides but is inactive towards substrates accepted by similar Ntn-hydrolases, such as taspase1, an enzyme implicated in some human leukemias. Structural comparisons of LlA and taspase1 provide interesting insights into the role of small inorganic ions in the latter enzyme.     

Structural and functional analysis of two glutamate racemase isozymes from Bacillus anthracis and implications for inhibitor design

Glutamate racemase (RacE) is responsible for converting l-glutamate to d-glutamate, which is an essential component of peptidoglycan biosynthesis, and the primary constituent of the poly-gamma-d-glutamate capsule of the pathogen Bacillus anthracis. RacE enzymes are essential for bacterial growth and lack a human homolog, making them attractive targets for the design and development of antibacterial therapeutics. We have cloned, expressed and purified the two glutamate racemase isozymes, RacE1 and RacE2, from the B. anthracis genome. Through a series of steady-state kinetic studies, and based upon the ability of both RacE1 and RacE2 to catalyze the rapid formation of d-glutamate, we have determined that RacE1 and RacE2 are bona fide isozymes. The X-ray structures of B. anthracis RacE1 and RacE2, in complex with d-glutamate, were determined to resolutions of 1.75 A and 2.0 A. Both enzymes are dimers with monomers arranged in a "tail-to-tail" orientation, similar to the B. subtilis RacE structure, but differing substantially from the Aquifex pyrophilus RacE structure. The differences in quaternary structures produce differences in the active sites of racemases among the various species, which has important implications for structure-based, inhibitor design efforts within this class of enzymes. We found a Val to Ala variance at the entrance of the active site between RacE1 and RacE2, which results in the active site entrance being less sterically hindered for RacE1. Using a series of inhibitors, we show that this variance results in differences in the inhibitory activity against the two isozymes and suggest a strategy for structure-based inhibitor design to obtain broad-spectrum inhibitors for glutamate racemases.     

pK(a) values for side-chain carboxyl groups of a PGB1 variant explain salt and pH-dependent stability

Determination of pK(a) values of titrating residues in proteins provides a direct means of studying electrostatic coupling as well as pH-dependent stability. The B1 domain of protein G provides an excellent model system for such investigations. In this work, we analyze the observed pK(a) values of all carboxyl groups in a variant of PGB1 (T2Q, N8D, N37D) at low and high ionic strength as determined using (1)H-(13)C heteronuclear NMR in a structural context. The pK(a) values are used to calculate the pH-dependent stability in low and high salt and to investigate electrostatic coupling in the system. The observed pK(a) values can explain the pH dependence of protein stability but require pK(a) shifts relative to model values in the unfolded state, consistent with persistent residual structure in the denatured state. In particular, we find that most of the deviations from the expected random coil values can be explained by a significantly upshifted pK(a) value. We show also that (13)C backbone carbonyl data can be used to study electrostatic coupling in proteins and provide specific information on hydrogen bonding and electrostatic potential at nontitrating sites.     

Thermodynamic and structural analysis of homodimeric proteins: Model of β-lactoglobulin

The energetics of protein homo-oligomerization was analyzed in detail with the application of a general thermodynamic model. We have studied the thermodynamic aspects of protein-protein interaction employing β-lactoglobulin A from bovine milk at pH = 6.7 where the protein is mainly in its dimeric form. We performed differential calorimetric scans at different total protein concentration and the resulting thermograms were analyzed with the thermodynamic model for oligomeric proteins previously developed. The thermodynamic model employed, allowed the prediction of the sign of the enthalpy of dimerization, the analysis of complex calorimetric profiles without transitions baselines subtraction and the obtainment of the thermodynamic parameters from the unfolding and the association processes and the compared with association parameters obtained with Isothermal Titration Calorimetry performed at different temperatures. The dissociation and unfolding reactions were also monitored by Fourier-transform infrared spectroscopy and the results indicated that the dimer of β-lactoglobulin (N₂) reversibly dissociates into monomeric units (N) which are structurally distinguishable by changes in their infrared absorbance spectra upon heating. Hence, it is proposed that β-lactoglobulin follows the conformational path induced by temperature:N₂ ? 2N ? 2D. The general model was validated with these results indicating that it can be employed in the study of the thermodynamics of other homo-oligomeric protein systems.     

Crystal structure and mechanism of tripeptidyl activity of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis

The crystal structure of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis was determined. Prolyl tripeptidyl aminopeptidase consists of beta-propeller and catalytic domains, and a large cavity between the domains; this structure is similar to dipeptidyl aminopeptidase IV. A catalytic triad (Ser603, His710, and Asp678) was located in the catalytic domain; this triad was virtually identical to that of the enzymes belonging to the prolyl oligopeptidase family. The structure of an inactive S603A mutant enzyme complexed with a substrate was also determined. The pyrrolidine ring of the proline residue appeared to fit into a hydrophobic pocket composed of Tyr604, Val629, Trp632, Tyr635, Tyr639, Val680, and Val681. There were characteristic differences in the residues of the beta-propeller domain, and these differences were related to the substrate specificity of tripeptidyl activity. The N-terminal amino group was recognized by salt bridges, with two carboxyl groups of Glu205 and Glu206 from a helix in dipeptidyl aminopeptidase IV. In prolyl tripeptidyl aminopeptidase, however, the Glu205 (located in the loop) and Glu636 were found to carry out this function. The loop structure provides sufficient space to accommodate three N-terminal residues (Xaa-Xaa-Pro) of substrates. This is the first report of the structure and substrate recognition mechanism of tripeptidyl peptidase.     

The anti-cancer drug chlorambucil as a substrate for the human polymorphic enzyme glutathione transferase P1-1: kinetic properties and crystallographic characterisation of allelic variants

The commonly used anti-cancer drug chlorambucil is the primary treatment for patients with chronic lymphocytic leukaemia. Chlorambucil has been shown to be detoxified by human glutathione transferase Pi (GST P1-1), an enzyme that is often found over-expressed in cancer tissues. The allelic variants of GST P1-1 are associated with differing susceptibilities to leukaemia and differ markedly in their efficiency in catalysing glutathione (GSH) conjugation reactions. Here, we perform detailed kinetic studies of the allelic variants with the aid of three representative co-substrates. We show that the differing catalytic properties of the variants are highly substrate-dependent. We show also that all variants exhibit the same temperature stability in the range 10 °C to 45 °C. We have determined the crystal structures of GST P1-1 in complex with chlorambucil and its GSH conjugate for two of these allelic variants that have different residues at positions 104 and 113. Chlorambucil is found to bind in a non-productive mode to the substrate-binding site (H-site) in the absence of GSH. This result suggests that under certain stress conditions where GSH levels are low, GST P1-1 can inactivate the drug by sequestering it from the surrounding medium. However, in the presence of GSH, chlorambucil binds in the H-site in a productive mode and undergoes a conjugation reaction with GSH present in the crystal. The crystal structure of the GSH-chlorambucil complex bound to the *C variant is identical with the *A variant ruling out the hypothesis that primary structure differences between the variants cause structural changes at the active site. Finally, we show that chlorambucil is a very poor inhibitor of the enzyme in contrast to ethacrynic acid, which binds to the enzyme in a similar fashion but can act as both substrate and inhibitor.     

Structural preference for changes in the direction of the Ca2+-induced transition: a study of the regulatory domain of skeletal troponin-C

The determinants for specificity in the Ca(2+)-dependent response of the regulatory N-terminal domain of skeletal troponin-C are a combination of intrinsic and induced properties. We characterized computationally the intrinsic propensity of this domain for structural changes similar to those observed experimentally in the Ca(2+)-induced transition. The preference for such changes was assessed by comparing the structural effect of the harmonic and quasiharmonic vibrations specific for each Ca(2+) occupancy with crystallographic data. Results show that only the Ca(2+)-saturated form of the protein features a slow vibrational motion preparatory for the transition. From the characteristics of this mode, we identified a molecular mechanism for transition, by which residues 42-51 of helix B and of the adjacent linker move toward helices (A, D), and bind to the surface used by the protein to interact with troponin-I. By obstructing the access of the target to hydrophobic residues important in the formation of the complex, helix B and the adjacent linker act as an autoinhibitory structural element. Specific properties of the methionines at the interaction surface were found to favor the binding of the autoinhibitory region. Located over hydrophobic residues critical for binding, the methionines are easily displaceable to increase the accessibility of these residues to molecular encounter.