Structural Determinants of Specificity and Catalytic Mechanism in mammalian 25-kDa Thiamine Triphosphatase

in Biochimica et Biophysica Acta - General Subjects (in press)

Background: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine ... [more ▼]

Background: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates. Methods: Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase. Results: Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine-tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds. Conclusion: The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals. General significance: We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins. [less ▲]

Towards a universal method for protein refolding: The trimeric beta barrel membrane Omp2a as a test case.

in Biotechnology and Bioengineering (2013), 110(2), 417-23

It has recently been reported that 2-methyl-2,4-pentanediol (MPD) can modulate the protein-binding properties of sodium dodecyl sulfate (SDS), turning it into a non-denaturing detergent. Indeed both alpha ... [more ▼]

It has recently been reported that 2-methyl-2,4-pentanediol (MPD) can modulate the protein-binding properties of sodium dodecyl sulfate (SDS), turning it into a non-denaturing detergent. Indeed both alpha (the lysozyme) and beta (the carbonic anhydrase II) soluble enzymes, as well as a beta membrane protein (PagP) have been successfully refolded into their native form by using this amphiphatic alcohol. In order to support the universal character of our MPD-based technique, we have extended its transferability to the Omp2a trimeric membrane porin. The far-UV circular dichroism signature of Omp2a refolded with our original procedure is identical to that obtained by classical techniques, clearly indicating a proper refolding. Moreover, we show that the optimal SDS/MPD ratio for refolding Omp2a is similar to what has been observed for other types of proteins. While the protocol allows refolding at higher protein concentration (up to 4 mg/mL) and ionic strength (up to 1 M NaCl) than other refolding methods, it is also more efficient at basic pH values and medium temperature (20-40 degrees C). Finally, the key role of the cosolvent was highlighted by a thorough study of the efficiency of MPD analogues, and a high variability was observed, as they can be able or unable to induce refolding at low or high salt concentrations. Biotechnol. Bioeng. 2013; 110: 417-423. (c) 2012 Wiley Periodicals, Inc. [less ▲]

Purification, refolding and characterization of the trimeric Omp2a outer membrane porin from Brucella melitensis.

in Protein Expression & Purification (2012), 83(2), 198-204

Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their ... [more ▼]

Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella beta-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-beta-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination. [less ▲]

Generation of camelid single-domain antibody fragments raised against proteins containing polyglutamine expansions

Poster (2011, January 25)

Stepwise adaptations to low temperature as revealed by multiple mutants of a psychrophilic alpha-amylase from an Antarctic bacterium

in Journal of Biological Chemistry (2011), 286(44), 3834838355

Effects of monopropanediamino-beta-cyclodextrin on the denaturation process of the hybrid protein BlaPChBD.

in Biochimica et biophysica acta (2011)

Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations ... [more ▼]

Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from diseases (such as Alzheimer's and Parkinson's diseases) to the production (e.g. inclusion bodies), stability, storage and delivery of protein drugs. beta-Cyclodextrin (beta-CD) is a circular heptasaccharide characterized by a hydrophilic exterior and a hydrophobic interior ring structure. In this research, we studied the effects of a chemically modified beta-CD (BCD07056), on the aggregating and refolding properties of BlaPChBD, a hybrid protein obtained by inserting the chitin binding domain of the human macrophage chitotriosidase into the class A beta-lactamase BlaP from Bacillus licheniformis 749/I during its thermal denaturation. The results show that BCD07056 strongly increases the refolding yield of BlaPChBD after thermal denaturation and constitutes an excellent additive to stabilize the protein over time at room temperature. Our data suggest that BCD07056 acts early in the denaturation process by preventing the formation of an intermediate which leads to an aggregated state. Finally, the role of beta-CD derivatives on the stability of proteins is discussed. [less ▲]

Three factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys 70

in Biochemical Journal (2010), 432(3), 495-504

Lys-70 carboxylation in the active site of class D β lactamases is essential for their activity. Structural, kinetic and affinity studies show that this post-translational modification can be affected by ... [more ▼]

Lys-70 carboxylation in the active site of class D β lactamases is essential for their activity. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val-117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys 70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate limiting step for the wild type OXA 10 β lactamase. [less ▲]

Interplay between the effects of the length of the polyglutamine tract and its structural context on the aggregation of polyglutamine (polyQ) proteins

Poster (2010, June 07)

1H, 13C and 15N backbone resonance assignments for the BS3 class A beta-lactamase from Bacillus licheniformis.

in Biomolecular NMR Assignments (2010), 4(2), 195-7

Class A beta-lactamases (260-280 amino acids; M ( r ) ~ 29,000) are among the largest proteins studied in term of their folding properties. They are composed of two structural domains: an all-alpha domain ... [more ▼]

Class A beta-lactamases (260-280 amino acids; M ( r ) ~ 29,000) are among the largest proteins studied in term of their folding properties. They are composed of two structural domains: an all-alpha domain formed by five to eight helices and an alpha/beta domain consisting of a five-stranded antiparallel beta-sheet covered by three to four alpha-helices. The alpha domain (~150 residues) is made up of the central part of the polypeptide chain whereas the alpha/beta domain (111-135 residues) is constituted by the N- and C-termini of the protein. Our goal is to determine in which order the different secondary structure elements are formed during the folding of BS3. With this aim, we will use pulse-labelling hydrogen/deuterium exchange experiments, in combination with 2D-NMR measurements, to monitor the time-course of formation and stabilization of secondary structure elements. Here we report the backbone resonance assignments as the requirement for further hydrogen/deuterium exchange studies. [less ▲]

Folding of class A beta-lactamases is rate-limited by peptide bond isomerization and occurs via parallel pathways.

in Biochemistry (2010), 49(19), 4264-75

Class A beta-lactamases (M(r) approximately 29000) provide good models for studying the folding mechanism of large monomeric proteins. In particular, the highly conserved cis peptide bond between residues ... [more ▼]

Class A beta-lactamases (M(r) approximately 29000) provide good models for studying the folding mechanism of large monomeric proteins. In particular, the highly conserved cis peptide bond between residues 166 and 167 at the active site of these enzymes controls important steps in their refolding reaction. In this work, we analyzed how conformational folding, reactivation, and cis/trans peptide bond isomerizations are interrelated in the folding kinetics of beta-lactamases that differ in the nature of the cis peptide bond, which involves a Pro167 in the BS3 and TEM-1 enzyme, a Leu167 in the NMCA enzyme, and which is missing in the PER-1 enzyme. The analysis of folding by spectroscopic probes and by the regain of enzymatic activity in combination with double-mixing procedures indicates that conformational folding can proceed when the 166-167 bond is still in the incorrect trans form. The very slow trans --> cis isomerization of the Glu166-Xaa167 peptide bond, however, controls the final step of folding and is required for the regain of the enzymatic activity. This very slow phase is absent in the refolding of PER-1, in which the Glu166-Ala167 peptide bond is trans. The double-mixing experiments revealed that a second slow kinetic phase is caused by the cis/trans isomerization of prolines that are trans in the folded proteins. The folding of beta-lactamases is best described by a model that involves parallel pathways. It highlights the role of peptide bond cis/trans isomerization as a kinetic determinant of folding. [less ▲]