Phage display identification of high-affinity ligands for human TSG101-UEV: A structural and thermodynamic study of PTAP recognition.

The ubiquitin E2 variant domain of TSG101 (TSG101-UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disrupting TSG101-UEV/PTAP interactions has emerged as a promising strategy for the development of novel host-oriented antivirals with a broad spectrum of action. Nonetheless, finding inhibitors with good properties as therapeutic agents remains a challenge since the key determinants of binding affinity and specificity are still poorly understood. Here we present a detailed thermodynamic, structural, and dynamic characterization viral PTAP Late domain recognition by TSG101-UEV, combining isothermal titration calorimetry, X-ray diffraction structural studies, molecular dynamics simulations, and computational analysis of intramolecular communication pathways. Our analysis highlights key contributions from conserved hydrophobic contacts and water-mediated hydrogen bonds at the PTAP binding interface. We have identified additional electrostatic hotspots adjacent to the core motif that modulate affinity. Using competitive phage display screening we have improved affinity by 1-2 orders of magnitude, producing novel peptides with low micromolar affinities that combine critical elements found in the best natural binders. Molecular dynamics simulations revealed that optimized peptides engage new pockets on the UEV domain surface. This study provides a comprehensive view of the molecular forces directing TSG101-UEV recognition of PTAP motifs, revealing that binding is governed by conserved structural elements yet tuneable through targeted optimization. These insights open new venues to design inhibitors targeting TSG101-dependent pathways with potential application as novel broad-spectrum antivirals.

Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.

N-Succinyl-amino acid racemase (NSAAR), long referred to as N-acyl- or N-acetyl-amino acid racemase, is an enolase superfamily member whose biotechnological potential was discovered decades ago, due to its use in the industrial dynamic kinetic resolution methodology first known as "Acylase Process". In previous works, an extended and enhanced substrate spectrum of the NSAAR from Geobacillus kaustophilus CECT4264 toward different N-substituted amino acids was reported. In this work, we describe the cloning, purification, and characterization of the NSAAR from Geobacillus stearothermophilus CECT49 (GstNSAAR). The enzyme has been extensively characterized, showing a higher preference toward N-formyl-amino acids than to N-acetyl-amino acids, thus confirming that the use of the former substrates is more appropriate for a biotechnological application of the enzyme. The enzyme showed an apparent thermal denaturation midpoint of 77.0 ± 0.1 °C and an apparent molecular mass of 184 ± 5 kDa, suggesting a tetrameric species. Optimal parameters for the enzyme activity were pH 8.0 and 55-65 °C, with Co(2+) as the most effective cofactor. Mutagenesis and binding experiments confirmed K166, D191, E216, D241, and K265 as key residues in the activity of GstNSAAR, but not indispensable for substrate binding.

Electrostatic effects in the folding of the SH3 domain of the c-Src tyrosine kinase: pH-dependence in 3D-domain swapping and amyloid formation.

The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6522 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P212121, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging ß-turn through a cluster of water molecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation.

Biochemical and mutational studies of allantoinase from Bacillus licheniformis CECT 20T.

Allantoinases (allantoin amidohydrolase, E.C. 3.5.2.5) catalyze the hydrolysis of the amide bond of allantoin to form allantoic acid, in those organisms where allantoin is not the final product of uric acid degradation. Despite their importance in the purine catabolic pathway, sequences of microbial allantoinases with proven activity are scarce, and only the enzyme from Escherichia coli (AllEco) has been studied in detail in the genomic era. In this work, we report the cloning, purification and characterization of the recombinant allantoinase from Bacillus licheniformis CECT 20T (AllBali). The enzyme was a homotetramer with an apparent Tm of 62 ± 1 °C. Optimal parameters for the enzyme activity were pH 7.5 and 50 °C, showing apparent Km and kcat values of 17.7 ± 2.7 mM and 24.4 ± 1.5 s(-1), respectively. Co(2+) proved to be the most effective cofactor, inverting the enantioselectivity of AllBali when compared to that previously reported for other allantoinases. The common ability of different cyclic amidohydrolases to hydrolyze distinct substrates to the natural one also proved true for AllBali. The enzyme was able to hydrolyze hydantoin, dihydrouracil and 5-ethyl-hydantoin, although at relative rates 3-4 orders of magnitude lower than with allantoin. Mutagenesis experiments suggest that S292 is likely implicated in the binding of the allantoin ring through the carbonyl group of the polypeptide main chain, which is the common mechanism observed in other members of the amidohydrolase family. In addition, our results suggest an allosteric effect of H2O2 toward allantoinase.

pH-dependent structural conformations of B-phycoerythrin from Porphyridium cruentum.

B-phycoerythrin from the red alga Porphyridium cruentum was crystallized using the technique of capillary counter-diffusion. Crystals belonging to the space group R3 with almost identical unit cell constants and diffracting to 1.85 and 1.70 Å were obtained at pH values of 5 and 8, respectively. The most important difference between structures is the presence of the residue His88α in two different conformations at pH 8. This residue is placed next to the chromophore phycoerythrobilin PEB82α and the new conformation results in the relocation of the hydrogen-bond network and hydration around PEB82α, which probably contributes to the observed pH dependence of the optical spectrum associated with this chromophore. Comparison with the structures of B-phycoerythrin from other red algae shows differences in the conformation of the A-ring of the chromophore PEB139α. This conformational difference in B-phycoerythrin from P. cruentum enables the formation of several hydrogen bonds that connect PEB139α with the chromophore PEB158ß at the (αß)(3) hexamer association interface. The possible influence of these structural differences on the optical spectrum and the ability of the protein to perform energy transfer are discussed, with the two pH-dependent conformations of His88α and PEB82α being proposed as representing critical structural features that are correlated with the pH dependence of the optical spectrum and transient optical states during energy transfer.

Thermodynamic impact of embedded water molecules in the unfolding of human CD2BP2-GYF domain.

GYF domains are small polyproline-recognition modules adopting a structural arrangement consisting of a single α-helix packed against a small ß-sheet. Although most families of proline-rich recognition modules have been extensively characterized in terms of function, structure, or conformational flexibility, little is known about GYF domain functionality and folding. We have undertaken the thermodynamic characterization of the unfolding of CD2BP2-GYF domain by combining differential scanning calorimetry and circular dichroism under different pH conditions. The experimental data can be well-described in terms of a two-state equilibrium, although an unusually high heat capacity of the native state reflects a considerable conformational flexibility and dynamics of CD2BP2-GYF domain. In addition, the normalized thermodynamic parameters of unfolding (enthalpy, entropy and heat capacity) are roughly a factor of two greater than expected. In contrast, stability curves reveal an ordinary unfolding behavior of CD2BP2-GYF domain in terms of Gibbs energies, incurring thus unusually strong enthalpy-entropy compensation. This phenomenon, previously described as "thermodynamic homeostasis", has been associated in different examples to the contribution of occluded water (solvent) molecules into the protein structure. By means of CASTp server, we have found seven cavities/pockets scattered throughout of the CD2BP2-GYF structure, each able to harbor at least one water molecule. This structural feature provides rationalization for the atypical enthalpy values observed for CD2BP2-GYF because each water molecule is able to organize an extra amount of hydrogen bonds in the native state. In addition, these bound waters increase the vibrational entropy of the protein, which could also be responsible for an increase in protein flexibility and may thus fully explain the homeostatic behavior experimentally observed.

Biochemical and mutational studies of the Bacillus cereus CECT 5050T formamidase support the existence of a C-E-E-K tetrad in several members of the nitrilase superfamily.

Formamidases (EC 3.5.1.49) are poorly characterized proteins. In spite of this scarce knowledge, ammonia has been described as playing a central role in the pathogenesis of human pathogens such as Helicobacter pylori, for which formamidase has been shown to participate in the nitrogen metabolic pathway. Sequence analysis has revealed that at least two different groups of formamidases are classified as EC 3.5.1.49: on the one hand, the derivatives of the FmdA-AmdA superfamily, which are the best studied to date, and on the other hand, the derivatives of Helicobacter pylori AmiF. Here we present the cloning, purification, and characterization of a recombinant formamidase from Bacillus cereus CECT 5050T (BceAmiF), the second member of the AmiF subfamily to be characterized, showing new features of the enzyme further supporting its relationship with aliphatic amidases. We also present homology modeling-based mutational studies confirming the importance of the Glu140 and Tyr191 residues in the enzymatic activities of the AmiF family. Moreover, we can conclude that a second glutamate residue is critical in several members of the nitrilase superfamily, meaning that what has consistently been identified as a C-E-K triad is in fact a C-E-E-K tetrad.

A thermodynamic characterization of the interaction of 8-anilino-1-naphthalenesulfonic acid with native globular proteins: the effect of the ligand dimerization in the analysis of the binding isotherms.

8-Anilino-1-naphthalenesulfonic acid (ANS) is a popular fluorescence probe, broadly used for the analysis of proteins, but the nature of its interaction with proteins and the high increase in the fluorescence intensity that takes place upon such process are still unclear. In the last few years, isothermal titration calorimetry has been used to characterize the nature of the interaction of this dye with proteins. The analysis of the binding isotherms of these studies has not considered the dimerization equilibrium of ANS, which is pH dependent, and it can result in serious errors in the data analysis. In the present work we have developed a suitable data analysis by which this process is taken into account. To study the binding of the dye to proteins at different pH values, we have used the Abl-SH3 domain. Our results suggest that at pH 3 and 5, where the dimerization of the ANS is important, electrostatic interactions are significant for the binding of ANS to the Abl-SH3 domain. However, at pH 7, ANS behaves mostly as monomer and the interaction with the protein is mainly hydrophobic. The pH dependent behavior of the ANS binding to proteins can be explained in terms of ionization states of both, the protein and the ANS.

Structure and conformational stability of a tetrameric thermostable N-succinylamino acid racemase.

The N-succinylamino acid racemases (NSAAR) belong to the enolase superfamily and they are large homooctameric/hexameric species that require a divalent metal ion for activity. We describe the structure and stability of NSAAR from Geobacillus kaustophilus (GkNSAAR) in the absence and in the presence of Co(2+) by using hydrodynamic and spectroscopic techniques. The Co(2+), among other assayed divalent ions, provides the maximal enzymatic activity at physiological pH. The protein seems to be a tetramer with a rather elongated shape, as shown by AU experiments; this is further supported by the modeled structure, which keeps intact the largest tetrameric oligomerization interfaces observed in other homooctameric members of the family, but it does not maintain the octameric oligomerization interfaces. The native functional structure is mainly formed by alpha-helix, as suggested by FTIR and CD deconvoluted spectra, with similar percentages of structure to those observed in other protomers of the enolase superfamily. At low pH, the protein populates a molten-globule-like conformation. The GdmCl denaturation occurs through a monomeric intermediate, and thermal denaturation experiments indicate a high thermostability. The presence of the cofactor Co(2+) did alter slightly the secondary structure, but it did not modify substantially the stability of the protein. Thus, GkNSAAR is one of the few members of the enolase family whose conformational propensities and stability have been extensively characterized.

Thermodynamic determination of the binding constants of angiotensin-converting enzyme inhibitors by a displacement method.

Somatic angiotensin I-converting enzyme (s-ACE) plays a central role in blood pressure regulation and has been the target of most antihypertensive drugs. A displacement isothermal titration calorimetry method has been used to accurately determine the binding constant of three strong s-ACE inhibitors. Under the experimental conditions studied in this work, the relative potency of the inhibitors was determined to be enalaprilat>lisinopril>captopril. We analyze the thermodynamic behaviour of the binding process using the new structural information provided by the ACE structures, as well as the conformational changes that occur upon binding.

Site-directed mutagenesis indicates an important role of cysteines 76 and 181 in the catalysis of hydantoin racemase from Sinorhizobium meliloti.

Hydantoin racemase enzyme plays a crucial role in the reaction cascade known as "hydantoinase process." In conjunction with a stereoselective hydantoinase and a stereospecific carbamoylase, it allows the total conversion from D,L-5-monosubstituted hydantoins, with a low rate of racemization, to optically pure D- or L-amino acids. Residues Cys76 and Cys181 belonging to hydantoin racemase from Sinorhizobium meliloti (SmeHyuA) have been proved to be involved in catalysis. Here, we report biophysical data of SmeHyuA Cys76 and Cys181 to alanine mutants, which point toward a two-base mechanism for the racemization of 5-monosubstituted hydantoins. The secondary and the tertiary structure of the mutants were not significantly affected, as shown by circular dichroism. Calorimetric and fluorescence experiments have shown that Cys76 is responsible for recognition and proton retrieval of D-isomers, while Cys181 is responsible for L-isomer recognition and racemization. This recognition process is further supported by measurements of protein stability followed by chemical denaturation in the presence of the corresponding compound.

Thermodynamic and mutational studies of l-N-carbamoylase from Sinorhizobium meliloti CECT 4114 catalytic centre.

Purified site-directed mutants of Sinorhizobium meliloti CECT 4114 l-N-carbamoylase (SmLcar) in which Glu132, His230, Asn279 and Arg292 were replaced have been studied by kinetic methods and isothermal titration calorimetry (ITC). The importance of His230, Asn279 and Arg292 residues in the recognition of N-carbamoyl-l-alpha-amino acids has been proved. The role of Glu132 has been confirmed in substrate hydrolysis. ITC has confirmed two Ni atoms per monomer of wild type enzyme, and two equal and independent substrate binding sites (one per monomer). Homology modelling of SmLcar supports the importance of His87, His194, His386, Glu133 and Asp98 in metal binding. A comprehensive reaction mechanism is proposed on the basis of binding experiments measured by ITC, kinetic assays, and homology of the active centre with beta-alanine synthase from Saccharomyces kluyveri and other enzymes.

Enzymatic activity assay of D-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates.

Isothermal titration calorimetry (ITC) has been applied to the determination of the activity of D-hydantoinase (EC 3.5.2.2) with several substrates by monitoring the heat released during the reaction. The method is based on the proportionality between the reaction rate and the thermal power (heat/time) generated. Microcalorimetric assays carried out at different temperatures provided the dependence of the catalytic rate constant on temperature. We show that ITC assay is a nondestructive method that allows the determination of the catalytic rate constant (kcat), Michaelis constant (KM), activation energy and activation Gibbs energy, enthalpy and entropy of this reaction.

Binding studies of hydantoin racemase from Sinorhizobium meliloti by calorimetric and fluorescence analysis.

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific d-carbamoylase guarantee the total conversion from d,l-5-monosubstituted hydantoins with a low velocity of racemization, to optically pure d-amino acids. Hydantoin racemase from Sinorhizobium meliloti was expressed in Escherichia coli. Calorimetric and fluorescence experiments were then carried out to obtain the thermodynamic binding parameters, deltaG, deltaH and DeltaS for the inhibitors L- and D-5-methylthioethyl-hydantoin. The number of active sites is four per enzyme molecule (one per monomer), and the binding of the inhibitor is entropically and enthalpically favoured under the experimental conditions studied. In order to obtain information about amino acids involved in the active site, four different mutants were developed in which cysteines 76 and 181 were mutated to Alanine and Serine. Their behaviour shows that these cysteines are essential for enzyme activity, but only cysteine 76 affects the binding to these inhibitors.

Crystallographic and thermodynamic analysis of the binding of S-octylglutathione to the Tyr 7 to Phe mutant of glutathione S-transferase from Schistosoma japonicum.

Glutathione S-transferases are a family of multifunctional enzymes involved in the metabolism of drugs and xenobiotics. Two tyrosine residues, Tyr 7 and Tyr 111, in the active site of the enzyme play an important role in the binding and catalysis of substrate ligands. The crystal structures of Schistosoma japonicum glutathione S-transferase tyrosine 7 to phenylalanine mutant [SjGST(Y7F)] in complex with the substrate glutathione (GSH) and the competitive inhibitor S-octylglutathione (S-octyl-GSH) have been obtained. These new structural data combined with fluorescence spectroscopy and thermodynamic data, obtained by means of isothermal titration calorimetry, allow for detailed characterization of the ligand-binding process. The binding of S-octyl-GSH to SjGST(Y7F) is enthalpically and entropically driven at temperatures below 30 degrees C. The stoichiometry of the binding is one molecule of S-octyl-GSH per mutant dimer, whereas shorter alkyl derivatives bind with a stoichiometry of two molecules per mutant dimer. The SjGST(Y7F).GSH structure showed no major structural differences compared to the wild-type enzyme. In contrast, the structure of SjGST(Y7F).S-octyl-GSH showed asymmetric binding of S-octyl-GSH. This lack of symmetry is reflected in the lower symmetry space group of the SjGST(Y7F).S-octyl-GSH crystals (P6(3)) compared to that of the SjGST(Y7F).GSH crystals (P6(3)22). Moreover, the binding of S-octyl-GSH to the A subunit is accompanied by conformational changes that may be responsible for the lack of binding to the B subunit.

A monomer form of the glutathione S-transferase Y7F mutant from Schistosoma japonicum at acidic pH.

Dissociation and unfolding of homodimeric glutathione S-transferase Y7F mutant from Schistosoma japonicum (SjGST-Y7F) were investigated at equilibrium using urea as denaturant. The conserved residue Tyr7 plays a central role in the catalytic mechanism and the mutation Tyr-Phe yields an inactive enzyme that is able to bind the substrate GSH with a higher binding constant than the wild type enzyme. Mutant SjGST-Y7F is a dimer at pH 6 or higher and a stable monomer at pH 5 that binds GSH (K value of 1.2x10(5)+/-6.4x10(3)M(-1) at pH 6.5 and 6.3x10(4)+/-1.25x10(3)M(-1) at pH 5). The stability of the SjGST-Y7F mutant was studied by urea induced unfolding techniques (DeltaG(W)=13.86+/-0.63kcalmol(-1) at pH 6.5 and DeltaG(W)=11.22+/-0.25kcalmol(-1) at pH 5) and the monomeric form characterized by means of size exclusion chromatography, fluorescence, and electrophoretic techniques.

Thermodynamics of glutathione binding to the tyrosine 7 to phenylalanine mutant of glutathione S-transferase from Schistosoma japonicum.

The binding of glutathione (GSH) to the tyrosine 7 to phenylalanine mutant of Schistosoma japonicum glutathione S-transferase (SjGST-Y7F) has been studied by isothermal titration calorimetry (ITC). At pH 6.5 and 25 degrees C this mutant shows a higher affinity for glutathione than wild type enzyme despite an almost complete loss of activity in the presence of 1-chloro-2,4-dinitrobenzene (CDNB) as second substrate. The enthalpy change upon binding of GSH is more negative for the mutant than for the wild type GST (SjGST). Changes in accessible solvent areas (ASA) have been calculated based on enthalpy and heat capacity changes. ASA values indicated the burial of apolar surfaces of protein and ligand upon binding. A more negative DeltaC(p) value has been obtained for the mutant enzyme, suggesting a more hydrophobic interaction, as may be expected from the change of a tyrosine residue to phenylalanine.