First 3-D structural evidence of a native-like intertwined dimer in the acylphosphatase family.

Acylphosphatase (AcP, EC 3.6.1.7) is a small model protein conformed by a ferredoxin-like fold, profoundly studied to get insights into protein folding and aggregation processes. Numerous studies focused on the aggregation and/or amyloidogenic properties of AcPs suggest the importance of edge-ß-strands in the process. In this work, we present the first crystallographic structure of Escherichia coli AcP (EcoAcP), showing notable differences with the only available NMR structure for this enzyme. EcoAcP is crystalised as an intertwined dimer formed by replacing a single C-terminal ß-strand between two protomers, suggesting a flexible character of the C-terminal edge of EcoAcP. Despite numerous works where AcP from different sources have been used as a model system for protein aggregation, our domain-swapped EcoAcP structure is the first 3-D structural evidence of native-like aggregated species for any AcP reported to date, providing clues on molecular determinants unleashing aggregation.

mRNA Levels of Aromatase, 5α-Reductase Isozymes, and Prostate Cancer-Related Genes in Plucked Hair from Young Men with Androgenic Alopecia.

Androgenic alopecia (AGA) is the most prevalent type of progressive hair loss and has psychological repercussions. Nevertheless, the effectiveness of current pharmacological treatments remains limited, in part because the molecular basis of the disease has not been fully elucidated. Our group previously highlighted the important roles of aromatase and 5α-reductase (5α-R) in alopecia in young women with female pattern hair loss. Additionally, an association has been proposed between AGA and prostate cancer (PCa), suggesting that genes implicated in PCa would also be involved in AGA. A low-invasive, sensitive, and precise method was used to determine mRNA levels of aromatase, 5α-R isozymes, and 84 PCa-related genes in samples of plucked hair from young men with AGA and controls. Samples were obtained with a trichogram from the vertex scalp, and mRNA levels were quantified using real-time RT-PCR. The men with AGA had significantly higher 5α-R2 mRNA levels in comparison to controls; interestingly, some of them also showed markedly elevated mRNA levels of 5α-R1 or 5α-R3 or of both, which may explain the varied response to 5α-R inhibitor treatments. The men with AGA also showed significant changes versus controls in 6 out of the 84 genes implicated in PCa. This study contributes greater knowledge of the molecular bases of AGA, facilitating early selection of the most appropriate pharmacological therapy and opening the way to novel treatments.

Impact of chronic exposure of rats to bisphenol A from perinatal period to adulthood on intraprostatic levels of 5α-reductase isozymes, aromatase, and genes implicated in prostate cancer development.

The synergetic effect of estrogens and androgens is known to play a crucial role in the physiopathology of the prostate gland. Bisphenol A (BPA) is an endocrine disrupting compound that can interfere with endocrine hormone functioning and thereby influence prostate development. The objective of this study was to examine the impact on prostate expression of aromatase, 5α-R isozymes, and prostate cancer-related genes of exposure to low doses of BPA from perinatal period to adulthood. Vehicle or BPA (2.5 µg/kg b.w./day) was administered to gestating Wistar rats from gestational day 12 (GD12) to parturition and then to their male pups from postnatal day 1 (PND1) until euthanization on PND90. Their prostate glands were examined by qRT-PCR, Western blot, PCR array, and morphological study. mRNA and protein levels of 5α-R2 were significantly reduced and mRNA and protein levels of aromatase were significantly increased in BPA-treated animals, which also showed modifications of 8 out of the 84 key genes implicated in the development of prostate cancer. Because BPA interferes with genes involved in intraprostatic androgen and estrogen production and others implicated in prostate cancer, research is warranted into the prostate disease risk associated with chronic low-dose BPA exposure throughout life.

The isolated GTPase-activating-protein-related domain of neurofibromin-1 has a low conformational stability in solution.

Neurofibromin-1 (NF1) is a large, multidomain tumour suppressor encoded by the NF1 gene. The gene is mutated in neurofibromatosis type I, a disease characterized by malignant tumours of the nervous system and benign neurofibromas. The best-known activity of NF1 is the down-regulation of the mitogen-activated protein kinase pathway via its three-hundred-residue-long GTPase-activating protein (GAP) domain (the so-called GAP-related domain (NF1-GRD)). The NF1-GRD stimulates Ras GTPase activity in turning off signalling. Despite this activity, NF1-GRD has been demonstrated to bind to other different proteins, such as SPRED1 or MC1R. We have embarked on the biophysical and conformational characterization of NF1-GRD in solution by using several spectroscopic (namely fluorescence and circular dichroism (CD)) and biophysical techniques (namely size exclusion chromatography (SEC) and differential scanning calorimetry (DSC)). This biophysical characterization is crucial in deciphering NF1-GRD interactome and in finding biochemical features, modulating possible protein interactions. The native-like structure of NF1-GRD (as monitored by intrinsic fluorescence and far-UV CD) was strongly pH-dependent showing a pH-titration causing a substantial increase in its helicity. NF1-GRD had a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by intrinsic and ANS fluorescence, and CD. Chemical denaturations showed that NF1-GRD unfolded through an intermediate which has a substantial amount of solvent-exposed hydrophobic patches.

The isolated armadillo-repeat domain of Plakophilin 1 is a monomer in solution with a low conformational stability.

Plakophilin 1 (PKP1) is a member of the armadillo repeat family of proteins. It serves as a scaffold component of desmosomes, which are key structural components for cell-cell adhesion. We have embarked on the biophysical and conformational characterization of the ARM domain of PKP1 (ARM-PKP1) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, analytical ultracentrifugation (AUC), dynamic light scattering (DLS) and differential scanning calorimetry (DSC)). ARM-PKP1 was a monomer in solution at physiological pH, with a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by fluorescence and CD. The presence or absence of disulphide bridges did not affect its low stability. The protein unfolded through an intermediate which has lost native-like secondary structure. ARM-PKP1 acquired a native-like structure in a narrow pH range (between pH 6.0 and 8.0), indicating that its adherent properties might only work in a very narrow pH range.

A novel cysteine carbamoyl-switch is responsible for the inhibition of formamidase, a nitrilase superfamily member.

Formamidases (EC 3.5.1.49) and amidases (EC 3.5.1.4) are paralogous cysteine-dependent enzymes which catalyze the conversion of amide substrates to ammonia and the corresponding carboxylic acid. Both enzymes have been suggested as an alternative pathway for ammonia production during urea shortage. Urea was proved key in the transcriptional regulation of formamidases/amidases, connecting urea level to amide metabolism. In addition, different amidases have also been shown to be inhibited by urea, pointing to urea-regulation at the enzymatic level. Although amidases have been widely studied due to its biotechnological application in the hydrolysis of aliphatic amides, up to date, only two formamidases have been extensively characterized, belonging to Helicobacter pylori (HpyAmiF) and Bacillus cereus (BceAmiF). In this work, we report the first structure of an acyl-intermediate of BceAmiF. We also report the inhibition of BceAmiF by urea, together with mass spectrometry studies confirming the S-carbamoylation of BceAmiF after urea treatment. X-ray studies of urea-soaked BceAmiF crystals showed short- and long-range rearrangements affecting oligomerization interfaces. Since cysteine-based switches are known to occur in the regulation of different metabolic and signaling pathways, our results suggest a novel S-carbamoylation-switch for the regulation of BceAmiF. This finding could relate to previous observations of unexplained modifications in the catalytic cysteine of different nitrilase superfamily members and therefore extending this regulation mechanism to the whole nitrilase superfamily.

Engineering ancestral protein hyperstability.

Many experimental analyses and proposed scenarios support that ancient life was thermophilic. In congruence with this hypothesis, proteins encoded by reconstructed sequences corresponding to ancient phylogenetic nodes often display very high stability. Here, we show that such 'reconstructed ancestral hyperstability' can be further engineered on the basis of a straightforward approach that uses exclusively information afforded by the ancestral reconstruction process itself. Since evolution does not imply continuous progression, screening of the mutations between two evolutionarily related resurrected ancestral proteins may identify mutations that further stabilize the most stable one. To explore this approach, we have used a resurrected thioredoxin corresponding to the last common ancestor of the cyanobacterial, Deinococcus and Thermus groups (LPBCA thioredoxin), which has a denaturation temperature of ∼123°C. This high value is within the top 0.1% of the denaturation temperatures in the ProTherm database and, therefore, achieving further stabilization appears a priori as a challenging task. Nevertheless, experimental comparison with a resurrected thioredoxin corresponding to the last common ancestor of bacteria (denaturation temperature of ∼115°C) immediately identifies three mutations that increase the denaturation temperature of LPBCA thioredoxin to ∼128°C. Comparison between evolutionarily related resurrected ancestral proteins thus emerges as a simple approach to expand the capability of ancestral reconstruction to search sequence space for extreme protein properties of biotechnological interest. The fact that ancestral sequences for many phylogenetic nodes can be derived from a single alignment of modern sequences should contribute to the general applicability of this approach.

Human COA3 Is an Oligomeric Highly Flexible Protein in Solution.

The assembly of the protein complex of cytochrome c oxidase (COX), which participates in the mitochondrial respiratory chain, requires a large number of accessory proteins (the so-called assembly factors). Human COX assembly factor 3 (hCOA3), also known as MITRAC12 or coiled-coil domain-containing protein 56 (CCDC56), interacts with the first subunit protein of COX to form its catalytic core and promotes its assemblage with the other units. Therefore, hCOA3 is involved in COX biogenesis in humans and can be exploited as a drug target in patients with mitochondrial dysfunctions. However, to be considered a molecular target, its structure and conformational stability must first be elucidated. We have embarked on the description of such features by using spectroscopic and hydrodynamic techniques, in aqueous solution and in the presence of detergents, together with computational methods. Our results show that hCOA3 is an oligomeric protein, forming aggregates of different molecular masses in aqueous solution. Moreover, on the basis of fluorescence and circular dichroism results, the protein has (i) its unique tryptophan partially shielded from solvent and (ii) a relatively high percentage of secondary structure. However, this structure is highly flexible and does not involve hydrogen bonding. Experiments in the presence of detergents suggest a slightly higher content of nonrigid helical structure. Theoretical results, based on studies of the primary structure of the protein, further support the idea that hCOA3 is a disordered protein. We suggest that the flexibility of hCOA3 is crucial for its interaction with other proteins to favor mitochondrial protein translocation and assembly of proteins involved in the respiratory chain.

Structure of the c-Src-SH3 domain in complex with a proline-rich motif of NS5A protein from the hepatitis C virus.

The non-structural hepatitis C virus proteins NS5A and NS5B form a complex through interaction with the SH2 and SH3 domains of the non-receptor Src tyrosine kinase, which seems essential for viral replication. We have crystallized the complex between the SH3 domain of the c-Src tyrosine kinase and the C-terminal proline rich motif of the NS5A protein (A349PPIPPPRRKR359). Crystals obtained at neutral pH belong to the space group I41, with a single molecule of the SH3/NS5A complex at the asymmetric unit. The NS5A peptide is bound in a reverse orientation (class II) and the comparison of this structure with those of the high affinity synthetic peptides APP12 and VSL12 shows some important differences at the salt bridge that drives the peptide orientation. Further conformational changes in residues placed apart from the binding site also seem to play an important role in the binding orientation of this peptide. Our results show the interaction of the SH3 domain of the c-Src tyrosine kinase with a proline rich motif in the NS5A protein and point to their potential interaction in vivo.

Crystal structure of the first WW domain of human YAP2 isoform.

The WW domains are the smallest modular domains known. The study of the structural basis of their stability is important to understand their physiological role. These domains are intrinsically flexible, which makes them difficult to crystallize. The first WW domain of the human Yes tyrosine kinase Associated Protein (YAP) has been crystallized and its structure has been solved by X-ray diffraction at 1.6 Å resolution. Crystals belong to the orthorhombic space group P21212 with unit cell parameters a=42.67, b=43.10 and c=21.30. The addition of proline and other small-molecule additives improves drastically the quality of the crystals. The interactions that stabilize this minimal modular domain have been analysed. This crystal structure reveals that, besides the stabilization of the hydrophobic core of the protein by the aromatic cluster formed by Trp177-Phe189-Pro202, some salt-bridges interactions might affect the stability of the domain.

Enzymatic dynamic kinetic resolution of racemic N-formyl- and N-carbamoyl-amino acids using immobilized L-N-carbamoylase and N-succinyl-amino acid racemase.

Taking advantage of the catalytic promiscuity of L-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) and N-succinyl-amino acid racemase from Geobacillus kaustophilus CECT4264 (GkNSAAR), we have evaluated the production of different optically pure L-α-amino acids starting from different racemic N-formyl- and N-carbamoyl-amino acids using a dynamic kinetic resolution approach. The enzymes were immobilized on two different solid supports, resulting in improved stability of the enzymes in terms of thermostability and storage when compared to the enzymes in solution. The bienzymatic system retained up to 80% conversion efficiency after 20 weeks at 4 °C and up to 90% after 1 week at 45 °C. The immobilization process also resulted in a great enhancement of the activity of BsLcar toward N-formyl-tryptophan, showing for the first time that substrate specificity of L-carbamoylases can be influenced by this approach. The system was effective for the biosynthesis of natural and unnatural L-amino acids (enantiomeric excess (e.e.) >99.5%), such as L-methionine, L-alanine, L-tryptophan, L-homophenylalanine, L-aminobutyric acid, and L-norleucine, with a higher performance toward N-formyl-α-amino acid substrates. Biocatalyst reuse was studied, and after 10 reaction cycles, over 75% activity remained.

3D domain swapping in a chimeric c-Src SH3 domain takes place through two hinge loops.

In the Src Homology 3 domain (SH3) the RT and n-Src loops form a pocket that accounts for the specificity and affinity in binding of proline rich motifs (PRMs), while the distal and diverging turns play a key role in the folding of the protein. We have solved the structure of a chimeric mutant c-Src-SH3 domain where specific residues at the RT- and n-Src-loops have been replaced by those present in the corresponding Abl-SH3 domain. Crystals of the chimeric protein show a single molecule in the asymmetric unit, which appears in an unfolded-like structure that upon generation of the symmetry related molecules reveals the presence of a domain swapped dimer where both, RT- and n-Src loops, act as hinge loops. In contrast, the fold of the diverging type II ß-turn and the distal loop are well conserved. Our results are the first evidence for the presence of a structured diverging type II ß-turn in an unfolded-like intermediate of the c-Src-SH3 domain, which can be stabilized by interactions from the ß-strands of the same polypeptide chain or from a neighboring one. Futhermore, this crystallographic structure opens a unique opportunity to study the effect of the amino acid sequence of the hinge loops on the 3D domain swapping process of c-Src-SH3.

Crystallization of the HigBA2 toxin-antitoxin complex from Vibrio cholerae.

The genome of Vibrio cholerae encodes two higBA toxin-antitoxin (TA) modules that are activated by amino-acid starvation. Here, the TA complex of the second module, higBA2, as well as the C-terminal domain of the corresponding HigA2 antitoxin, have been purified and crystallized. The HigBA2 complex crystallized in two crystal forms. Crystals of form I belonged to space group P2(1)2(1)2, with unit-cell parameters a = 129.0, b = 119.8, c = 33.4 Å, and diffracted to 3.0 Šresolution. The asymmetric unit is likely to contain a single complex consisting of two toxin monomers and one antitoxin dimer. The second crystal form crystallized in space group P3(2)21, with unit-cell parameters a = 134.5, c = 55.4 Å. These crystals diffracted to 2.2 Šresolution and probably contain a complex with a different stoichiometry. Crystals of the C-terminal domain of HigA2 belonged to space group C2, with unit-cell parameters a = 115.4, b = 61.2, c = 73.8 Å, ß = 106.7°, and diffracted to 1.8 Šresolution.

Mutational and structural analysis of L-N-carbamoylase reveals new insights into a peptidase M20/M25/M40 family member.

N-Carbamoyl-L-amino acid amidohydrolases (L-carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures of N-carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the first L-carbamoylase structure belonging to Geobacillus stearothermophilus CECT43 (BsLcar), at a resolution of 2.7 Å. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family, and site-directed mutagenesis revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg(234) in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a "small-molecule binding domain," allowing further evolutionary considerations for this enzyme family.

Peptides as inhibitors of the first phosphorylation step of the Streptomyces coelicolor phosphoenolpyruvate: sugar phosphotransferase system.

The phosphotransferase system (PTS) controls the use of sugars in bacteria. The PTS is ubiquitous in bacteria, but it does not occur in plants and animals; it modulates catabolite repression, intermediate metabolism, gene expression, and chemotaxis. Its uniqueness and pleiotropic function make the PTS an attractive target for new antibacterial drugs. The PTS is constituted of two general proteins, namely, enzyme I (EI) and the histidine phosphocarrier (HPr), and various sugar-specific permeases. EI has two domains: the N-terminal domain (EIN), which binds to HPr, and the C-terminal domain (EIC), which contains the dimerization interface. In this work, we determined the binding affinities of peptides derived from EIN of Streptomyces coelicolor (EIN(sc)) against HPr of the same organism (HPr(sc)), by using nuclear magnetic resonance and isothermal titration calorimetry techniques. Furthermore, we measured the affinity of EIN(sc) for (i) a peptide derived from HPr(sc), containing the active-site histidine, and (ii) other peptides identified previously by phage display and combinatorial chemistry in Escherichia coli [Mukhija, S. L., et al (1998) Eur. J. Biochem. 254, 433-438; Mukhija, S., and Erni, B. (1997) Mol. Microbiol. 25, 1159-1166]. The affinities were in the range of ~10 µM, being slightly higher for the binding of EIN(sc) with peptides derived from HPr(sc), phage display, or combinatorial chemistry (K(D) ~ 5 µM). Because the affinity of intact EIN(sc) for the whole HPr(sc) is 12 µM, we suggest that the assayed peptides might be considered as good hit compounds for inhibiting the interaction between HPr(sc) and EIN(sc).

The plasticity of the ß-trefoil fold constitutes an evolutionary platform for protease inhibition.

Proteases carry out a number of crucial functions inside and outside the cell. To protect the cells against the potentially lethal activities of these enzymes, specific inhibitors are produced to tightly regulate the protease activity. Independent reports suggest that the Kunitz-soybean trypsin inhibitor (STI) family has the potential to inhibit proteases with different specificities. In this study, we use a combination of biophysical methods to define the structural basis of the interaction of papaya protease inhibitor (PPI) with serine proteases. We show that PPI is a multiple-headed inhibitor; a single PPI molecule can bind two trypsin units at the same time. Based on sequence and structural analysis, we hypothesize that the inherent plasticity of the ß-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition.

Stability and binding of the phosphorylated species of the N-terminal domain of enzyme I and the histidine phosphocarrier protein from the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system.

The phosphotransferase system (PTS) is involved in the use of carbon sources in bacteria. It is formed by two general proteins: enzyme I (EI) and the histidine phosphocarrier (HPr), and various sugar-specific permeases. EI is formed by two domains, with the N-terminal domain (EIN) being responsible for the binding to HPr. In low-G+C Gram-positive bacteria, HPr becomes phosphorylated not only by phosphoenolpyruvate (PEP) at the active-site histidine, but also by ATP at a serine. In this work, we have characterized: (i) the stability and binding affinities between the active-site-histidine phosphorylated species of HPr and the EIN from Streptomyces coelicolor; and (ii) the stability and binding affinities of the species involving the phosphorylation at the regulatory serine of HPr(sc). Our results show that the phosphorylated active-site species of both proteins are less stable than the unphosphorylated counterparts. Conversely, the Hpr-S47D, which mimics phosphorylation at the regulatory serine, is more stable than wild-type HPr(sc) due to helical N-capping effects, as suggested by the modeled structure of the protein. Binding among the phosphorylated and unphosphorylated species is always entropically driven, but the affinity and the enthalpy vary widely.

Structural insights into choline-O-sulfatase reveal the molecular determinants for ligand binding.

Choline-O-sulfatase (COSe; EC 3.1.6.6) is a member of the alkaline phosphatase (AP) superfamily, and its natural function is to hydrolyze choline-O-sulfate into choline and sulfate. Despite its natural function, the major interest in this enzyme resides in the landmark catalytic/substrate promiscuity of sulfatases, which has led to attention in the biotechnological field due to their potential in protein engineering. In this work, an in-depth structural analysis of wild-type Sinorhizobium (Ensifer) meliloti COSe (SmeCOSe) and its C54S active-site mutant is reported. The binding mode of this AP superfamily member to both products of the reaction (sulfate and choline) and to a substrate-like compound are shown for the first time. The structures further confirm the importance of the C-terminal extension of the enzyme in becoming part of the active site and participating in enzyme activity through dynamic intra-subunit and inter-subunit hydrogen bonds (Asn146A-Asp500B-Asn498B). These residues act as the `gatekeeper' responsible for the open/closed conformations of the enzyme, in addition to assisting in ligand binding through the rearrangement of Leu499 (with a movement of approximately 5 Å). Trp129 and His145 clamp the quaternary ammonium moiety of choline and also connect the catalytic cleft to the C-terminus of an adjacent protomer. The structural information reported here contrasts with the proposed role of conformational dynamics in promoting the enzymatic catalytic proficiency of an enzyme.

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.

Electrokinetic techniques applied to electrochemical DNA biosensors.

Electrokinetic techniques are contact-free methods currently used in many applications, where precise handling of biological entities, such as cells, bacteria or nucleic acids, is needed. These techniques are based on the effect of electric fields on molecules suspended in a fluid, and the corresponding induced motion, which can be tuned according to some known physical laws and observed behaviours. Increasing interest on the application of such strategies in order to improve the detection of DNA strands has appeared during the recent decades. Classical electrode-based DNA electrochemical biosensors with combined electrokinetic techniques present the advantage of being able to improve the working electrode's bioactive part during their fabrication and also the hybridization yield during the sensor detection phase. This can be achieved by selectively manipulating, driving and directing the molecules towards the electrodes increasing the speed and yield of the floating DNA strands attached to them. On the other hand, this technique can be also used in order to make biosensors reusable, or reconfigurable, by simply inverting its working principle and pulling DNA strands away from the electrodes. Finally, the combination of these techniques with nanostructures, such as nanopores or nanochannels, has recently boosted the appearance of new types of electrochemical sensors that exploit the time-varying position of DNA strands in order to continuously scan these molecules and to detect their properties. This review gives an insight into the main forces involved in DNA electrokinetics and discusses the state of the art and uses of these techniques in recent years.