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Adenosine deaminase acting on transfer RNA (ADAT) is an essential eukaryotic enzyme that catalyzes the deamination of adenosine to inosine at the first position of tRNA anticodons. Mammalian ADATs modify eight different tRNAs, having increased their substrate range from a bacterial ancestor that likely deaminated exclusively tRNAArg. Here we investigate the recognition mechanisms of tRNAArg and tRNAAla by human ADAT to shed light on the process of substrate expansion that took place during the evolution of the enzyme. We show that tRNA recognition by human ADAT does not depend on conserved identity elements, but on the overall structural features of tRNA. We find that ancestral-like interactions are conserved for tRNAArg, while eukaryote-specific substrates use alternative mechanisms. These recognition studies show that human ADAT can be inhibited by tRNA fragments in vitro, including naturally occurring fragments involved in important regulatory pathways.

Variolin B is a rare marine alkaloid that showed promising anti-cancer activity soon after its isolation. It acts as a cyclin-dependent kinase inhibitor, although the precise mechanism through which it exerts the cytotoxic effects is still unknown. The crystal structure of a variolin B bound to a DNA forming a pseudo-Holliday junction shows that this compound can also contribute, through intercalative binding, to either the formation or stabilization of multi-stranded DNA forms., This work was supported by the Spanish Ministry of Science and Innovation (grants BFU2008-02372/BMC, CONSOLIDER CSD 2006-23, BFU2011-22588, BFU2014-53550-P, CTQ2012-30930 and Unidad de Excelencia Maria de Maeztu MDM-2014-0435), the Generalitat de Catalunya (grants SGR2009-1309 and 2009SGR1024). Synchrotron data collection was supported by the ESRF and the EC.

Transcription factors modulate gene expression by distinct, barely understood mechanisms. The crystal structure of a bacterial transcription subcomplex comprising the effector domain of factor PhoB, its target DNA and the σ 4 domain of the RNA polymerase σ 70 subunit supports the notion that a stronger grip on the promoter-factor complex results in an enhanced RNAP architecture. © 2012 Landes Bioscience., This work was supported by the Spanish Ministry of Science and Innovation (grants BFU2008-02372/BMC, CONSOLIDER CSD 2006-23 and BFU2011-22588), the Generalitat de Catalunya (grant SGR2009-1309) and the European Commission (FP7 Cooperation Project SILVER—GA No. 260644).

PhoB is a two-component response regulator that activates transcription by interacting with the σ70 subunit of the E. coli RNA polymerase in promoters in which the −35 σ70-recognition element is replaced by the pho box. The crystal structure of a transcription initiation subcomplex that includes the σ4 domain of σ70 fused with the RNA polymerase β subunit flap tip helix, the PhoB effector domain and the pho box DNA reveals how σ4 recognizes the upstream pho box repeat. As with the −35 element, σ4 achieves this recognition through the N-terminal portion of its DNA recognition helix, but contact with the DNA major groove is less extensive. Unexpectedly, the same recognition helix contacts the transactivation loop and helices α2 and α3 of PhoB. This result shows a simple and elegant mechanism for polymerase recruitment to pho box promoters in which the lost −35 element contacts are compensated by new ones with the activator. In addition, σ4 is reoriented, thereby suggesting a remodelling mechanism for transcription initiation.

ubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α-tubulin-β-tubulin dissociation. We used electron microscopy and image processing to determine the three-dimensional structure of the human TBCE, TBCB and α-tubulin (αEB) complex, which is formed upon α-tubulin-β-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the aEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the α-tubulin-β-tubulin interface that is caused by a steric interaction between b-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucine-rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the aEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating α-tubulin degradation. © 2015. Published by The Company of Biologists Ltd., This work was supported by the Spanish Ministry of Science and Innovation [grant numbers CONSOLIDER CSD 2006‐23 to M.C., J.C.Z. and J.M.V., BFU2011‐22588 to M.C., BFU2011‐25090 to J.M.B., BFU2013‐44202 to J.M.V., and BFU2010‐18948 to J.C.Z.]; the Madrid Regional Government [grant number S2013/MIT‐2807 to J.M.V.]; the Generalitat de Catalunya [grant number SGR2009‐1309 to M.C.]; the Instituto de Investigación Marqués de Valdecilla (IDIVAL); the Universidad de Cantabria [grant number 02.VP01.64005 to J.C.Z.]; and the European Commission FP7 Cooperation Project SILVER ‐ GA [grant number 260644 to M.C.]

Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α-tubulin-β-tubulin dissociation. We used electron microscopy and image processing to determine the three-dimensional structure of the human TBCE, TBCB and α-tubulin (αEB) complex, which is formed upon α-tubulin-β-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the αEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the α-tubulin-β-tubulin interface that is caused by a steric interaction between β-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucine-rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the αEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating α-tubulin degradation.

Background: Domain swapping has been proposed as a mechanism that explains the evolution from monomeric to oligomeric proteins. Bovine and human pancreatic ribonucleases are monomers with no biological properties other than their RNA cleavage ability. In contrast, the closely related bovine seminal ribonuclease is a natural domain-swapped dimer that has special biological properties, such as cytotoxicity to tumour cells. Several recombinant ribonuclease variants are domain-swapped dimers, but a structure of this kind has not yet been reported for the human enzyme. Results: The crystal structure at 2 A resolution of an engineered ribonuclease variant called PM8 reveals a new kind of domain-swapped dimer, based on the change of N-terminal domains between the two subunits. The swapping is fastened at both hinge peptides by the newly introduced Gln101, involved in two intermolecular hydrogen bonds and in a stacking interaction between residues of different chains. Two antiparallel salt bridges and water-mediated hydrogen bonds complete a new interface between subunits, while the hinge loop becomes organized in a 3 10 helix structure. Conclusions: Proteins capable of domain swapping may quickly evolve toward an oligomeric form. As shown in the present structure, a single residue substitution reinforces the quaternary structure by forming an open interface. An evolutionary advantage derived from the new oligomeric state will fix the mutation and favour others, leading to a more extended complementary dimerization surface, until domain swapping is no longer necessary for dimer formation. The newly engineered swapped dimer reported here follows this hypothetical pathway for the rapid evolution of proteins.

Human pancreatic ribonuclease, the homolog of bovine pancreatic ribonuclease, has a significant therapeutic potential. Its study has been hindered by the difficulty of obtaining the enzyme in a pure and homogeneous form, either from human source or using heterologous expression. Engineering of different variants of human pancreatic ribonuclease has allowed us to study and overcome some problems encountered during its heterologous production in an Escherichia coli system and its purification from inclusion bodies. The 5'-end region of the mRNA that encodes the enzyme is critical for obtaining high expression levels. The results also suggest the importance of the proline 50 residue in the recovery yields of human pancreatic ribonuclease. All the variants produced are pure and homogeneous. Their homogeneity has been demonstrated by cation-exchange and reversed-phase chromatography and by mass spectrometry analysis. Moreover, enhancement of human pancreatic ribonuclease thermal stability is observed when residues R4, K6, Q9, D16, and S17 are changed to the corresponding residues of bovine seminal ribonuclease.

et al., In the search for structural models of integral-membrane metallopeptidases (MPs), we discovered three related proteins from thermophilic prokaryotes, which we grouped into a novel family called minigluzincins. We determined the crystal structures of the zymogens of two of these (Pyrococcus abyssi proabylysin and Methanocaldococcus jannaschii projannalysin) which are soluble and, with ~100 residues, constitute the shortest structurally characterized MPs to date. Despite relevant sequence and structural similarity, the structures revealed two unique mechanisms of latency maintenance through the C-terminal segments hitherto unseen in MPs: intra-molecular, through an extended tail, in proabylysin, and crosswise inter-molecular, through a helix swap, in projannalysin. In addition, structural and sequence comparisons, as well as phylogenetic and bioinformatics analyses, revealed large similarity with MPs of the gluzincin tribe such as thermolysin, leukotriene A4 hydrolase relatives, and cowrins. Interestingly, gluzincins mostly have a glutamate as third characteristic zinc ligand while minigluzincins have a histidine. Sequence and structure similarity further allowed us to ascertain that minigluzincins are very similar to the catalytic domains of integral-membrane MPs of MEROPS database families M48 and M56, such as FACE1, HtpX, Oma1, and BlaR1/MecR1, which are provided with trans-membrane helices flanking or inserted into a minigluzincin-like catalytic domain. In a time where structural biochemistry of integral-membrane proteins in general still faces formidable challenges, the minigluzincin soluble minimal scaffold may contribute to our understanding of the working mechanisms of these membrane MPs and to the design of novel inhibitors through structure-aided rational drug design approaches., This work was supported in part by European, Spanish, and Catalan Agency Grants FP7-HEALTH-F3-2009-223101 “AntiPathoGN,” FP7-HEALTH-2010-261460 “Gums&Joints,” FP7-PEOPLE-2011-ITN-290246 “RAPID,” FP7-HEALTH-2012-306029-2 “TRIGGER,” BFU2010-19310, BFU2012-32862, CSD2006-00015, Fundació “La Marató de TV3” Grants 2009-100732 and 2009SGR1036, postdoctoral JAE contract from Consejo Superior de Investigaciones Científicas (co-funded by FSE), and two FPI Ph.D. fellowships from the Spanish Ministry for Science and Technology, currently part of the Ministry of Economy and Competitiveness.

The PhoR-PhoB phosphorelay is a bacterial two-component system that activates the transcription of several genes involved in phosphate uptake and assimilation. The response begins with the autophosphorylation of the sensor kinase PhoR, which activates the response regulator PhoB. Upon binding to the pho box DNA sequence, PhoB recruits the RNA polymerase and thereby activates the transcription of specific genes. To unveil hitherto unknown molecular mechanisms along the activation pathway, we report biochemical data characterizing the PhoB binding to promoters containing multiple pho boxes and describe the crystal structure of two PhoB DNA-binding domains bound in tandem to a 26-mer DNA. Copyright © 2011-2012 by Walter de Gruyter., This work was supported by the Spanish Ministry of Science and Innovation (grants BFU2008-02372/BMC, CONSOLIDER CSD 2006-23 and BFU2011-22588), the Generalitat de Catalunya (grant SGR2009-1309) and the European Commission (FP7 Cooperation Project SILVER-GA No. 260644).

Mouse liver glutathione transferase P1-1 has three cysteine residues at positions 14, 47 and 169. We have constructed the single, double and triple cysteine to alanine mutants to define the behaviour of all three thiols. We confirm that C47 is the 'fast' thiol (pK 7.4), and define C169 as the alkaline reactive residue with a pKa of 8.6. Only a small proportion of C14 is reactive with 5,5′-dithiobis-(2-nitrobenoic acid) (DTNB) at pH 9 in the C47A/C169A double mutant. The native enzyme and the C169A mutant exhibited Michaelis-Menten kinetics, but all other thiol to alanine mutants exhibited sigmoidal kinetics to varying degrees. The C169A mutant exhibited 'ping pong' kinetics, consistent with a mechanism whereby liberation of a proton from a reduced enzyme-glutathione (GSH) complex to form an enzyme-GS- (unprotonated) complex is essentially irreversible. Intriguingly, similar behaviour has recently been reported for a mutant of the yeast prion Ure2p. This cooperative behaviour is 'mirrored' in the crystal structure of the C47A mutant, which binds the p-nitrobenzyl moiety of p-nitrobenzyglutathione in distinct orientations in the two crystallographic subunits. The asymmetry seen in this structure for product binding is associated with absence of a water molecule W0 in the standard wild-type conformation of product binding that is clearly identifiable in the new structure, which may represent a structural model for binding of incoming GSH prior to displacement of W0. Elimination of W0 as a hydroxonium ion may be the mechanism for the initial proton extrusion from the active site. © 2010 FEBS., This study was supported by the Ministerio de Ciencia e Innovación (grants BFU2008-02372 ⁄BMC, PET 2007_0319_03, BFU2005-24122-E and CSD2006-23 to M.C.), the Generalitat de Catalunya (grant 2009 SGR 1309 to M.C.), the European Union (LSHG-CT-2006-031220) and the Health Research Board of Ireland (HRB 144 ⁄ 96).

The Rho factor is a ring-shaped ATP-dependent helicase that mediates transcription termination in most prokaryotic cells by disengaging the transcription elongation complex formed by the RNA polymerase, DNA, and the nascent RNA transcript. The crystal structures of key intermediates along the kinetic pathway of RNA binding to Rho unveiled an unprecedented mode of helicase loading and provided a model for the ATP turnover coupled to coordinated strand movement. Here we report the structure of the early RNA-free state of Rho, which had eluded crystallization for many years but now completes the series. The structure allows the characterization of the apo-form Rho from Thermotoga maritima to 2.3 Å resolution, reveals an RNA-recruiting site that becomes hidden after occupancy of the adjacent specific primary RNA-binding site, and suggests an enriched model for mRNA capture that is consistent with previous data. © 2010 Elsevier Ltd., This work was supported by the “Ministerio de Educación y Ciencia de España” (Grants BFU2005-24123-E and BFU2008-02372/BMC to M.C. and Grants BIO2006-14139 and BIO2009-10576 to I.U.), by the “Generalitat de Catalunya” (Grant 2009SGR-1309), by the EU (Spine2-Complexes LSHG-2006-031220 and 3DRepertoire LSHG-CT-2005-512028 projects), and by the “Fundació La Marató de TV3” (Grant 052810). Synchrotron data collection was supported by the European Synchrotron Radiation Facility. A.C. acknowledges a postdoctoral fellowship from the “Ministerio de Educación y Ciencia de España”.

Rho is an essential ATP-dependant homohexameric helicase that is found in the vast majority of bacterial species. It is responsible for transcription termination at factor-dependent terminators. Rho binds to a specific region of the newly-synthesised mRNA and translocates along the chain until it reaches and disassembles the transcription complex. Basically, two crystallographic structures of Rho hexamer from Escherichia coli have been reported: an open ring with RNA (or ssDNA) bound to the RNA-binding domain, and a closed ring with the RNA bound to both the RNA-binding domain and the ATP-ase domain. The structure of the protein free from RNA is still unknown, but thermophilic bacteria enable an alternative approach to its characterization as their proteins often crystallize more easily than those of their mesophilic homologs. We report here the heterologous expression in E. coli of full-length Rho from the thermophile Thermotoga maritima, a simple protocol for the purification of its hexameric nucleic acid-free form, and the obtainment of 2.4 Å-diffracting crystals. © 2009 Elsevier Inc. All rights reserved., This work was supported by the Ministerio de Educación y Ciencia de España (Grants BFU2005-06758/BMC and BFU2008-02372/BMC), by the Generalitat de Catalunya (Grant 2005SGR-0028) and the Fundació La Marató de TV3 (Grant 052810)

In this paper, we review recent DNA-binding agents that are expected to influence the field of DNA-targeting. We restrict ourselves to binders for which the three-dimensional structure in complex with DNA or RNA has been determined by X-ray crystallography or NMR. Furthermore, we primarily focus on unprecedented ways of targeting peculiar DNA structures, such as junctions, quadruplexes, and duplex DNAs different from the B-form. Classical binding modes of small molecular weight compounds to DNA, i.e. groove binding, intercalation and covalent addition are discussed in those cases where the structures represent a novelty. In addition, we review 3D structures of triple-stranded DNA, of the so-called Peptide Nucleic Acids (PNAs), which are oligonucleotide bases linked by a polypeptide backbone, and of aptamers, which are DNA or RNA receptors that are designed combinatorially. A discussion on perspectives in the field of DNA-targeting and on sequence recognition is also provided. © 2009 The Royal Society of Chemistry.