Albert Canals

The anticancer agent ellipticine unwinds DNA by intercalative binding in an orientation parallel to base pairs.

Ellipticine is a natural plant product that has been found to be a powerful anticancer drug. Although still unclear, its mechanism of action is considered to be mainly based on DNA intercalation and/or the inhibition of topoisomerase II. Many experimental data suggest an intercalation based on stacking interactions along the major base-pair axis, but alternative binding modes have been proposed, in particular for ellipticine derivatives. The 1.5 A resolution structure of ellipticine complexed to a 6 bp oligonucleotide unveils its mode of binding and enables a detailed analysis of the distorting effects of the drug on the DNA.

The structure of a transcription activation subcomplex reveals how σ70 is recruited to PhoB promoters

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.

The Structure of an Engineered Domain-Swapped Ribonuclease Dimer and Its Implications for the Evolution of Proteins toward Oligomerization

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.

PhoB transcriptional activator binds hierarchically to pho box promoters

Abstract 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.

Site-directed mutagenesis of mouse glutathione transferase P1-1 unlocks masked cooperativity, introduces a novel mechanism for 'ping pong' kinetic behaviour, and provides further structural evidence for participation of a water molecule in proton abstraction from glutathione

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.

The Structure of RNA-Free Rho Termination Factor Indicates a Dynamic Mechanism of Transcript Capture

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 A 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.

The structure of the complex between α-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism.

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 &agr;‐tubulin–&bgr;‐tubulin dissociation. We used electron microscopy and image processing to determine the three‐dimensional structure of the human TBCE, TBCB and &agr;‐tubulin (&agr;EB) complex, which is formed upon &agr;‐tubulin–&bgr;‐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 &agr;EB complex. We found that heterodimer dissociation is an energy‐independent process that takes place through a disruption of the &agr;‐tubulin–&bgr;‐tubulin interface that is caused by a steric interaction between &bgr;‐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 &agr;EB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating &agr;‐tubulin degradation.

The structure of the complex between a-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism

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 &agr;‐tubulin–&bgr;‐tubulin dissociation. We used electron microscopy and image processing to determine the three‐dimensional structure of the human TBCE, TBCB and &agr;‐tubulin (&agr;EB) complex, which is formed upon &agr;‐tubulin–&bgr;‐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 &agr;EB complex. We found that heterodimer dissociation is an energy‐independent process that takes place through a disruption of the &agr;‐tubulin–&bgr;‐tubulin interface that is caused by a steric interaction between &bgr;‐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 &agr;EB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating &agr;‐tubulin degradation.

σ70 and PhoB activator: getting a better grip.

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 σ4 domain of the RNA polymerase σ70 subunit supports the notion that a stronger grip on the promoter-factor complex results in an enhanced RNAP architecture.

Cloning, expression, purification and crystallization of the Rho transcription termination factor from Thermotoga maritima

Rho is an essential ATP-dependent 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 A-diffracting crystals.