The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein

The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a highmobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4- dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria

Structure and biophysical studies of mitochondrial Transcription Factor A in complex with DNA

[eng] The mitochondrial transcription factor A, TFAM, has a dual function in the organelle: it activates mitochondrial DNA transcription by binding to the HSP and LSP promoters, while in higher concentrations compacts the mtDNA. In this thesis the mechanism of complex formation between the mitochondrial transcription factor A (TFAM) and its cognate DNA binding sequences is analysed. TFAM is a DNA binding protein that belongs to the HMG- box family. Previous crystallographic works have shown that, by its two HMG-boxes and the intervening linker, TFAM binds to the DNA minor groove of mitochondrial DNA promoters imposing severe DNA distortions. These include two sharp 90-degree kinks that bend the cognate DNA into a U-turn. We here present the crystallographic structure of TFAM in complex with site Y, which together with site X are protein binding sites alternative to the promoter binding regions at the control region of mitochondrial DNA (mtDNA). The structure of the TFAM/site Y complex shows the two HMG-box domains (HMG-box1 and 2) organized in an “L”-shape fold that bends the contacted DNA by 90 degrees. Each HMG-box domain inserts a leucine, Leu 58 from HMG-box1 and Leu182 from HMG-box2, into a base-pair step from respective DNA contacted regions. The two DNA steps are separated by a DNA helix turn. Each insertion disrupts the DNA stacking and, together with additional interactions, facilitates the 90º DNA bending, the two bends resulting in the U-turn conformation. A structural comparison between available TFAM/DNA complexes shows that the linker between HMG-domains is instrumental for the protein to adapt to a conformation variability induced by the different DNA sequences. In addition, while all other crystal structures are unambiguous in the assigned DNA sequence, TFAM/site Y electron density maps indicated a surprising DNA disorder that suggested to trace the DNA in an alternative, not predicted, orientation. Thus in order to better characterize the binding mechanism of TFAM to the DNA and the role of the DNA properties in this process, we further studied the TFAM/site Y, TFAM/site X and TFAM/LSP complexes by molecular dynamics (MD) simulations, isothermal titration calorimetry (ITC) and electrophoretic mobility shift assays (EMSA). All these techniques showed a recurrent result, which is that TFAM has a clear preference in binding and bending site Y over site X and LSP. The three DNAs present intrinsic distortions that facilitate binding, which occurs by a mechanism in all cases endothermic and spontaneous and TFAM presents similar affinities to all of them. However, site Y is intrinsically more rigid but easier to distort into the shape found in the crystal, it competes better for TFAM binding, and the enthalpy and entropy of binding are much higher than for the other two sequences. These results suggest a specific binding and bending mechanism significantly dependent on the DNA sequence. Finally, by multi-angle laser light scattering (MALLS) and analytical ultracentrifugation the multimerization ability of TFAM detected by EMSA and size exclusion chromatography was analysed. The results indicate multimerization of the protein either alone or on the DNA in a cooperative manner at increased complex concentrations, which is consistent with the alternative function of TFAM as an mtDNA packaging protein. Altogether, our results suggest that the DNA sequence properties mediate TFAM binding, involving either specific interactions at the mtDNA control region, or non-specific contacts during mtDNA compaction. For this latter, the regulation of TFAM binding exerted by the DNA sequence might be combined with regulation of protein multimerization processes, all together determining mtDNA compaction, which is essential for cell life.[spa] Este trabajo de tesis doctoral está centrado en el análisis del mecanismo de unión del factor A de transcripción mitocondrial (TFAM) con sus secuencias de reconocimiento en la región control del ADN mitocondrial (mtADN). En la mitocondria TFAM está implicado en dos procesos fundamentales: la regulación de la trascripción del mtADN, cuando está unido a las secuencias promotoras del filamento ligero y pesado (HSP y LSP), y la compactación del mismo ADN cuando está presente en alta concentración. TFAM pertenece a la familia de los HMG-box y está constituida por dos dominios HMG conectados por un “linker” de 20 residuos. En este trabajo se presenta la estructura cristalográfica de TFAM en complejo con su sitio de reconocimiento alternativo a los promotores, site Y. Desde el análisis de la estructura se ha evidenciado que TFAM presenta el mismo plegamiento observado también cuando está en complejo con LSP, HSP, ADN no específico (nsADN) y su otro sitio de unión site X. Además en todos estos complejos el ADN resulta doblado 180° por medio de dos inserciones mediadas por LEU58 y 182, cada una responsable de un “kink” de 90°. La diferencia principal entre todas las estructuras se observa a nivel del linker que presenta una desviación en respuesta a las diferentes propiedades de los ADNs que contacta. Para caracterizar mejor el mecanismo de unión de TFAM con sus secuencias de reconocimiento en la región control del mtADN (LSP, site Y and site X), se realizaron diferentes análisis de tipo biofísico y bioquímico. La flexibilidad de estas secuencias se estudió primero por dinámica molecular. Estudios de “isothermal titration calorimetry” y “electrophoresis mobility shift assays” permitieron evidenciar que también si TFAM tiene el mismo mecanismo de unión y la misma afinidad por las tres secuencias, la cinética de formación de los complejos parece ser diferente. Para el análisis de la estequiometria de la unión de TFAM a los diferentes ADN fueron empleadas las técnicas de “multi angle laser light scattering” y “analytical ultracentrifugation”. Estos estudios evidenciaron la tendencia de TFAM de multimerizar, en presencia y ausencia de ADN, en respuesta a aumento de su concentración

The pCri system: A vector collection for recombinant protein expression and purification

A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5′end and multiple cloning sites at the 3′end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N-and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies.This study was supported in part by grants from European, Spanish, and Catalan agencies (FP7-HEALTH-2010-261460 “Gums&Joints”; FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; BFU2012-32862; CSD2006-00015; and 2014SGR9)Peer Reviewe

Biochemical and biophysical parameters influencing macromolecular crystallization and X-ray diffraction quality

Póster presentado en el Congrés Internacional de Biologia de Catalunya "Global Questions on Advanced Biology : an international congress on interdisciplinary frontiers in biology", organizado por la Societat Catalana de Biologia, del 9 al 12 de julio de 2012 en Barcelona (España)One way to investigate and properly understand the function of a protein and its interaction with partners is to know its three-dimensional structure. Macromolecular crystallography is a tool that provides the three-dimensional structure at atomic level of a protein that has been previously crystallized. A protein crystal consists of a very large number of repeating units where each individual unit is known as the unit cell, with no internal crystalline symmetry and which contains the crystallized sample. In general, crystallization starts with the formation of nuclei of protein molecules in supersaturated chemical conditions. There are several techniques available for bringing a pure protein solution gradually to a supersaturated state, such as batch, microbatch, vapour diffusion by hanging or sitting drops, and seeding. Once obtained a protein crystal, a potential bottleneck is to obtain a wellordered crystal that will diffract X-rays strongly. Sometimes co-crystallization of a protein with a substrate may help the crystal quality, because the protein is structurally stabilized by the ligand, the crystal packing is more regular and this improves the X-ray diffraction pattern. In this case, a protein in complex with different substrates may result in different crystals that yield X-ray diffraction patterns of variable quality. We will present an example of crystal quality improvement of a protein/DNA complex in which we changed the design of the oligonucleotides harboring the DNA binding site, including the sequence, the length and the type of ends, blunt or cohesive. These changes modified the crystallization, as assessed by the macroscopic aspect of the crystals and the corresponding X-ray diffraction qualityPeer Reviewe

Multiple architectures and mechanisms of latency in metallopeptidase zymogens

Metallopeptidases cleave polypeptides bound in the active-site cleft of catalytic domains through a general base/acid mechanism. This involves a solvent molecule bound to a catalytic zinc and general regulation of the mechanism through zymogen-based latency. Sixty reported structures from 11 metallopeptidase families reveal that prosegments, mostly N-terminal of the catalytic domain, block the cleft regardless of their size. Prosegments may be peptides (5−14 residues), which are only structured within the zymogens, or large moieties (<227 residues) of one or two folded domains. While some prosegments globally shield the catalytic domain through a few contacts, others specifically run across the cleft in the same or opposite direction as a substrate, making numerous interactions. Some prosegments block the zinc by replacing the solvent with particular side chains, while others use terminal α-amino or carboxylate groups. Overall, metallopeptidase zymogens employ disparate mechanisms that diverge even within families, which supports that latency is less conserved than catalysisThis study was funded in part by grants from Spanish and Catalan public agencies (BFU2015-64487-R, MDM-2014-0435, and 2017SGR00003). T.G. acknowledges a “Juan de la Cierva” research contract (JCI-2012-13573) from the Spanish Ministry of Economy and Competitiveness. The Structural Biology Unit of IBMB is a“María de Maeztu”Unit of Excellence of the Spanish Ministry of Economy, Industry and Competitiveness.Peer reviewe

DNA-binding proteins analysed by SAXS

Small Angle X-ray Scattering (SAXS) of macromolecules in solution is a technique that allows lowresolution structural analysis of proteins and their complexes, including protein-DNA complexes. The number of SAXS studies on macromolecular particles has increased significantly in the last years, due to improvement of algorithms and beamtime availability in synchrotron facilities. One advantage of this technique is that is no limited by factors like particle size or flexibility; on the contrary, it is possible to assess these or other features, like the coexistence of particles of different sizes in the same preparation. The experimental SAXS curve allows to fit against it a theoretical curve calculated using a crystallographic atomic model or an electron microscopy shape, which might be modified to optimise the curve fitting. Fitting optimisation can be attempted by exploring the conformational space of the particle on the basis of the available model, using techniques like simple manual displacement of domains or more sophisticated ones like normal mode analysis, and select those models that altogether yield a curve that fits best; such a methodology can be useful in determining the degree of flexibility of domains or interdomain segments. Macromolecular complexes are reconstructed using static threedimensional models of the complexes or by docking the individual components; by playing with the proportion of non-interacting vs interacting partners, is possible to establish the dynamics of the complex. Furthermore, by introducing the conformational-space analysis algorithms within macromolecular complexes is possible to explore the conformational changes induced by ligands, substrates or interacting partners. We are going to present a SAXS analysis of three phylogenetically-related proteins whose structure is based on homology models, and in one case involving a protein/DNA complex. The strategies applied will be analysed and the results discussedPeer Reviewe

Genetic Control of Immune Response in Carriers of the 8.1 Ancestral Haplotype: Correlation with Levels of IgG Subclasses: Its Relevance in the Pathogenesis of Autoimmune Diseases

Ancestral haplotype (AH) 8.1(HLA-A1, Cw7, B8, TNFAB*a2b3, TNFN*S, C2*C, Bf*s, C4A*Q0, C4B*1, DRB1*0301, DRB3*0101, DQA1*0501, DQB1*0201) seems to be associated with susceptibility to autoimmune diseases. Different mechanisms are probably involved in increasing autoimmunity, such as unbalanced cytokine production and the lack of C4A protein. So AH 8.1 modifies immune response in many ways. In this study we demonstrate that IgG2 serum levels were significantly lower in 8.1 AH carriers than in 8.1 AH non-carriers. On the contrary, as regards IgG1, IgG3, IgG4 serum levels, no significant differences were observed between the two groups. In AH 8.1 carriers low IgG2 levels might take to slower clearance of the infectious agent and hence to a lasting presence of it. The persistence of infectious antigens could determine an increased production of autoantibodies with a higher risk of cross-reactions

Protein flexibility and synergy of HMG domains underlie U-turn bending of DNA by TFAM in solution

Human mitochondrial transcription factor A (TFAM) distorts DNA into a U-turn, as shown by crystallographic studies. The relevance of this U-turn is associated with transcription initiation at the mitochondrial light strand promoter (LSP). However, it has not been yet discerned whether a tight U-turn or an alternative conformation, such as a V-shape, is formed in solution. Here, single-molecule FRET experiments on freely diffusing TFAM/LSP complexes containing different DNA lengths show that a DNA U-turn is induced by progressive and cooperative binding of the two TFAM HMG-box domains and the linker between them. SAXS studies further show compaction of the protein upon complex formation. Finally, molecular dynamics simulations reveal that TFAM/LSP complexes are dynamic entities, and the HMG boxes induce the U-turn against the tendency of the DNA to adopt a straighter conformation. This tension is resolved by reversible unfolding of the linker, which is a singular mechanism that allows a flexible protein to stabilize a tight bending of DNA.This work was supported by the Ministry of Economy and Competitiveness (MINECO) (BFU2012-33516 and BFU2015-70645-R to M.S., and BIO2012-32868 and BFU2014-61670-EXP to M.O.); Generalitat de Catalunya (SGR2009-1366 and 2014-SGR-997 to M.S., and SGR2009- 1348, 2014 SGR-134 to M.O.); the Instituto Nacional de Bioinforma´tica; the European Union (FP7-HEALTH-2010-261460, FP7-PEOPLE-2011- 290246, and FP7-HEALTH-2012-306029-2 to M.S., and H2020-EINFRA-2015-1-675728 and H2020-EINFRA-2015-676556 to M.O.); and the European Research Council (ERC-2011-ADG_20110209-291433 to M.O.). A.R.-C. was awarded with a ‘‘Junta para la Ampliacio´n de Estudios’’ (Programa JAE) fellowship from Consejo Superior de Investigaciones Cientı´ficas (CSIC). The Structural Biology Unit at IBMB-CSIC is a ‘‘Maria de Maeztu’’ Unit of Excellence awarded by the Ministry of Economy and Competitiveness (MINECO) under MDM-2014-0435. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from the Ministry of Economy and Competitiveness (MINECO). The CBS is a member of the French Infrastructure for Integrated Structural Biology (FRISBI), a national infrastructure supported by the French National Research Agency (ANR-10-INBS-05).Peer reviewe