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

Structure of eIF4E in Complex with an eIF4G Peptide Supports a Universal Bipartite Binding Mode for Protein Translation

The association-dissociation of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) with eIF4G is a key control step in eukaryotic translation. The paradigm on the eIF4E-eIF4G interaction states that eIF4G binds to the dorsal surface of eIF4E through a single canonical alpha-helical motif, while metazoan eIF4E-binding proteins (m4E-BPs) advantageously compete against eIF4G via bimodal interactions involving this canonical motif and a second noncanonical motif of the eIF4E surface. Metazoan eIF4Gs share this extended binding interface with m4E-BPs, with significant implications on the understanding of translation regulation and the design of therapeutic molecules. Here we show the high-resolution structure of melon (Cucumis melo) eIF4E in complex with a melon eIF4G peptide and propose the first eIF4E-eIF4G structural model for plants. Our structural data together with functional analyses demonstrate that plant eIF4G binds to eIF4E through both the canonical and noncanonical motifs, similarly to metazoan eIF4E-eIF4G complexes. As in the case of metazoan eIF4E-eIF4G, this may have very important practical implications, as plant eIF4E-eIF4G is also involved in a significant number of plant diseases. In light of our results, a universal eukaryotic bipartite mode of binding to eIF4E is proposed.Work in Murcia was financially supported by grants AGL2015-65838 and PCIN-2013-043 (MINECO, Spain-FEDER). Work in Barcelona was supported by grants BIO2014-54588-P (MINECO, Spain-FEDER) and Maria de Maeztu Unit of Excellence MDM-2014-0435. X-ray data were collected at ALBA-CELLS (beamline XALOC), Cerdanyola del Valles, Barcelona, Spain, with the collaboration of ALBA staff and at ESRF (Grenoble, France). Financial support was also provided by ALBA and ESRF.Peer reviewe

Structural analysis of a RNA viral element and its translation initiation factor partner, both controlling cap-independent translation of viral RNAs

Póster presentado en el 35th Annual Meeting American Society for Virology, celebrado del 11 al 15 de julio de 2015 en London, Ontario (Canadá)In animal viruses, internal ribosome entry sites at the 5¿-unstranslated regions (UTRs) of viral RNAs are the most frequently found translation control elements. In contrast to animal viruses, plant positive-strand RNA viruses, in the absence of a 5¿cap, have evolved 3¿ cap-independent translational enhancers (3¿-CITEs) in their 3¿-UTRs. It has been reported that these 3¿-CITEs require and directly bind eukaryotic translation initiation factors (eIFs) for their function. We have shown that capindependent translation of Melon necrotic spot virus (MNSV) RNAs is controlled by a 3¿-CITE in cis (Truniger et al., 2008). Remarkably, MNSV 3¿-CITEs are diverse, including at least M¿5TE, M264TE and CXTE (Truniger et al., 2008; Miras et al., 2014). Genetic evidence indicates that the eIF4E subunit of melon eIF4F is necessary for cap-independent translation of MNSV RNAs harboring M¿5TE (Nieto et al., 2006); in contrast, M264TE and CXTE are both eIF4E independent, conferring in cis translational competence to viral RNAs in the absence of this factor (Miras et al., 2014). In this study, we have performed structural and functional analysis of M¿5TE. Thus, we defined the minimal size of the 3¿-CITE in ¿in vivo¿ translation assays to a sequence of 45 nucleotides. Its secondary structure in solution was studied by Selective 2¿-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) and compared with those of the other two types of 3¿-CITEs. On the other hand, we have expressed, crystalized and determined by X-ray crystallography the 3D structure of melon eIF4E and a truncated version of eIF4F complex (eIF4E-eIF4G1003-1092). Binding to eIF4G1003-1092 significantly alters eIF4E conformation. UV-crosslinking assays indicated the formation revealed eIF4F-binding sites on a bulge of M¿5TE.Mutational analyses of Ma5TE revealed residues involved in (inside the bulge associated with loss of) binding to eIF4F. Further structural and functional analyses of M¿5TE:eIF4F are in progress. References: Miras, M., Sempere, R.N., Kraft, J., Miller, A.W., Aranda, M.A. and Truniger, V. 2014. New Phytologist 202: 233-246. Nieto, C., Rodriguez-Moreno, L., Rodriguez-Hernández, A.M., Aranda., M.A. and Truniger, V. 2011. The Plant Journal, 66, 492-501. Truniger, V., Nieto, C., González-Ibeas, D. and Aranda, M.A. 2008. The Plant Journal 56:716-727Peer 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