Étude de complexes à forte diffusion anomale pour la détermination rapide de la structure de protéines par la méthode MAD

For the de novo structure resolution of proteins by crystallography, it is necessary to calculate the phases of the structure factors from the diffracted X-ray intensities. For this purpose, the SAD and MAD methods take advantage of the anomalous scattering properties of atoms inside the protein crystals. The introduction of the anomalous scatterers into the crystals is one of the key steps of protein structure resolution.We have investigated a class of eight gadolinium complexes used to introduce the anomalous scatterers into the protein crystals. Gadolinium exhibits high anomalous scattering with a laboratory X-ray generator as well as in its LIII absorption edge.A crystallographic study carried out with the class of complexes and eight different proteins allowed to demonstrate the great potential of the complexes for the preparation of derivatives with high phasing power. Indeed, for a great number of derivatives, the calculated experimental phases have led to experimental electron-density maps of excellent quality, allowing the easy construction of the protein model.The refinement of the structure of the complexes bound to the proteins allowed to gain understanding of the interaction between the different complexes and the proteins.The utilization of the complexes allowed the structure resolution of four new proteins.Besides the crystallographic studies we attempted to detect the binding of a complex to a protein with different physico-chemical methods taking into consideration the weak binding constant that characterizes the interaction.Pour résoudre de novo la structure de protéines par cristallographie, il est nécessaire de calculer les phases des facteurs de structure à partir des intensités des rayons X diffractés. Pour ce but, les méthodes SAD et MAD se servent de la diffusion anomale d'atomes présents dans le cristal de la protéine. L'introduction de ces diffuseurs anomaux dans les cristaux est une des étapes clés de la résolution de structure des protéines.Nous avons étudié une classe de huit complexes de gadolinium servant à insérer les diffuseurs anomaux dans les cristaux de protéine. Le gadolinium présente une forte diffusion anomale et avec le rayonnement X d'un générateur de laboratoire et dans son seuil d'absorption LIII.Une étude cristallographique menée avec cette classe de complexes et avec huit protéines différentes a permis de démontrer le potentiel élevé des complexes pour la préparation de dérivés à fort pouvoir de phasage. En effet, pour un grand nombre des dérivés testés, les phases expérimentales calculées ont mené à des cartes de densité expérimentale d'excellente qualité, permettant la construction aisée du modèle de la protéine.L'affinement de la structure des complexes liés à la protéine a permis de comprendre l'interaction des différents complexes avec les protéines.L'utilisation des complexes a permis de résoudre la structure de quatre nouvelles protéines.Nous avons également étudié des méthodes physico-chimiques alternatives à la cristallographie dans le dessein de détecter la fixation d'un complexe sur une protéine en tenant compte de la particularité de l'interaction qui est caractérisée par une constante de d'association faible

Structural and mechanistic insight into DNA unwinding by Deinococcus radiodurans UvrD.

DNA helicases are responsible for unwinding the duplex DNA, a key step in many biological processes. UvrD is a DNA helicase involved in several DNA repair pathways. We report here crystal structures of Deinococcus radiodurans UvrD (drUvrD) in complex with DNA in different nucleotide-free and bound states. These structures provide us with three distinct snapshots of drUvrD in action and for the first time trap a DNA helicase undergoing a large-scale spiral movement around duplexed DNA. Our structural data also improve our understanding of the molecular mechanisms that regulate DNA unwinding by Superfamily 1A (SF1A) helicases. Our biochemical data reveal that drUvrD is a DNA-stimulated ATPase, can translocate along ssDNA in the 3'-5' direction and shows ATP-dependent 3'-5', and surprisingly also, 5'-3' helicase activity. Interestingly, we find that these translocase and helicase activities of drUvrD are modulated by the ssDNA binding protein. Analysis of drUvrD mutants indicate that the conserved β-hairpin structure of drUvrD that functions as a separation pin is critical for both drUvrD's 3'-5' and 5'-3' helicase activities, whereas the GIG motif of drUvrD involved in binding to the DNA duplex is essential for the 5'-3' helicase activity only. These special features of drUvrD may reflect its involvement in a wide range of DNA repair processes in vivo

A complement to the modern crystallographer's toolbox: caged gadolinium complexes with versatile binding modes.

International audienceA set of seven caged gadolinium complexes were used as vectors for introducing the chelated Gd(3+) ion into protein crystals in order to provide strong anomalous scattering for de novo phasing. The complexes contained multidentate ligand molecules with different functional groups to provide a panel of possible interactions with the protein. An exhaustive crystallographic analysis showed them to be nondisruptive to the diffraction quality of the prepared derivative crystals, and as many as 50% of the derivatives allowed the determination of accurate phases, leading to high-quality experimental electron-density maps. At least two successful derivatives were identified for all tested proteins. Structure refinement showed that the complexes bind to the protein surface or solvent-accessible cavities, involving hydrogen bonds, electrostatic and CH-π interactions, explaining their versatile binding modes. Their high phasing power, complementary binding modes and ease of use make them highly suitable as a heavy-atom screen for high-throughput de novo structure determination, in combination with the SAD method. They can also provide a reliable tool for the development of new methods such as serial femtosecond crystallography

Conformational changes associated with ATP hydrolysis and nucleotide release.

<p>A-C. Domain movements. The AMPPNP-bound form is colored in red, while the apo-form is colored in blue. A. Upon ATP hydrolysis and nucleotide release, domain 2B along with the dsDNA rotates by ~15° and domain 1A and 1B by 8° relative to domain 2A. B. Close up view of the rotation of domain 2B and duplex DNA. C. Domains 1A and 1B undergo a 15° twist relative to domain 2A around the ssDNA axis (orange). D. Conformational changes occurring at the ssDNA gateway (circled in green). The linker between domains 2B and 2A adopts a short helix (α25) and loop in the AMPPNP-bound form and interacts tightly with the 3′-end of the ssDNA via Ser546, while it consists of an unstructured loop (dashed line) in the apo-form. In the AMPPNP form, the ssDNA gateway is more closed: the distance between the carboxyl oxygen of Phe65 (motif Ia) and the hydroxyl group of Ser546 is 4.5 Å in the AMPPNP-bound form versus 9.9 Å in the apo-form. The represented DNA corresponds to the AMPPNP bound form.</p

Crystal structures of <i>dr</i>UvrD-DNA complexes.

<p>A ribbon illustration of the AMPPNP-bound <i>dr</i>UvrD^FL is shown in A, the AMPPNP-bound <i>dr</i>UvrD^∆C form I is shown in B, the mixed AMPPNP-bound (red) and apo- (blue) <i>dr</i>UvrD^∆C form II is shown in C. The DNA and AMPPNP are shown in sticks. D-E. Large-scale conformational changes. D. Overlay of chains A (red) of <i>dr</i>UvrD^FL, <i>dr</i>UvrD^∆C form I and apo-<i>dr</i>UvrD^∆C form II, illustrating the large spiral movement of chains B colored respectively yellow, grey and blue. The DNA is shown as an orange ribbon. E. As in (D) but viewed down the DNA axis, and for clarity <i>dr</i>UvrD^∆C form I has been removed. </p

DNA binding of <i>dr</i>UvrD.

<p>Illustrations of <i>dr</i>UvrD binding to dsDNA with a 3′-ssDNA tail in form I (A,D and G), form II with AMPPNP bound (B, E and H) and in the apo-form of form II (C, F and I). A-C. Schematic diagrams (top) illustrating the translocation of form I (A), form II with AMPPNP bound (B) and the apo-form of form II (C) of <i>dr</i>UvrD^∆C along the ssDNA. The ssDNA nucleotides are illustrated as black bars and are numbered as in the crystal structures. The grey oval shape representing <i>dr</i>UvrD covers the nucleotides bound in the ssDNA binding pocket. Surface representations of the ssDNA binding pockets of these three forms of <i>dr</i>UvrD^∆C bound to ssDNA (orange sticks) are shown below. The important residues are labeled and the bases are numbered as in the schematic diagrams. D-F. Binding of <i>dr</i>UvrD^∆C to dsDNA in form I (D), form II with AMPPNP bound (E) and in the apo-form of form II (F). The dsDNA is illustrated in sticks with the translocated strand in grey. Domains of <i>dr</i>UvrD are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077364#pone-0077364-g002" target="_blank">Figure 2A</a>. The helices belonging to the HLH motifs and the β-hairpin structure (orange) are shown and labeled according to the secondary structure succession (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077364#pone.0077364.s005" target="_blank">Figure S1</a>). The positively charged residues in contact with dsDNA are illustrated in sticks and the GIG motif is indicated. The number of base-pairs formed between the ss-dsDNA junction and the contact point with the <i>dr</i>UvrD GIG motif is shown to the left of each panel. This number differs significantly between the two crystal forms. G-I. Schematic representation of <i>dr</i>UvrD's DNA binding in the different crystal structures as indicated below the models. The four protein-DNA contact points that are critical for the wrench-and-inchworm unwinding mechanism are indicated with circled numbers in all panels: HLH motifs interact with dsDNA (1), the β-hairpin motif with the ss-dsDNA junction (2), motif III with the ssDNA (3) and the ssDNA gateway with the exiting ssDNA (4). G. In AMPPNP bound Form I, contact points 1, 3 and 4 are tight. H. In AMPPNP bound Form II, <i>dr</i>UvrD's GIG motif (1) has slided along the DNA duplex and pushes the DNA junction against the β-hairpin motif (2), which now stacks tightly against the first base-pair. I. In the apo molecule of Form II, the ssDNA gateway (4) has opened and ssDNA exited the helicase. Domains of <i>dr</i>UvrD are colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077364#pone-0077364-g002" target="_blank">Figure 2A</a>.</p

DNA binding ability and helicase activity of drUvrD mutants.

<p>Comparison of DNA binding ability and helicase activity of wild type (WT) and <i>dr</i>UvrD mutants: β-hairpin deletion mutant (ΔHairpin), and mutants of the GIG motif from domain 2B involved in dsDNA binding (G424T, G426T and double mutant G424T/G426T). A. DNA binding affinities (K_d values) of WT and mutant <i>dr</i>UvrD for either 3'-tailed (blue) or 5'-tailed (red) dsDNA determined by fluorescence anisotropy measurements. B. Helicase activity of WT and mutant <i>dr</i>UvrD (250 nM) on 3'-tailed (blue) or 5'-tailed (red) dsDNA (20 nM). Initial reaction rates were determined from reaction time courses and were normalized with respect to the activity of WT <i>dr</i>UvrD. Standard deviations are shown as vertical bars.</p