RESUMEN

RTEL1 is an essential DNA helicase which plays an important role in various aspects of genome stability, from telomere metabolism to DNA replication, repair and recombination. RTEL1 has been implicated in a number of genetic diseases and cancer development, including glioma, breast, lung and gastrointestinal tumors. RTEL1 is a FeS helicase but, in addition to the helicase core, it comprises a long C-terminal region which includes a number of folded domains connected by intrinsically disordered loops and mediates RTEL1 interaction with factors involved in pivotal cellular pathways. However, information on the architecture and the function of this region is still limited. We expressed and purified a variety of fragments encompassing the folded domains and the unstructured regions. We determined the crystal structure of the second repeat, confirming that it has a fold similar to the harmonin homology domains. SAXS data provide low-resolution information on all the fragments and suggest that the presence of the RING domain affects the overall architecture of the C-terminal region, making the structure significantly more compact. NMR data provide experimental information on the interaction between PCNA and the RTEL1 C-terminal region, revealing a putative low-affinity additional site of interaction. A biochemical analysis shows that the C-terminal region, in addition to a preference for telomeric RNA and DNA G-quadruplexes, has a high affinity for R-loops and D-loops, consistent with the role played by the RTEL1 helicase in homologous recombination, telomere maintenance and preventing replication-transcription conflicts. We further dissected the contribution of each domain in binding different substrates.

Asunto(s)

ADN Helicasas , Humanos , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Helicasas/genética , Cristalografía por Rayos X , Modelos Moleculares , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Dominios Proteicos , Dispersión del Ángulo Pequeño

RESUMEN

Protein crystallization is one of the major bottlenecks in protein structure elucidation with new strategies being constantly developed to improve the chances of crystallization. Generally, well-ordered epitopes possessing complementary surface and capable of producing stable inter-protein interactions generate a regular three-dimensional arrangement of protein molecules which eventually results in a crystal lattice. Metals, when used for crystallization, with their various coordination numbers and geometries, can generate such epitopes mediating protein oligomerization and/or establish crystal contacts. Some examples of metal-mediated oligomerization and crystallization together with our experience on metal-mediated crystallization of a putative rRNA methyltransferase from Sinorhizobium meliloti are presented. Analysis of crystal structures from protein data bank (PDB) using a non-redundant data set with a 90% identity cutoff, reveals that around 67% of proteins contain at least one metal ion, with ∼14% containing combination of metal ions. Interestingly, metal containing conditions in most commercially available and popular crystallization kits generally contain only a single metal ion, with combinations of metals only in a very few conditions. Based on the results presented in this review, it appears that the crystallization screens need expansion with systematic screening of metal ions that could be crucial for stabilizing the protein structure or for establishing crystal contact and thereby aiding protein crystallization.

Asunto(s)

Cristalización/métodos , Metales/química , Metales/metabolismo , Proteínas/química , Proteínas/metabolismo , Cristalografía por Rayos X , Modelos Moleculares

RESUMEN

Single-wavelength anomalous dispersion (SAD) utilizing anomalous signal from native S atoms, or other atoms with Z ≤ 20, generally requires highly redundant data collected using relatively long-wavelength X-rays. Here, the results from two proteins are presented where the anomalous signal from serendipitously acquired surface-bound Ca atoms with an anomalous data multiplicity of around 10 was utilized to drive de novo structure determination. In both cases, the Ca atoms were acquired from the crystallization solution, and the data-collection strategy was not optimized to exploit the anomalous signal from these scatterers. The X-ray data were collected at 0.98 Šwavelength in one case and at 1.74 Šin the other (the wavelength was optimized for sulfur, but the anomalous signal from calcium was exploited for structure solution). Similarly, using a test case, it is shown that data collected at ∼1.0 Šwavelength, where the f'' value for sulfur is 0.28 e, are sufficient for structure determination using intrinsic S atoms from a strongly diffracting crystal. Interestingly, it was also observed that SHELXD was capable of generating a substructure solution from high-exposure data with a completeness of 70% for low-resolution reflections extending to 3.5 Šresolution with relatively low anomalous multiplicity. Considering the fact that many crystallization conditions contain anomalous scatterers such as Cl, Ca, Mn etc., checking for the presence of fortuitous anomalous signal in data from well diffracting crystals could prove useful in either determining the structure de novo or in accurately assigning surface-bound atoms.

Asunto(s)

Proteínas Arqueales/química , Proteínas Bacterianas/química , Calcio/química , Proteínas del Huevo/química , Muramidasa/química , Azufre/química , Animales , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cationes Bivalentes , Pollos/metabolismo , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Proteínas del Huevo/genética , Proteínas del Huevo/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Muramidasa/genética , Muramidasa/metabolismo , Conformación Proteica , Pseudomonas syringae/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermoplasmales/química , Difracción de Rayos X , Rayos X

RESUMEN

The crystal structures of two oligopeptides containing di-n-propylglycine (Dpg) residues, Boc-Gly-Dpg-Gly-Leu-OMe (1) and Boc-Val-Ala-Leu-Dpg-Val-Ala-Leu-Val-Ala-Leu-Dpg-Val-Ala-Leu-OMe (2) are presented. Peptide 1 adopts a type I'beta-turn conformation with Dpg(2)-Gly(3) at the corner positions. The 14-residue peptide 2 crystallizes with two molecules in the asymmetric unit, both of which adopt alpha-helical conformations stabilized by 11 successive 5 --> 1 hydrogen bonds. In addition, a single 4 --> 1 hydrogen bond is also observed at the N-terminus. All five Dpg residues adopt backbone torsion angles (phi, psi) in the helical region of conformational space. Evaluation of the available structural data on Dpg peptides confirm the correlation between backbone bond angle N-C(alpha)-C' (tau) and the observed backbone phi,psi values. For tau > 106 degrees, helices are observed, while fully extended structures are characterized by tau < 106 degrees. The mean tau values for extended and folded conformations for the Dpg residue are 103.6 degrees +/- 1.7 degrees and 109.9 degrees +/- 2.6 degrees, respectively.

Asunto(s)

Oligopéptidos/química , Péptidos/química , Valina/análogos & derivados , Secuencia de Aminoácidos , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Complejos Multiproteicos/química , Oligopéptidos/síntesis química , Péptidos/síntesis química , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Valina/química

RESUMEN

Single crystal X-ray diffraction studies on the water soluble, synthetic tetrapeptide Tyr(1)-Aib(2)-Tyr(3)-Val(4) with a non-coded amino acid residue (Aib: [small alpha]-amino isobutyric acid) reveal that the peptide adopts an "S"-shaped molecular structure which self-assembles to form a supramolecular triple helix using various non-covalent interactions including water mediated hydrogen bonds in the solid state.

Asunto(s)

Péptidos/química , Agua/química , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Péptidos/síntesis química , Conformación Proteica , Solubilidad