RESUMEN
Cancer will directly affect the lives of over one-third of the population. The DNA Damage Response (DDR) is an intricate system involving damage recognition, cell cycle regulation, DNA repair, and ultimately cell fate determination, playing a central role in cancer etiology and therapy. Two primary therapeutic approaches involving DDR targeting include: combinatorial treatments employing anticancer genotoxic agents; and synthetic lethality, exploiting a sporadic DDR defect as a mechanism for cancer-specific therapy. Whereas, many DDR proteins have proven "undruggable", Fragment- and Structure-Based Drug Discovery (FBDD, SBDD) have advanced therapeutic agent identification and development. FBDD has led to 4 (with â¼50 more drugs under preclinical and clinical development), while SBDD is estimated to have contributed to the development of >200, FDA-approved medicines. Protein X-ray crystallography-based fragment library screening, especially for elusive or "undruggable" targets, allows for simultaneous generation of hits plus details of protein-ligand interactions and binding sites (orthosteric or allosteric) that inform chemical tractability, downstream biology, and intellectual property. Using a novel high-throughput crystallography-based fragment library screening platform, we screened five diverse proteins, yielding hit rates of â¼2-8% and crystal structures from â¼1.8 to 3.2 Å. We consider current FBDD/SBDD methods and some exemplary results of efforts to design inhibitors against the DDR nucleases meiotic recombination 11 (MRE11, a.k.a., MRE11A), apurinic/apyrimidinic endonuclease 1 (APE1, a.k.a., APEX1), and flap endonuclease 1 (FEN1).
Asunto(s)
Descubrimiento de Drogas , Preparaciones Farmacéuticas , Cristalografía por Rayos X , Daño del ADN , Reparación del ADNRESUMEN
BACKGROUND: N-terminal domains of BVU_4064 and BF1687 proteins from Bacteroides vulgatus and Bacteroides fragilis respectively are members of the Pfam family PF12985 (DUF3869). Proteins containing a domain from this family can be found in most Bacteroides species and, in large numbers, in all human gut microbiome samples. Both BVU_4064 and BF1687 proteins have a consensus lipobox motif implying they are anchored to the membrane, but their functions are otherwise unknown. The C-terminal half of BVU_4064 is assigned to protein family PF12986 (DUF3870); the equivalent part of BF1687 was unclassified. RESULTS: Crystal structures of both BVU_4064 and BF1687 proteins, solved at the JCSG center, show strikingly similar three-dimensional structures. The main difference between the two is that the two domains in the BVU_4064 protein are connected by a short linker, as opposed to a longer insertion made of 4 helices placed linearly along with a strand that is added to the C-terminal domain in the BF1687 protein. The N-terminal domain in both proteins, corresponding to the PF12985 (DUF3869) domain is a ß-sandwich with pre-albumin-like fold, found in many proteins belonging to the Transthyretin clan of Pfam. The structures of C-terminal domains of both proteins, corresponding to the PF12986 (DUF3870) domain in BVU_4064 protein and an unclassified domain in the BF1687 protein, show significant structural similarity to bacterial pore-forming toxins. A helix in this domain is in an analogous position to a loop connecting the second and third strands in the toxin structures, where this loop is implicated to play a role in the toxin insertion into the host cell membrane. The same helix also points to the groove between the N- and C-terminal domains that are loosely held together by hydrophobic and hydrogen bond interactions. The presence of several conserved residues in this region together with these structural determinants could make it a functionally important region in these proteins. CONCLUSIONS: Structural analysis of BVU_4064 and BF1687 points to possible roles in mediating multiple interactions on the cell-surface/extracellular matrix. In particular the N-terminal domain could be involved in adhesive interactions, the C-terminal domain and the inter-domain groove in lipid or carbohydrate interactions.
Asunto(s)
Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Bacteroides/química , Moléculas de Adhesión Celular/metabolismo , Lípidos/química , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/química , Cristalografía por Rayos X , Humanos , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
YeaZ is involved in a protein network that is essential for bacteria. The crystal structure of YeaZ from Thermotoga maritima was determined to 2.5â Å resolution. Although this protein belongs to a family of ancient actin-like ATPases, it appears that it has lost the ability to bind ATP since it lacks some key structural features that are important for interaction with ATP. A conserved surface was identified, supporting its role in the formation of protein complexes.
Asunto(s)
Proteínas Bacterianas/química , Thermotoga maritima/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
BT2081 from Bacteroides thetaiotaomicron (GenBank accession code NP_810994.1) is a member of a novel protein family consisting of over 160 members, most of which are found in the different classes of Bacteroidetes. Genome-context analysis lends support to the involvement of this family in carbohydrate metabolism, which plays a key role in B. thetaiotaomicron as a predominant bacterial symbiont in the human distal gut microbiome. The crystal structure of BT2081 at 2.05â Å resolution represents the first structure from this new protein family. BT2081 consists of an N-terminal domain, which adopts a ß-sandwich immunoglobulin-like fold, and a larger C-terminal domain with a ß-sandwich jelly-roll fold. Structural analyses reveal that both domains are similar to those found in various carbohydrate-active enzymes. The C-terminal ß-jelly-roll domain contains a potential carbohydrate-binding site that is highly conserved among BT2081 homologs and is situated in the same location as the carbohydrate-binding sites that are found in structurally similar glycoside hydrolases (GHs). However, in BT2081 this site is partially occluded by surrounding loops, which results in a deep solvent-accessible pocket rather than a shallower solvent-exposed cleft.
Asunto(s)
Proteínas Bacterianas/química , Bacteroides/química , Metabolismo de los Hidratos de Carbono , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Bacteroides/metabolismo , Sitios de Unión , Carbohidratos/química , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
Approximately 30% of the human genome, and likewise for other genomes, encodes membrane proteins. Also, the majority of known human pharmaceutical targets are membrane proteins. As a consequence, the future success of structure-based drug-design efforts will rely heavily on membrane-protein structural information. While a number of techniques are available to determine the structure of membrane proteins, crystallographic methods (either using two-dimensional or three-dimensional crystals) have been the most productive. Nonetheless, membrane-protein structure determination using crystallographic methods has encountered at least three serious bottlenecks: protein production, purification and crystallization. While a number of crystallization strategies for membrane proteins are available today, they all must ensure that the membrane protein of interest is thermodynamically stable for crystallization to be feasible. Thermodynamic stability is so fundamental to protein crystallization that it is often overlooked experimentally. Here, simple and effective protocols for determining the relative stabilities of membrane proteins using commercially available instruments and reagents are demonstrated. The results demonstrate suitability for the rapid screening of conditions that maximize protein stability using minimal amounts of reagents and protein.
Asunto(s)
Proteínas de la Membrana/química , Desnaturalización Proteica , Transportadoras de Casetes de Unión a ATP/química , Animales , Pollos , Cristalización/métodos , Cristalografía por Rayos X , Detergentes/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Colorantes Fluorescentes/química , Calor , Concentración de Iones de Hidrógeno , Canales Iónicos/química , Muramidasa/química , Conformación Proteica , Pliegue de Proteína , Receptores Colinérgicos/química , Termodinámica , Torpedo , Temperatura de TransiciónRESUMEN
The [2Fe-2S] ferredoxin (Fd4) from Aquifex aeolicus adopts a thioredoxin-like polypeptide fold that is distinct from other [2Fe-2S] ferredoxins. Crystal structures of the Cys-55 --> Ser (C55S) and Cys-59 --> Ser (C59S) variants of this protein have been determined to 1.25 A and 1.05 A resolution, respectively, whereas the resolution of the wild type (WT) has been extended to 1.5 A. The improved WT structure provides a detailed description of the [2Fe-2S] cluster, including two features that have not been noted previously in any [2Fe-2S] cluster-containing protein, namely, pronounced distortions in the cysteine coordination to the cluster and a Calpha-H-Sgamma hydrogen bond between cluster ligands Cys-55 and Cys-9. These features may contribute to the unusual electronic and magnetic properties of the [2Fe-2S] clusters in WT and variants of this ferredoxin. The structures of the two variants of Fd4, in which single cysteine ligands to the [2Fe-2S] cluster are replaced by serine, establish the metric details of serine-ligated Fe-S active sites with unprecedented accuracy. Both the cluster and its surrounding protein matrix change in subtle ways to accommodate this ligand substitution, particularly in terms of distortions of the Fe(2)S(2) inorganic core from planarity and displacements of the polypeptide chain. These high resolution structures illustrate how the interactions between polypeptide chains and Fe-S active sites reflect combinations of flexibility and rigidity on the part of both partners; these themes are also evident in more complex systems, as exemplified by changes associated with serine ligation of the nitrogenase P cluster.
Asunto(s)
Proteínas Bacterianas/química , Ferredoxinas/química , Cristalización , Hierro , Mutación , Relación Estructura-Actividad , AzufreRESUMEN
In the photosynthetic bacterium Rhodobacter sphaeroides, a water soluble cytochrome c2 (cyt c2) is the electron donor to the reaction center (RC), the membrane-bound pigment-protein complex that is the site of the primary light-induced electron transfer. To determine the interactions important for docking and electron transfer within the transiently bound complex of the two proteins, RC and cyt c2 were co-crystallized in two monoclinic crystal forms. Cyt c2 reduces the photo-oxidized RC donor (D+), a bacteriochlorophyll dimer, in the co-crystals in approximately 0.9 micros, which is the same time as measured in solution. This provides strong evidence that the structure of the complex in the region of electron transfer is the same in the crystal and in solution. X-ray diffraction data were collected from co-crystals to a maximum resolution of 2.40 A and refined to an R-factor of 22% (R(free)=26%). The structure shows the cyt c2 to be positioned at the center of the periplasmic surface of the RC, with the heme edge located above the bacteriochlorophyll dimer. The distance between the closest atoms of the two cofactors is 8.4 A. The side-chain of Tyr L162 makes van der Waals contacts with both cofactors along the shortest intermolecular electron transfer pathway. The binding interface can be divided into two domains: (i) A short-range interaction domain that includes Tyr L162, and groups exhibiting non-polar interactions, hydrogen bonding, and a cation-pi interaction. This domain contributes to the strength and specificity of cyt c2 binding. (ii) A long-range, electrostatic interaction domain that contains solvated complementary charges on the RC and cyt c2. This domain, in addition to contributing to the binding, may help steer the unbound proteins toward the right conformation.