RESUMEN
BACKGROUND: Many receptors function by binding to multiple ligands, each eliciting a distinct biological output. The extracellular domain of the human prolactin receptor (hPRL-R) uses an identical epitope to bind to both prolactin (hPRL) and growth hormone (hGH), yet little is known about how each hormone binding event triggers the appropriate response. FINDINGS: Here, we utilized a phage display library to generate synthetic antibodies (sABs) that preferentially modulate hPRL-R function in a hormone-dependent fashion. We determined the crystal structure of a sAB-hPRL-R complex, which revealed a novel allosteric mechanism of antagonism, whereby the sAB traps the receptor in a conformation more suitable for hGH binding than hPRL. This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway. CONCLUSIONS: The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding. We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.
Asunto(s)
Epítopos , Receptores de Prolactina , Transducción de Señal , Anticuerpos de Cadena Única , Epítopos/química , Epítopos/genética , Hormona de Crecimiento Humana/química , Hormona de Crecimiento Humana/genética , Humanos , Prolactina/química , Prolactina/genética , Estructura Terciaria de Proteína , Receptores de Prolactina/química , Receptores de Prolactina/genética , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genéticaRESUMEN
With the rapid rise of methicillin-resistant Staphylococcus aureus infections, new strategies against S. aureus are urgently needed. De novo purine biosynthesis is a promising yet unexploited target, insofar as abundant evidence has shown that bacteria with compromised purine biosynthesis are attenuated. Fundamental differences exist within the process by which humans and bacteria convert 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). In bacteria, this transformation occurs through a two-step conversion catalyzed by PurK and PurE; in humans, it is mediated by a one-step conversion catalyzed by class II PurE. Thus, these bacterial enzymes are potential targets for selective antibiotic development. Here, the first comprehensive structural and biochemical characterization of PurK and PurE from S. aureus is presented. Structural analysis of S. aureus PurK reveals a nonconserved phenylalanine near the AIR-binding site that occupies the putative position of the imidazole ring of AIR. Mutation of this phenylalanine to isoleucine or tryptophan reduced the enzyme efficiency by around tenfold. The K(m) for bicarbonate was determined for the first time for a PurK enzyme and was found to be â¼18.8â mM. The structure of PurE is described in comparison to that of human class II PurE. It is confirmed biochemically that His38 is essential for function. These studies aim to provide foundations for future structure-based drug-discovery efforts against S. aureus purine biosynthesis.
Asunto(s)
Carboxiliasas/química , Staphylococcus aureus/enzimología , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Biocatálisis , Carboxiliasas/metabolismo , Dominio Catalítico , Escherichia coli/enzimología , Humanos , Cinética , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Homología Estructural de ProteínaRESUMEN
SlyA is a master virulence regulator that controls the transcription of numerous genes in Salmonella enterica. We present here crystal structures of SlyA by itself and bound to a high-affinity DNA operator sequence in the slyA gene. SlyA interacts with DNA through direct recognition of a guanine base by Arg-65, as well as interactions between conserved Arg-86 and the minor groove and a large network of non-base-specific contacts with the sugar phosphate backbone. Our structures, together with an unpublished structure of SlyA bound to the small molecule effector salicylate (Protein Data Bank code 3DEU), reveal that, unlike many other MarR family proteins, SlyA dissociates from DNA without large conformational changes when bound to this effector. We propose that SlyA and other MarR global regulators rely more on indirect readout of DNA sequence to exert control over many genes, in contrast to proteins (such as OhrR) that recognize a single operator.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , ADN/metabolismo , Salmonella enterica/metabolismo , Secuencia de Aminoácidos , Apoproteínas/química , Apoproteínas/metabolismo , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , ADN/química , ADN/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica/efectos de los fármacos , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Ácido Salicílico/farmacología , Salmonella enterica/genética , Salmonella enterica/patogenicidadRESUMEN
We describe a phage display methodology for engineering synthetic antigen binders (sABs) that recognize either the apo or the ligand-bound conformation of maltose-binding protein (MBP). sABs that preferentially recognize the maltose-bound form of MBP act as positive allosteric effectors by substantially increasing the affinity for maltose. A crystal structure of a sAB bound to the closed form of MBP reveals the basis for this allosteric effect. We show that sABs that recognize the bound form of MBP can rescue the function of a binding-deficient mutant by restoring its natural affinity for maltose. Furthermore, the sABs can enhance maltose binding in vivo, as they provide a growth advantage to bacteria under low-maltose conditions. The results demonstrate that structure-specific sABs can be engineered to dynamically control ligand-binding affinities by modulating the transition between different conformations.
Asunto(s)
Regulación Alostérica , Ingeniería de Proteínas , Ligandos , Modelos Moleculares , Mutación , Unión Proteica , Conformación ProteicaRESUMEN
RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab-ligase complex at 3.1-Å resolution using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone's role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix, and this sequence retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of a smaller loop and Fabs' high molecular weight, large surface area and phasing power.
Asunto(s)
Cristalización/métodos , Fragmentos Fab de Inmunoglobulinas/química , ARN Catalítico/química , Secuencia de Bases , Sitios de Unión , Ligasas/química , Ligasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Biblioteca de Péptidos , ARN Catalítico/inmunología , ARN no Traducido/químicaRESUMEN
Escherichia coli AlkB and its human homologues ABH2 and ABH3 repair DNA/RNA base lesions by using a direct oxidative dealkylation mechanism. ABH2 has the primary role of guarding mammalian genomes against 1-meA damage by repairing this lesion in double-stranded DNA (dsDNA), whereas AlkB and ABH3 preferentially repair single-stranded DNA (ssDNA) lesions and can repair damaged bases in RNA. Here we show the first crystal structures of AlkB-dsDNA and ABH2-dsDNA complexes, stabilized by a chemical cross-linking strategy. This study reveals that AlkB uses an unprecedented base-flipping mechanism to access the damaged base: it squeezes together the two bases flanking the flipped-out one to maintain the base stack, explaining the preference of AlkB for repairing ssDNA lesions over dsDNA ones. In addition, the first crystal structure of ABH2, presented here, provides a structural basis for designing inhibitors of this human DNA repair protein.
Asunto(s)
Enzimas Reparadoras del ADN/química , ADN/metabolismo , Dioxigenasas/química , Dioxigenasas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , ARN/metabolismo , Adenina/análogos & derivados , Adenina/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB , Sitios de Unión , Reactivos de Enlaces Cruzados/química , Cristalografía por Rayos X , ADN/química , Daño del ADN , Reparación del ADN , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Unión ProteicaRESUMEN
Staphylococcus aureus is a human pathogen responsible for most wound and hospital-acquired infections. The protein MgrA is both an important virulence determinant during infection and a regulator of antibiotic resistance in S. aureus. The crystal structure of the MgrA homodimer, solved at 2.86 A, indicates the presence of a unique cysteine residue located at the interface of the protein dimer. We discovered that this cysteine residue can be oxidized by various reactive oxygen species, such as hydrogen peroxide and organic hydroperoxide. Cysteine oxidation leads to dissociation of MgrA from DNA and initiation of signaling pathways that turn on antibiotic resistance in S. aureus. The oxidation-sensing mechanism is typically used by bacteria to counter challenges of reactive oxygen and nitrogen species. Our study reveals that in S. aureus, MgrA adopts a similar mechanism but uses it to globally regulate different defensive pathways.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Represoras/metabolismo , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/química , Cisteína/química , Cisteína/efectos de los fármacos , Cisteína/metabolismo , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/farmacología , Oxidación-Reducción , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Represoras/químicaAsunto(s)
Daño del ADN , Reparación del ADN , ADN/metabolismo , Alquilación , Secuencia de Aminoácidos , Animales , ADN/química , ADN/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Relación Estructura-ActividadRESUMEN
O6-Alklyguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that protects cells from mutagenesis and toxicity arising from alkylating agents. We present an X-ray crystal structure of the wild-type human protein (hAGT) bound to double-stranded DNA with a chemically modified cytosine base. The protein binds at two different sites: one at the modified base, and the other across a sticky-ended DNA junction. The protein molecule that binds the modified cytosine base flips the base and recognizes it in its active site. The one that binds ends of neighboring DNA molecules partially flips an overhanging thymine base. This base is not inserted into the active-site pocket of the protein. These two different hAGT/DNA interactions observed in the structure suggest that hAGT may not detect DNA lesions by searching for the adduct itself, but rather for weakened and/or distorted base-pairs caused by base damage in the duplex DNA. We propose that hAGT imposes a strain on the DNA duplex and searches for DNA regions where the native structure is destabilized. The structure provides implications for pyrimidine recognition, improved inhibitor design, and a possible protein/protein interaction patch on hAGT.
Asunto(s)
Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Daño del ADN/fisiología , ADN/metabolismo , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Oligonucleótidos/metabolismo , Estructura Terciaria de Proteína , Timina/metabolismo , Zinc/metabolismoRESUMEN
O(6)-alkylguanine-DNA alkyltransferases directly reverse the alkylation on the O(6) position of guanine in DNA. This group of proteins has been proposed to repair the damaged base in an extrahelical manner; however, the detailed mechanism is not understood. Here we applied a chemical disulfide crosslinking method to probe the damage-searching mechanism of two O(6)-alkylguanine-DNA alkyltransferases, the Escherichia coli C-Ada and the human AGT. Crosslinking reactions with different efficiency occur between the reactive Cys residues of both proteins and a modified cytosine bearing a thiol tether in various DNA probes. Our results indicate that it is not necessary for these proteins to actively flip out every base to find damage. Instead they can locate potential lesions by simply capturing a lesioned base that is transiently extrahelical or sensing the unstable nature of a damaged base pair.