Small angle scattering reveals the orientation of cytochrome P450 19A1 in lipoprotein nanodiscs.

Human aromatase (CYP19A1), the cytochrome P450 enzyme responsible for conversion of androgens to estrogens, was incorporated into lipoprotein nanodiscs (NDs) and interrogated by small angle X-ray and neutron scattering (SAXS/SANS). CYP19A1 was associated with the surface and centered at the edge of the long axis of the ND membrane. In the absence of the N-terminal anchor, the amphipathic A'- and G'-helices were predominately buried in the lipid head groups, with the possibly that their hydrophobic side chains protrude into the hydrophobic, aliphatic tails.

Induced Fit Describes Ligand Binding to Membrane-Associated Cytochrome P450 3A4.

Cytochrome P450 3A4 (CYP3A4) is the dominant P450 involved in human xenobiotic metabolism. Competition for CYP3A4 therefore underlies several adverse drug-drug interactions. Despite its clinical significance, the mechanisms CYP3A4 uses to bind diverse ligands remain poorly understood.

The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states.

The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state.

Dynamics and Mechanism of Binding of Androstenedione to Membrane-Associated Aromatase.

Aromatase (CYP19A1) catalyzes the synthesis of estrogens from androgens and is an invaluable target of pharmacotherapy for estrogen-dependent cancers. CYP19A1 is also one of the most primordial human CYPs and, to the extent that its fundamental dynamics are conserved, is highly relevant to understanding those of the more recently evolved and promiscuous enzymes. A complementary approach employing molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to interrogate the changes in CYP19A1 dynamics coupled to binding androstenedione (ASD).

Conformational selection is present in ligand binding to cytochrome P450 19A1 lipoprotein nanodiscs.

Cytochromes P450 (CYPs) display remarkable plasticity in their ability to bind substrates and catalyze a broad array of chemical reactions. Herein we evaluate binding of androstenedione, testosterone, and 7-hydroxyflavone to CYP19A1, also known as aromatase, in phospholipid nanodiscs by stopped-flow UV-vis spectroscopy. Exponential fitting of the kinetic traces supports the possibility of a multi-step binding mechanism.

Active-Site Tryptophan, the Target of Antineoplastic C-Terminal Binding Protein Inhibitors, Mediates Inhibitor Disruption of CtBP Oligomerization and Transcription Coregulatory Activities.

C-terminal binding proteins (CtBP1/2) are oncogenic transcriptional coregulators and dehydrogenases often overexpressed in multiple solid tumors, including breast, colon, and ovarian cancer, and associated with poor survival. CtBPs act by repressing expression of genes responsible for apoptosis (e.g.

Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus.

Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase.

Biophysical characterization of Aptenodytes forsteri cytochrome P450 aromatase.

Cytochrome P450 19 (CYP19, aromatase) catalyzes the conversion of androgens to estrogens in a sequence of three reactions that each depend on NADPH and O. Aromatase is a phylogenetically-ancient enzyme and its breadth of expression in other species has highlighted distinct physiological functions. In songbirds, estrogen production is required for programming the neural circuits controlling song and in the determination of sex in fish and reptiles.

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting.

Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy.

Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the integration site using fluorescent anisotropy. This protocol can be used to measure the binding constants of any DNA binding protein provided the substrate DNA is fluorescently labeled.

Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface.

Unlabelled: The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The large Rep proteins, Rep78 and Rep68, are remarkably multifunctional and display a range of biochemical activities, including DNA binding, nicking, and unwinding. Functionally, Rep78 and Rep68 are involved in transcriptional regulation, DNA replication, and genomic integration.

Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1.

Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker.

Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation.

Adeno-associated virus (AAV) nonstructural proteins Rep78 and Rep68 carry out all DNA transactions that regulate the AAV life cycle. They share two multifunctional domains: an N-terminal origin binding/nicking domain (OBD) from the HUH superfamily and a SF3 helicase domain. A short linker of ∼20 amino acids that is critical for oligomerization and function connects the two domains.

Oligomeric properties of adeno-associated virus Rep68 reflect its multifunctionality.

The adeno-associated virus (AAV) encodes four regulatory proteins called Rep. The large AAV Rep proteins Rep68 and Rep78 are essential factors required in almost every step of the viral life cycle. Structurally, they share two domains: a modified version of the AAA(+) domain that characterizes the SF3 family of helicases and an N-terminal domain that binds DNA specifically.

The interdomain linker of AAV-2 Rep68 is an integral part of its oligomerization domain: role of a conserved SF3 helicase residue in oligomerization.

The four Rep proteins of adeno-associated virus (AAV) orchestrate all aspects of its viral life cycle, including transcription regulation, DNA replication, virus assembly, and site-specific integration of the viral genome into the human chromosome 19. All Rep proteins share a central SF3 superfamily helicase domain. In other SF3 members this domain is sufficient to induce oligomerization.

Different catalytic properties of two highly homologous triosephosphate isomerase monomers.

It is assumed that amino acid sequence differences in highly homologous enzymes would be found at the peripheral level, subtle changes that would not necessarily affect catalysis. Here, we demonstrate that, using the same set of mutations at the level of the interface loop 3, the activity of a triosephosphate isomerase monomeric enzyme is ten times higher than that of a homologous enzyme with 74% identity and 86% similarity, whereas the activity of the native, dimeric enzymes is essentially the same. This is an example of how the dimeric biological unit evolved to compensate for the intrinsic differences found at the monomeric species level.