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Significance Metalloproteins are ubiquitous, accounting for about 30–50% of all proteins. Their functions are wide-ranging, but metalloproteins are frequently used to carry out challenging molecular transformations. Metalloprotein reactivity comes at a price, however, often requiring specialized molecular machinery for holoenzyme assembly. G-protein metallochaperones are an important part of this assembly apparatus, but an understanding of their molecular mechanisms has been hindered by a lack of structural data. Here, we describe crystal structures of a G-protein metallochaperone together with a target enzyme, in this case an adenosylcobalamin-dependent radical enzyme, thereby providing a visualization of the molecular architecture of the G-protein:target enzyme complex. G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. Here, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms of IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.

The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore ™ T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor. In addition, functional activity against p38α was assessed in a biochemical assay using a mobility shift platform (LC3000, Caliper LifeSciences). A selection of fragments was also evaluated using fluorescence lifetime (FLEXYTE ™ ) and microscale thermophoresis (Nanotemper) technologies. A good correlation between the data for the different assays was found. Crystal structures were solved for four of the small molecules complexed to p38α. Interestingly, as determined both by X-ray analysis and SPR competition experiments, three of the complexes involved the fragment at the ATP binding site, while the fourth compound bound in a distal site that may offer potential as a novel drug target site. A first round of optimization around the remotely bound fragment has led to the identification of a series of triazole-containing compounds. This approach could form the basis for developing novel and active p38α inhibitors. More broadly, it illustrates the power of combining a range of biophysical and biochemical techniques to the discovery of fragments that facilitate the development of novel modulators of kinase and other drug targets.

Metalloproteins are ubiquitous, accounting for about 30–50% of all proteins. Their functions are wide-ranging, but metalloproteins are frequently used to carry out challenging molecular transformations. Metalloprotein reactivity comes at a price, however, often requiring specialized molecular machinery for holoenzyme assembly. G-protein metallochaperones are an important part of this assembly apparatus, but an understanding of their molecular mechanisms has been hindered by a lack of structural data. Here, we describe crystal structures of a G-protein metallochaperone together with a target enzyme, in this case an adenosylcobalamin-dependent radical enzyme, thereby providing a visualization of the molecular architecture of the G-protein:target enzyme complex. G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. Here, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms of IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.

G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. Here, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms of IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.

G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. Here, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms of IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.

PIWI proteins and their associated PIWI-interacting RNAs (piRNAs) play crucial roles in proper gametogenesis in animal gonads. Partner of PIWIs (PAPI) is one of the important piRNA biogenesis factors. PAPI contains a Tudor domain and tandem KH domains. The tudor domain specifically recognizes symmetrical-dimethylarginines (sDMAs) on PIWI proteins. BmPAPI, a Bombyx mori homolog of PAPI, is localized at the outer membrane of mitochondria and supports exonucleolytic trimming of piRNA precursors to form mature 3-prime-end of piRNAs. To understand the structural basis of piRNA processing by BmPAPI, we present crystal structures of the apo- and sDMA-liganded Tudor domain of BmPAPI. Footnotes * http://www.rcsb.org/structure/5VY1

Issue Title: Special Issue: Fragment-Based Ligand Design 2011 The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore^sup (TM)^ T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor. In addition, functional activity against p38α was assessed in a biochemical assay using a mobility shift platform (LC3000, Caliper LifeSciences). A selection of fragments was also evaluated using fluorescence lifetime (FLEXYTE^sup (TM)^) and microscale thermophoresis (Nanotemper) technologies. A good correlation between the data for the different assays was found. Crystal structures were solved for four of the small molecules complexed to p38α. Interestingly, as determined both by X-ray analysis and SPR competition experiments, three of the complexes involved the fragment at the ATP binding site, while the fourth compound bound in a distal site that may offer potential as a novel drug target site. A first round of optimization around the remotely bound fragment has led to the identification of a series of triazole-containing compounds. This approach could form the basis for developing novel and active p38α inhibitors. More broadly, it illustrates the power of combining a range of biophysical and biochemical techniques to the discovery of fragments that facilitate the development of novel modulators of kinase and other drug targets.[PUBLICATION ABSTRACT]

This dissertation demonstrates the application of macromolecular crystallography to three enzymes involved in the metabolism of various endogenous compounds or xenobiotics within the cell. The structures provide detailed information on the catalytic mechanism of each enzyme and give insight into the structural and functional evolution of their respective protein families. The first group of structures were determined to investigate how electrons are transferred from NADPH to microsomal cytochrome P450s using the two-flavin containing enzyme NADPH-cytochrome P450 oxidoreductase. Here, the solution of crystal structures of various catalytically significant mutant forms of the enzyme, in conjunction with traditional enzymological approaches, give conclusive evidence as to how this enzyme regulates electron transfer through its two flavin cofactors by use of an amino acid “gating” mechanism. In addition, comparison of the these structures show significant mobility between the two domains of the enzyme, suggesting conformational change is associated with electron transfer and subsequent reduction of cytochrome P454s. The two other enzymes focus on the mechanism of metabolism of unsaturated fatty acids. The first, a bacterial 2,4-dienoyl-CoA reductase, is an enzyme which contains two flavin cofactors and a 4Fe-4S cluster. This enzyme utilizes electrons provided by NADPH to reduce the double bond of certain unsaturated fatty acyl-CoA's which would otherwise be unable to enter the fatty acid β-oxidation pathway to produce acetyl-CoA. The crystal structure of the protein in the presence of modified substrate has been solved to atomic resolution using single-wavelength anomalous scattering and multiple isomorphous replacement phasing techniques. The structure shows the relative arrangement of NADP +, the two flavin molecules, the metal cluster, and modified substrate within the protein fold, allowing us to propose an electron transfer pathway from NADPH to substrate via the flavin cofactors and metal cluster. A catalytic dyad which provides a proton relay system to complete reduction of substrate is also proposed. The topology of the region surrounding the active site shows the substrate binding site to have a large sock-shaped cavity, explaining how the enzyme is able to catalyze reduction of both 2-cis and 2-trans isomers with similar efficiency. The final crystal structure presented is of a mammalian Δ 3,Δ2-enoyl-CoA isomerase, another enzyme required for the metabolism of unsaturated fatty acids. This enzyme works in conjunction with 2,4-dienoyl-CoA reductase in the isomerization of double bonds within the acyl chain of the CoA, allowing the product to reenter the fatty acid β-oxidation pathway. Sequence alignment shows the enzyme to belong to the crotonase superfamily of enzymes, proteins which catalyze a diverse range of reactions. Using the molecular replacement technique, the crystal structure of the rat mitochondrial isoform has been solved. The overall structure illustrates the conservation of catalytic and substrate-binding residues within a group of the crotonase superfamily, yet contrasts the functional/structural homolog found in both peroxisomes and mitochondria which appears to use an alternative catalytic base. Substrate modeling suggests how the binding pocket may accommodate various substrate acyl chain lengths using a tunnel which passes through the core the enzyme via the active site. Also, correlations in the evolutionary aspects of this superfamily of enzymes are shown.

Surf zones are highly dynamic marine ecosystems that are subject to increasing anthropogenic and climatic pressures, posing multiple challenges for biomonitoring. Traditional methods such as seines and hook and line surveys are often labor intensive, taxonomically biased, and can be physically hazardous. Emerging techniques, such as baited remote underwater video (BRUV) and environmental DNA (eDNA) are promising nondestructive tools for assessing marine biodiversity in surf zones of sandy beaches. Here we compare the relative performance of beach seines, BRUV, and eDNA in characterizing community composition of bony (teleost) and cartilaginous (elasmobranch) fishes of surf zones at 18 open coast sandy beaches in southern California. Seine and BRUV surveys captured overlapping, but distinct fish communities with 50% (18/36) of detected species shared. BRUV surveys more frequently detected larger species (e.g. sharks and rays) while seines more frequently detected one of the most abundant species, barred surfperch ( Amphistichus argenteus ). In contrast, eDNA metabarcoding captured 88.9% (32/36) of all fishes observed in seine and BRUV surveys plus 57 additional species, including 15 that frequent surf zone habitats. On average, eDNA detected over 5 times more species than BRUVs and 8 times more species than seine surveys at a given site. eDNA approaches also showed significantly higher sensitivity than seine and BRUV methods and more consistently detected 31 of the 32 (96.9%) jointly observed species across beaches. The four species detected by BRUV/seines, but not eDNA were only resolved at higher taxonomic ranks (e.g. Embiotocidae surfperches and Sygnathidae pipefishes). In frequent co-detection of species between methods limited comparisons of richness and abundance estimates, highlighting the challenge of comparing biomonitoring approaches. Despite potential for improvement, results overall demonstrate that eDNA can provide a cost-effective tool for long-term surf zone monitoring that complements data from seine and BRUV surveys, allowing more comprehensive surveys of vertebrate diversity in surf zone habitats.

An inability to estimate absolute DNA methylation levels has slowed progress in understanding the role of this epigenetic modification in health and disease. Down et al . describe an algorithm for analyzing methylated DNA immunoprecipitation profiles generated using either high-throughput sequencing or oligonucleotide arrays. DNA methylation is an indispensible epigenetic modification required for regulating the expression of mammalian genomes. Immunoprecipitation-based methods for DNA methylome analysis are rapidly shifting the bottleneck in this field from data generation to data analysis, necessitating the development of better analytical tools. In particular, an inability to estimate absolute methylation levels remains a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling. To address this issue, we developed a cross-platform algorithm—Bayesian tool for methylation analysis (Batman)—for analyzing methylated DNA immunoprecipitation (MeDIP) profiles generated using oligonucleotide arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). We developed the latter approach to provide a high-resolution whole-genome DNA methylation profile (DNA methylome) of a mammalian genome. Strong correlation of our data, obtained using mature human spermatozoa, with those obtained using bisulfite sequencing suggest that combining MeDIP-seq or MeDIP-chip with Batman provides a robust, quantitative and cost-effective functional genomic strategy for elucidating the function of DNA methylation.