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Pathway docking ( in silico docking of metabolites to several enzymes and binding proteins in a metabolic pathway) enables the discovery of a catabolic pathway for the osmolyte trans -4-hydroxy- l -proline betaine. Structural key to predicting enzyme function Overprediction and database annotation errors in genome-sequencing projects have caused much confusion because of the difficulty of assigning valid functions to the proteins identified. These authors use structure-guided approaches for predicting the substrate specificities of several enzymes encoded by a bacterial gene cluster to correctly predict the in vitro activity of an enzyme of unknown function and identify the catabolic pathway in which it participates in cells. The substrate-liganded pose predicted by virtual library screening was confirmed experimentally, enzyme activities in the predicted pathway were confirmed by in vitro assays and genetic analyses, the intermediates were identified by metabolomics, and repression of the genes encoding the pathway by high salt concentrations was established by transcriptomics. This study establishes the utility of structure-guided functional predictions for the discovery of new metabolic pathways. Assigning valid functions to proteins identified in genome projects is challenging: overprediction and database annotation errors are the principal concerns 1 . We and others 2 are developing computation-guided strategies for functional discovery with ‘metabolite docking’ to experimentally derived 3 or homology-based 4 three-dimensional structures. Bacterial metabolic pathways often are encoded by ‘genome neighbourhoods’ (gene clusters and/or operons), which can provide important clues for functional assignment. We recently demonstrated the synergy of docking and pathway context by ‘predicting’ the intermediates in the glycolytic pathway in Escherichia coli 5 . Metabolite docking to multiple binding proteins and enzymes in the same pathway increases the reliability of in silico predictions of substrate specificities because the pathway intermediates are structurally similar. Here we report that structure-guided approaches for predicting the substrate specificities of several enzymes encoded by a bacterial gene cluster allowed the correct prediction of the in vitro activity of a structurally characterized enzyme of unknown function (PDB 2PMQ), 2-epimerization of trans -4-hydroxy- l -proline betaine (tHyp-B) and cis -4-hydroxy- d -proline betaine (cHyp-B), and also the correct identification of the catabolic pathway in which Hyp-B 2-epimerase participates. The substrate-liganded pose predicted by virtual library screening (docking) was confirmed experimentally. The enzymatic activities in the predicted pathway were confirmed by in vitro assays and genetic analyses; the intermediates were identified by metabolomics; and repression of the genes encoding the pathway by high salt concentrations was established by transcriptomics, confirming the osmolyte role of tHyp-B. This study establishes the utility of structure-guided functional predictions to enable the discovery of new metabolic pathways.

Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin β2 or Kapβ2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kapβ2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kapβ2 is conserved throughout eukaryotes. Kap104, the Kapβ2 homolog in Saccharomyces cerevisiae , recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kapβ2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kapβ2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kapβ2·PY-NLS structures.

High-resolution structural information is important for improving our understanding of protein function in vitro and in vivo and providing information to enable drug discovery. The process leading to X-ray structure determination is often time consuming and labor intensive. It requires informed decisions in expression construct design, expression host selection, and strategies for protein purification, crystallization and structure determination. Previously published studies have demonstrated that compact globular domains defined by limited proteolysis represent good candidates for production of diffraction quality crystals [1-7]. Integration of mass spectrometry and proteolysis experiments can provide accurate definition of domain boundaries at unprecedented rates. We have conducted a critical evaluation of this approach with 400 target proteins produced by SGX (Structural GenomiX, Inc.) for the New York Structural GenomiX Research Consortium (NYSGXRC; http://www.nysgxrc.org) under the auspices of the National Institute of General Medical Sciences Protein Structure Initiative (http://www.nigms.nih.gov/psi). The objectives of this study were to develop parallel/automated protocols for proteolytic digestion and data acquisition for multiple proteins, and to carry out a systematic study to correlate domain definition via proteolysis with outcomes of crystallization and structure determination attempts. Initial results from this work demonstrate that proteins yielding diffraction quality crystals are typically resistant to proteolysis. Large-scale sub cloning and subsequent testing of expression, solubility, and crystallizability of proteolytically defined truncations is currently underway.

Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin [beta]2 or Kap[beta]2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kap[beta]2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kap[beta]2 is conserved throughout eukaryotes. Kap104, the Kap[beta]2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kap[beta]2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kap[beta]2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kap[beta]2·PY-NLS structures.[PUBLICATION ABSTRACT]

Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin beta 2 or Kap beta 2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kap beta 2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kap beta 2 is conserved throughout eukaryotes. Kap104, the Kap beta 2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kap beta 2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Aa resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kap beta 2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kap beta 2.PY-NLS structures.

Histone H3 arginine 2 (H3R2) is post-translationally modified in three different states by \"writers\" of the protein arginine methyltransferase (PRMT) family. H3R2 methylarginine isoforms include PRMT5-catalyzed mono- and symmetric di-methylation (me1, me2s), and PRMT6-catalyzed me1 and asymmetric dimethylation (me2a). WD-40 repeat-containing protein 5 (WDR5) is an epigenetic \"reader\" protein that interacts with H3R2 and is a subunit of numerous chromatin-modifying complexes, such as the Mixed Lineage Leukemia (MLL) H3 lysine 4 methyltransferase complex. Previous studies suggested that MLL recruitment to chromatin was specified by the high-affinity interaction between WDR5 and H3R2me2s. However, our prior biological data prompted the hypothesis that WDR5 may also interact with H3R2me1 to recruit MLL activity. Here, using highly accurate quantitative binding analysis combined with high-resolution crystal structures of WDR5 in complex with unmodified (me0) and me1/me2s L-Arginine amino acids and in complex with H3R2me1 peptide, we provide a rigorous biochemical study of this important biological interaction. Despite modest structural differences at the binding interface, our study supports an interaction model regulated by a binary arginine methylation switch: H3R2me2a prevents interaction with WDR5, whereas H3R2me0/me1/me2s are equally permissive.