Lit Lunch-February 13, 2013 Conformational Selection in Substrate Recognition by Hsp70 Chaperones

Hsp70s are molecular chaperones involved in the folding and assembly of proteins. They recognize hydrophobic amino acid stretches in their substrate binding groove. However, a detailed understanding of substrate specificity is still missing. Here, we use the endoplasmic reticulum-resident Hsp70 BiP to identify binding sites in a natural client protein. Two sites are mutually recognized and form stable Hsp70–substrate complexes. In silico and in vitro analyses revealed an extended substrate conformation as a crucial factor for interaction and show an unexpected plasticity of the is conserved among different Hsp70s. Hsp70/Hsp90 chaperone machinery is involved in the assembly of the purinosome Jarrod B. Frencha,1, Hong Zhaoa,1, Songon Ana,2, Sherry Niessenb, Yijun Denga, Benjamin F. Cravattb,3, and Stephen J. Benkovica,3 aDepartment of Chemistry, Pennsylvania State University, University Park, PA 16802; and bThe Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037 Contributed by Stephen J. Benkovic, January 4, 2013 (sent for review December 7, 2012) The de novo biosynthesis of purines is carried out by a highly conserved metabolic pathway that includes several validated targets for anticancer, immunosuppressant, and anti-inflammatory chemotherapeutics. The six enzymes in humans that catalyze the 10 chemical steps from phosphoribosylpyrophosphate to inosine monophosphate were recently shown to associate into a dynamic multiprotein complex called the purinosome. Here, we demonstrate that heat shock protein 90 (Hsp90), heat shock protein 70 (Hsp70), and several cochaperones functionally colocalize with this protein complex. Knockdown of expression levels of the identified cochaperones leads to disruption of purinosomes. In addition, small molecule inhibitors of Hsp90 and Hsp70 reversibly disrupt purinosomes and are shown to have a synergistic effect with methotrexate, an anticancer agent that targets purine biosynthesis. These data implicate the Hsp90/Hsp70 chaperone machinery in the assembly of the purinosome and provide a strategy for the development of improved anticancer therapies that disrupt purine biosynthesis. Nature and Structural and Molecular Biology Interaction between FIP200 and ATG16L1 distinguishes ULK1 complex–dependent and –independent autophagy Noor Gammoh, Oliver Florey, Michael Overholtzer & Xuejun Jiang Cell Biology Department, Memorial Sloan Kettering Cancer Center, New York, New York, USA. Autophagy is a finely orchestrated cellular catabolic process that requires multiple autophagy-related gene products (ATG proteins). The ULK1 complex functions to integrate upstream signals to downstream ATG proteins through an unknown mechanism. Here we have identified an interaction between mammalian FIP200 and ATG16L1, essential components of the ULK1 and ATG5 complexes, respectively. Further analyses show this is a direct interaction mediated by a short domain of ATG16L1 that we term the FIP200-binding domain (FBD). The FBD is not required for ATG16L1 self-dimerization or interaction with ATG5. Notably, an FBD-deleted ATG16L1 mutant is defective in mediating amino acid starvation– induced autophagy, which requires the ULK1 complex. However, this mutant retains its function in supporting glucose deprivation–induced autophagy, a ULK1 complex–independent process. This study therefore identifies a previously uncharacterized interaction between the ULK1 and ATG5 complexes that can distinguish ULK1-dependent and -independent autophagy processes. Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a structure compatible with RNA degradation Mikalai Lapkouski, Lan Tian, Jennifer T Miller, Stuart F J Le Grice & Wei Yang Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA. HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, National Cancer Institute, Frederick, Maryland, USA. Hundreds of structures of type 1 human immunodeficiency virus (HIV-1) reverse transcriptase (RT) have been determined, but only one contains an RNA/DNA hybrid. Here we report three structures of HIV-1 RT complexed with a non-nucleotide RT inhibitor (NNRTI) and an RNA/DNA hybrid. In the presence of an NNRTI, the RNA/DNA structure differs from all prior nucleic acid–RT structures including the RNA/DNA hybrid. The enzyme structure also differs from all previous RT–DNA complexes. Thus, the hybrid has ready access to the RNase-H active site. These observations indicate that an RT–nucleic acid complex may adopt two structural states, one competent for DNA polymerization and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites often map to the unique RT-hybrid interface that undergoes conformational changes between two catalytic states. Journal of Biological Chemistry Glycosyltransferases from Oat (Avena) Implicated in the Acylation of Avenacins Amorn Owatworakit‡,1, Belinda Townsend§,2, Thomas Louveau, Helen Jenner§,3, Martin Rejzek, Richard K. Hughes, Gerhard Saalbach, Xiaoquan Qi‡,4, Saleha Bakht‡,4, Abhijeet Deb Roy‖, Sam T. Mugford, Rebecca J. M. Goss‖, Robert A. Field¶,5 and Anne Osbourn‡,5,6 ‡Department of Metabolic Biology and Department of Biological Chemistry, The John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom, The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, United Kingdom, and the ‖School of Chemical Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid.