Publications by authors named "Joseph D Koos"

4 Publications

  • Page 1 of 1

Heterologous and in Vitro Reconstitution of Fuscanodin, a Lasso Peptide from Thermobifida fusca.

J Am Chem Soc 2019 01 27;141(2):928-935. Epub 2018 Dec 27.

Lasso peptides are a class of ribosomally derived natural products typified by their threaded rotaxane structure. The conversion of a linear precursor peptide into a lasso peptide structure requires two enzymatic activities: cleavage of the precursor via a cysteine protease and cyclization via isopeptide bond formation. In vitro studies of lasso peptide enzymology have been hampered by difficulties in obtaining pure, soluble enzymes. We reasoned that thermophilic bacteria would be a good source for well-behaved lasso peptide biosynthetic enzymes. The genome of the thermophilic actinobacterium Thermobifida fusca encodes for a lasso peptide with an unprecedented Trp residue at its N-terminus, a peptide we have named fuscanodin. Here we reconstitute fuscanodin biosynthesis in vitro with purified components, establishing a minimal fuscanodin synthetase. These experiments have allowed us to probe the kinetics of lasso peptide biosynthesis for the first time, and we report initial rates of fuscanodin biosynthesis. The fuscanodin biosynthetic enzymes are insensitive to substrate concentration and operate in a near single-turnover regime in vitro. While lasso peptides are often touted for their stability to both chaotropic and thermal challenges, fuscanodin is found to undergo a conformational change consistent with lasso peptide unthreading in organic solvents at room temperature.
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http://dx.doi.org/10.1021/jacs.8b10724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475475PMC
January 2019

Structure of the Lasso Peptide Isopeptidase Identifies a Topology for Processing Threaded Substrates.

J Am Chem Soc 2016 12 7;138(50):16452-16458. Epub 2016 Dec 7.

Department of Biochemistry, ‡Institute for Genomic Biology and §Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States and.

Lasso peptides are a class of bioactive ribosomally synthesized and post-translationally modified peptides (RiPPs), with a threaded knot structure that is formed by an isopeptide bond attaching the N-terminus of the peptide to a side chain carboxylate. Some lasso peptide biosynthetic clusters harbor an enzyme that specifically hydrolyzes the isopeptide bond to yield the linear peptide. We describe here the 2.4 Å resolution structure of a lasso peptide isopeptidase revealing a topologically novel didomain architecture consisting of an open β-propeller appended to an α/β hydrolase domain. The 2.2 Å resolution cocrystal structure of an inactive variant in complex with a lasso peptide reveals deformation of the substrate, and reorganization of the enzyme active site, which exposes and orients the isopeptide bond for hydrolysis. Structure-based mutational analysis reveals how this enzyme recognizes the lasso peptide substrate by shape complementarity rather than through sequence specificity. The isopeptidase gene can be used to facilitate genome mining, as a network-based mining strategy queried with this sequence identified 87 putative lasso peptide biosynthetic clusters, 65 of which have not been previously described. Lastly, we validate this mining approach by heterologous expression of two clusters encoded within the genome of Asticcaucalis benevestitus, and demonstrate that both clusters produce lasso peptides.
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http://dx.doi.org/10.1021/jacs.6b10389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5245167PMC
December 2016

Elucidating the Specificity Determinants of the AtxE2 Lasso Peptide Isopeptidase.

J Biol Chem 2015 Dec 3;290(52):30806-12. Epub 2015 Nov 3.

From the Departments of Chemical and Biological Engineering, Molecular Biology, and

Lasso peptide isopeptidase is an enzyme that specifically hydrolyzes the isopeptide bond of lasso peptides, rendering these peptides linear. To carry out a detailed structure-activity analysis of the lasso peptide isopeptidase AtxE2 from Asticcacaulis excentricus, we solved NMR structures of its substrates astexin-2 and astexin-3. Using in vitro enzyme assays, we show that the C-terminal tail portion of these peptides is dispensable with regards to isopeptidase activity. A collection of astexin-2 and astexin-3 variants with alanine substitutions at each position within the ring and the loop was constructed, and we showed that all of these peptides except for one were cleaved by the isopeptidase. Thus, much like the lasso peptide biosynthetic enzymes, lasso peptide isopeptidase has broad substrate specificity. Quantitative analysis of the cleavage reactions indicated that alanine substitutions in loop positions of these peptides led to reduced cleavage, suggesting that the loop is serving as a recognition element for the isopeptidase.
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http://dx.doi.org/10.1074/jbc.M115.694083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692210PMC
December 2015

mTOR kinase structure, mechanism and regulation.

Nature 2013 May 1;497(7448):217-23. Epub 2013 May 1.

Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

The mammalian target of rapamycin (mTOR), a phosphoinositide 3-kinase-related protein kinase, controls cell growth in response to nutrients and growth factors and is frequently deregulated in cancer. Here we report co-crystal structures of a complex of truncated mTOR and mammalian lethal with SEC13 protein 8 (mLST8) with an ATP transition state mimic and with ATP-site inhibitors. The structures reveal an intrinsically active kinase conformation, with catalytic residues and a catalytic mechanism remarkably similar to canonical protein kinases. The active site is highly recessed owing to the FKBP12-rapamycin-binding (FRB) domain and an inhibitory helix protruding from the catalytic cleft. mTOR-activating mutations map to the structural framework that holds these elements in place, indicating that the kinase is controlled by restricted access. In vitro biochemistry shows that the FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with substrates to grant them access to the restricted active site. Rapamycin-FKBP12 inhibits the kinase by directly blocking substrate recruitment and by further restricting active-site access. The structures also reveal active-site residues and conformational changes that underlie inhibitor potency and specificity.
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http://dx.doi.org/10.1038/nature12122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4512754PMC
May 2013