Browsing by Author "Phillips, George N.Jr."
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Item A collagen glucosyltransferase drives lung adenocarcinoma progression in mice(Springer Nature, 2021) Guo, Hou-Fu; Bota-Rabassedas, Neus; Terajima, Masahiko; Leticia Rodriguez, B.; Gibbons, Don L.; Chen, Yulong; Banerjee, Priyam; Tsai, Chi-Lin; Tan, Xiaochao; Liu, Xin; Yu, Jiang; Tokmina-Roszyk, Michal; Stawikowska, Roma; Fields, Gregg B.; Miller, Mitchell D.; Wang, Xiaoyan; Lee, Juhoon; Dalby, Kevin N.; Creighton, Chad J.; Phillips, George N.Jr.; Tainer, John A.; Yamauchi, Mitsuo; Kurie, Jonathan M.Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen’s amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interactions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH-independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyltransferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A. CRISPR/Cas-9-mediated deletion of exon 13 A sharply reduces the growth and metastasis of LH2b-expressing LUADs in mice. These findings identify a previously unrecognized collagen GGT activity that drives LUAD progression.Item A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation(Oxford University Press, 2014) Desai, Kevin K.; Cheng, Chin L.; Bingman, Craig A.; Phillips, George N.Jr.; Raines, Ronald T.Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2′,3′-cyclic phosphate or 3′-phosphate termini to 5′-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3′-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal structure of P. horikoshii Archease that reveals a metal-binding site consisting of conserved carboxylates located at the protein tip. Substitution of the Archease metal-binding residues drastically reduced Archease-dependent activation of RtcB. Thus, evolution has sought to co-express archease and rtcB by creating a tRNA splicing operon.Item Characterization and Crystal Structure of a Nonheme Diiron Monooxygenase Involved in Platensimycin and Platencin Biosynthesis(American Chemical Society, 2019) Dong, Liao-Bin; Liu, Yu-Chen; Cepeda, Alexis J.; Kalkreuter, Edward; Deng, Ming-Rong; Rudolf, Jeffrey D.; Chang, Changsoo; Joachimiak, Andrzej; Phillips, George N.Jr.; Shen, BenNonheme diiron monooxygenases make up a rapidly growing family of oxygenases that are rarely identified in secondary metabolism. Herein, we report the in vivo, in vitro, and structural characterizations of a nonheme diiron monooxygenase, PtmU3, that installs a C-5 β-hydroxyl group in the unified biosynthesis of platensimycin and platencin, two highly functionalized diterpenoids that act as potent and selective inhibitors of bacterial and mammalian fatty acid synthases. This hydroxylation sets the stage for the subsequent A-ring cleavage step key to the unique diterpene-derived scaffolds of platensimycin and platencin. PtmU3 adopts an unprecedented triosephosphate isomerase (TIM) barrel structural fold for this class of enzymes and possesses a noncanonical diiron active site architecture with a saturated six-coordinate iron center lacking a μ-oxo bridge. This study reveals the first member of a previously unidentified superfamily of TIM-barrel-fold enzymes for metal-dependent dioxygen activation, with the majority predicted to act on CoA-linked substrates, thus expanding our knowledge of nature’s repertoire of nonheme diiron monooxygenases and TIM-barrel-fold enzymes.Item Crystal Structure of Human Protein N-Terminal Glutamine Amidohydrolase, an Initial Component of the N-End Rule Pathway(Public Library of Science, 2014) Park, Mi Seul; Bitto, Eduard; Kim, Kyung Rok; Bingman, Craig A.; Miller, Mitchell D.; Kim, Hyun-Jung; Han, Byung Woo; Phillips, George N.Jr.The N-end rule states that half-life of protein is determined by their N-terminal amino acid residue. N-terminal glutamine amidohydrolase (Ntaq) converts N-terminal glutamine to glutamate by eliminating the amine group and plays an essential role in the N-end rule pathway for protein degradation. Here, we report the crystal structure of human Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule at 1.5 Å resolution. The structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1. Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases.Item Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027(Springer Nature, 2016) Huang, Tingting; Chang, Chin-Yuan; Lohman, Jeremy R.; Rudolf, Jeffrey D.; Kim, Youngchang; Chang, Changsoo; Yang, Dong; Ma, Ming; Yan, Xiaohui; Crnovcic, Ivana; Bigelow, Lance; Clancy, Shonda; Bingman, Craig A.; Yennamalli, Ragothaman M.; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.Jr.; Shen, BenComparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us to propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase-associated enzymes, into an enzyme-sequestered enediyne core intermediate. These findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in nine-membered enediyne core biosynthesis.Item Crystal structure of SsfS6, the putative C-glycosyltransferase involved in SF2575 biosynthesis(Wiley, 2013) Wang, Fengbin; Zhou, Maoquan; Singh, Shanteri; Yennamalli, Ragothaman M.; Bingman, Craig A.; Thorson, Jon S.; Phillips, George N.Jr.The molecule known as SF2575 from Streptomyces sp. is a tetracycline polyketide natural product that displays antitumor activity against murine leukemia P388 in vivo. In the SF2575 biosynthetic pathway, SsfS6 has been implicated as the crucial C-glycosyltransferase (C-GT) that forms the CC glycosidic bond between the sugar and the SF2575 tetracycline-like scaffold. Here, we report the crystal structure of SsfS6 in the free form and in complex with TDP, both at 2.4 Å resolution. The structures reveal SsfS6 to adopt a GT-B fold wherein the TDP and docked putative aglycon are consistent with the overall C-glycosylation reaction. As one of only a few existing structures for C-glycosyltransferases, the structures described herein may serve as a guide to better understand and engineer C-glycosylation.Item Crystal structure of the protein At3g01520, a eukaryotic universal stress protein-like protein from arabidopsis thaliana in complex with AMP(Wiley, 2015) Kim, Do Jin; Bitto, Eduard; Bingman, Craig A.; Kim, Hyun-Jung; Han, Byung Woo; Phillips, George N.Jr.Members of the universal stress protein (USP) family are conserved in a phylogenetically diverse range of prokaryotes, fungi, protists, and plants and confer abilities to respond to a wide range of environmental stresses. Arabidopsis thaliana contains 44 USP domain-containing proteins, and USP domain is found either in a small protein with unknown physiological function or in an N-terminal portion of a multi-domain protein, usually a protein kinase. Here, we report the first crystal structure of a eukaryotic USP-like protein encoded from the gene At3g01520. The crystal structure of the protein At3g01520 was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 21.8% (Rfree = 26.1%) at 2.5 Å resolution. The crystal structure includes three At3g01520 protein dimers with one AMP molecule bound to each protomer, comprising a Rossmann-like α/β overall fold. The bound AMP and conservation of residues in the ATP-binding loop suggest that the protein At3g01520 also belongs to the ATP-binding USP subfamily members.Item Crystal Structure of the Zorbamycin-Binding Protein ZbmA, the Primary Self-Resistance Element in Streptomyces flavoviridis ATCC21892(American Chemical Society, 2015) Rudolf, Jeffrey D.; Bigelow, Lance; Chang, Changsoo; Cuff, Marianne E.; Lohman, Jeremy R.; Chang, Chin-Yuan; Ma, Ming; Yang, Dong; Clancy, Shonda; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.Jr.; Shen, BenThe bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity, while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at high resolutions of 1.90 and 1.65 Å, respectively. A comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein–ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM family of antitumor antibiotics.Item Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis(American Chemical Society, 2017) Annaval, Thibault; Rudolf, Jeffrey D.; Chang, Chin-Yuan; Lohman, Jeremy R.; Kim, Youngchang; Bigelow, Lance; Jedrzejczak, Robert; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.Jr.; Shen, BenEnediynes are potent natural product anticancer antibiotics, and are classified as 9- or 10-membered according to the size of their enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase (TE), and PKSE-associated enzymes. Although the divergence between 9- and 10-membered enediyne core biosynthesis remains unclear, it has been observed that nascent polyketide intermediates, tethered to the acyl carrier protein (ACP) domain of PKSE, could be released by TE in the absence of the PKSE-associated enzymes. In this study, we determined the crystal structure of SgcE10, the TE that participates in the biosynthesis of the 9-membered enediyne C-1027. Structural comparison of SgcE10 with CalE7 and DynE7, two TEs that participate in the biosynthesis of the 10-membered enediynes calicheamicin and dynemicin, respectively, revealed that they share a common α/β hot-dog fold. The amino acids involved in both substrate binding and catalysis are conserved among SgcE10, CalE7, and DynE7. The volume and the shape of the substrate-binding channel and active site in SgcE10, CalE7, and DynE7 confirm that TEs from both 9- and 10-membered enediyne biosynthetic machineries bind the linear form of similar ACP-tethered polyene intermediates. Taken together, these findings further support the proposal that the divergence between 9- and 10-membered enediyne core biosynthesis occurs beyond PKSE and TE catalysis.Item Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers(Nature Publishing Group, 2017) Fuller, Franklin D.; Gul, Sheraz; Chatterjee, Ruchira; Burgie, E.Sethe; Young, Iris D.; Lebrette, Hugo; Srinivas, Vivek; Brewster, Aaron S.; Michels-Clark, Tara; Clinger, Jonathan A.; Andi, Babak; Ibrahim, Mohamed; Pastor, Ernest; de Lichtenberg, Casper; Hussein, Rana; Pollock, Christopher J.; Zhang, Miao; Stan, Claudiu A.; Kroll, Thomas; Fransson, Thomas; Weninger, Clemens; Kubin, Markus; Aller, Pierre; Lassalle, Louise; Bräuer, Philipp; Miller, Mitchell D.; Amin, Muhamed; Koroidov, Sergey; Roessler, Christian G.; Allaire, Marc; Sierra, Raymond G.; Docker, Peter T.; Glownia, James M.; Nelson, Silke; Koglin, Jason E.; Zhu, Diling; Chollet, Matthieu; Song, Sanghoon; Lemke, Henrik; Liang, Mengning; Sokaras, Dimosthenis; Alonso-Mori, Roberto; Zouni, Athina; Messinger, Johannes; Bergmann, Uwe; Boal, Amie K.; Bollinger, J.Martin Jr.; Krebs, Carsten; Högbom, Martin; Phillips, George N.Jr.; Vierstra,, Richard D.; Sauter, Nicholas K.; Orville, Allen M.; Kern, Jan; Yachandra, Vittal K.; Yano, JunkoX-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.Item Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography(Springer Nature, 2018) Olmos, Jose Luis Jr.; Pandey, Suraj; Martin-Garcia, Jose M.; Calvey, George; Katz, Andrea; Knoska, Juraj; Kupitz, Christopher; Hunter, Mark S.; Liang, Mengning; Oberthuer, Dominik; Yefanov, Oleksandr; Wiedorn, Max; Heyman, Michael; Holl, Mark; Pande, Kanupriya; Barty, Anton; Miller, Mitchell D.; Stern, Stephan; Roy-Chowdhury, Shatabdi; Coe, Jesse; Nagaratnam, Nirupa; Zook, James; Verburgt, Jacob; Norwood, Tyler; Poudyal, Ishwor; Xu, David; Koglin, Jason E.; Seaberg, Matthew H.; Zhao, Yun; Bajt, Saša; Grant, Thomas; Mariani, Valerio; Nelson, Garrett; Subramanian, Ganesh; Bae, Euiyoung; Fromme, Raimund; Fung, Russell; Schwander, Peter; Frank, Matthias; White, Thomas A.; Weierstall, Uwe; Zatsepin, Nadia; Spence, John; Fromme, Petra; Chapman, Henry N.; Pollack, Lois; Tremblay, Lee; Ourmazd, Abbas; Phillips, George N.Jr.; Schmidt, MariusBACKGROUND: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. RESULTS: Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. CONCLUSIONS: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.Item Expression platforms for producing eukaryotic proteins: a comparison ofﾠE. coliﾠcell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies(Springer, 2015) Aceti, David J.; Bingman, Craig A.; Wrobel, Russell L.; Frederick, Ronnie O.; Makino, Shin-ichi; Nichols, Karl W.; Sahu, Sarata C.; Bergeman, Lai F.; Blommel, Paul G.; Cornilescu, Claudia C.; Gromek, Katarzyna A.; Seder, Kory D.; Hwang, Soyoon; Primm, John G.; Sabat, Grzegorz; Vojtik, Frank C.; Volkman, Brian F.; Zolnai, Zsolt; Phillips, George N.Jr.; Markley, John L.; Fox, Brian G.Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed.Item Functional Evolution of Ribonuclease Inhibitor: Insights from Birds and Reptiles(Elsevier, 2014) Lomax, Jo E.; Bianchetti, Christopher M.; Chang, Aram; Phillips, George N.Jr.; Fox, Brian G.; Raines, Ronald T.Ribonuclease inhibitor (RI) is a conserved protein of the mammalian cytosol. RI binds with high affinity to diverse secretory ribonucleases (RNases) and inhibits their enzymatic activity. Although secretory RNases are found in all vertebrates, the existence of a non-mammalian RI has been uncertain. Here, we report on the identification and characterization of RI homologs from chicken and anole lizard. These proteins bind to RNases from multiple species but exhibit much greater affinity for their cognate RNases than for mammalian RNases. To reveal the basis for this differential affinity, we determined the crystal structure of mouse, bovine, and chicken RI·RNase complexes to a resolution of 2.20, 2.21, and 1.92 Å, respectively. A combination of structural, computational, and bioinformatic analyses enabled the identification of two residues that appear to contribute to the differential affinity for RNases. We also found marked differences in oxidative instability between mammalian and non-mammalian RIs, indicating evolution toward greater oxygen sensitivity in RIs from mammalian species. Taken together, our results illuminate the structural and functional evolution of RI, along with its dynamic role in vertebrate biology.Item Improving the efficiency of molecular replacement by utilizing a new iterative transform phasing algorithm(International Union of Crystallography, 2016) He, Hongxing; Fang, Hengrui; Miller, Mitchell D.; Phillips, George N.Jr.; Su, Wu-PeiAn iterative transform method proposed previously for direct phasing of high-solvent-content protein crystals is employed for enhancing the molecular-replacement (MR) algorithm in protein crystallography. Target structures that are resistant to conventional MR due to insufficient similarity between the template and target structures might be tractable with this modified phasing method. Trial calculations involving three different structures are described to test and illustrate the methodology. The relationship of the approach toﾠPHENIX Phaser-MRﾠandﾠMR-Rosettaﾠis discussed.Item Loop dynamics of thymidine diphosphate-rhamnose 3′-O-methyltransferase (CalS11), an enzyme in calicheamicin biosynthesis(AIP Publishing, 2016) Han, Lu; Singh, Shanteri; Thorson, Jon S.; Phillips, George N.Jr.Structure analysis and ensemble refinement of the apo-structure of thymidine diphosphate (TDP)-rhamnose 3′-O-methyltransferase reveal a gate for substrate entry and product release. TDP-rhamnose 3′-O-methyltransferase (CalS11) catalyses a 3′-O-methylation of TDP-rhamnose, an intermediate in the biosynthesis of enediyne antitumor antibiotic calicheamicin. CalS11 operates at the sugar nucleotide stage prior to glycosylation step. Here, we present thecrystal structure of the apo form of CalS11 at 1.89 Å resolution. We propose that the L2 loop functions as a gate facilitating and/or providing specificity for substrate entry or promoting product release. Ensemble refinement analysis slightly improves the crystallographic refinement statistics and furthermore provides a compelling way to visualize the dynamic model of loop L2, supporting the understanding of its proposed role in catalysis.Item LucY: A Versatile New Fluorescent Reporter Protein(Public Library of Science, 2015) Auldridge, Michele E.; Cao, Hongnan; Sen, Saurabh; Franz, Laura P.; Bingman, Craig A.; Yennamalli, Ragothaman M.; Phillips, George N.Jr.; Mead, David; Steinmetz, Eric J.We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276 nm, 377 nm, and 460 nm and a single emission peak at 530 nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.Item Moving beyond static snapshots: Protein dynamics and the Protein Data Bank(Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology, 2021) Miller, Mitchell D.; Phillips, George N.Jr.Proteins are the molecular machines of living systems. Their dynamics are an intrinsic part of their evolutionary selection in carrying out their biological functions. Although the dynamics are more difficult to observe than a static, average structure, we are beginning to observe these dynamics and form sound mechanistic connections between structure, dynamics, and function. This progress is highlighted in case studies from myoglobin and adenylate kinase to the ribosome and molecular motors where these molecules are being probed with a multitude of techniques across many timescales. New approaches to time-resolved crystallography are allowing simple “movies” to be taken of proteins in action, and new methods of mapping the variations in cryo-electron microscopy are emerging to reveal a more complete description of life’s machines. The results of these new methods are aided in their dissemination by continual improvements in curation and distribution by the Protein Data Bank and their partners around the world.Item Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme(Springer Nature, 2018) Wang, Nan L.; Rudolf, Jeffrey D.; Dong, Liao-Bin; Osipiuk, Jerzy; Hatzos-Skintges, Catherine; Endres, Michael; Chang, Chin-Yuan; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.Jr.; Shen, BenAcyl-coenzyme A (CoA) ligases catalyze the activation of carboxylic acids via a two-step reaction of adenylation followed by thioesterification. Here, we report the discovery of a non-adenylating acyl-CoA ligase PtmA2 and the functional separation of an acyl-CoA ligase reaction. Both PtmA1 and PtmA2, two acyl-CoA ligases from the biosynthetic pathway of platensimycin and platencin, are necessary for the two steps of CoA activation. Gene inactivation of ptmA1 and ptmA2 resulted in the accumulation of free acid and adenylate intermediates, respectively. Enzymatic and structural characterization of PtmA2 confirmed its ability to only catalyze thioesterification. Structural characterization of PtmA2 revealed it binds both free acid and adenylate substrates and undergoes the established mechanism of domain alternation. Finally, site-directed mutagenesis restored both the adenylation and complete CoA activation reactions. This study challenges the currently accepted paradigm of adenylating enzymes and inspires future investigations on functionally separated acyl-CoA ligases and their ramifications in biology.Item Pro-metastatic collagen lysyl hydroxylase dimer assemblies stabilized by Fe2+-binding(Springer Nature, 2018) Guo, Hou-Fu; Tsai, Chi-Lin; Terajima, Masahiko; Tan, Xiaochao; Banerjee, Priyam; Miller, Mitchell D.; Liu, Xin; Yu, Jiang; Byemerwa, Jovita; Alvarado, Sarah K.; Kaoud, Tamer S.; Dalby, Kevin N.; Bota-Rabassedas, Neus; Chen, Yulong; Yamauchi, Mitsuo; Tainer, John A.; Phillips, George N.Jr.; Kurie, Jonathan M.Collagen lysyl hydroxylases (LH1-3) are Fe2+- and 2-oxoglutarate (2-OG)-dependent oxygenases that maintain extracellular matrix homeostasis. High LH2 levels cause stable collagen cross-link accumulations that promote fibrosis and cancer progression. However, developing LH antagonists will require structural insights. Here, we report a 2 Å crystal structure and X-ray scattering on dimer assemblies for the LH domain of L230 in Acanthamoeba polyphaga mimivirus. Loop residues in the double-stranded β-helix core generate a tail-to-tail dimer. A stabilizing hydrophobic leucine locks into an aromatic tyrosine-pocket on the opposite subunit. An active site triad coordinates Fe2+. The two active sites flank a deep surface cleft that suggest dimerization creates a collagen-binding site. Loss of Fe2+-binding disrupts the dimer. Dimer disruption and charge reversal in the cleft increase Km and reduce LH activity. Ectopic L230 expression in tumors promotes collagen cross-linking and metastasis. These insights suggest inhibitor targets for fibrosis and cancer.Item Pyridine-2,6-Dithiocarboxylic Acid and Its Metal Complexes: New Inhibitors of New Delhi Metallo -Lactamase-1(MDPI, 2020) Thomas, Chris S.; Braun, Doug R.; Olmos, Jose Luis Jr.; Rajski, Scott R.; Phillips, George N.Jr.; Andes, David N.; Bugni, Tim S.Carbapenem-resistant Enterobacteriaceae continue to threaten human health worldwide with few effective treatment options. New Delhi metallo--lactamase (NDM) enzymes are a contributing element that drive resistance to many -lactam- and carbapenem-based antimicrobials. Many NDM inhibitors are known, yet none are clinically viable. In this study, we present and characterize a new class of NDM-1 inhibitors based on a pyridine-2,6-dithiocarboxylic acid metal complex scaffold. These complexes display varied and unique activity profiles against NDM-1 in kinetic assays and serve to increase the effectiveness of meropenem, an established antibacterial, in assays using clinical Enterobacteriaceae isolates.