Publications by authors named "Thomas B Acton"

98 Publications

High-Throughput Production of Proteins in E. coli for Structural Studies.

Methods Mol Biol 2017 ;1586:359-371

Evotec (U.S.) Inc., Building 303B, College Road East, Princeton, NJ, 08540, USA.

We have developed a standardized and efficient workflow for high-throughput (HT) protein expression in E. coli and parallel purification which can be tailored to the downstream application of the target proteins. It includes a one-step purification for the purposes of functional assays and a two-step protocol for crystallographic studies, with the option of on-column tag removal.
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http://dx.doi.org/10.1007/978-1-4939-6887-9_24DOI Listing
February 2018

X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition.

Proteins 2017 04 3;85(4):647-656. Epub 2017 Feb 3.

Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway, New Jersey, 08854.

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1-44) and an HHCC zinc-finger NTD (residues 45-105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1-105 (NTR ) and 8-105 (NTR ) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR packs as a dimer and NTR packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel β-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three β-strands form an extended β-sheet with another β-strand in the HHCC Zn binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647-656. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/prot.25245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5357174PMC
April 2017

Codon influence on protein expression in E. coli correlates with mRNA levels.

Nature 2016 Jan 13;529(7586):358-363. Epub 2016 Jan 13.

Department of Biological Sciences, 702A Fairchild Center, MC2434, Columbia University, New York, NY 10027, USA.

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054687PMC
http://dx.doi.org/10.1038/nature16509DOI Listing
January 2016

A community resource of experimental data for NMR / X-ray crystal structure pairs.

Protein Sci 2016 Jan 22;25(1):30-45. Epub 2015 Sep 22.

Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers, the State University of New Jersey, Piscataway, New Jersey, 08854, USA.

We have developed an online NMR / X-ray Structure Pair Data Repository. The NIGMS Protein Structure Initiative (PSI) has provided many valuable reagents, 3D structures, and technologies for structural biology. The Northeast Structural Genomics Consortium was one of several PSI centers. NESG used both X-ray crystallography and NMR spectroscopy for protein structure determination. A key goal of the PSI was to provide experimental structures for at least one representative of each of hundreds of targeted protein domain families. In some cases, structures for identical (or nearly identical) constructs were determined by both NMR and X-ray crystallography. NMR spectroscopy and X-ray diffraction data for 41 of these "NMR / X-ray" structure pairs determined using conventional triple-resonance NMR methods with extensive sidechain resonance assignments have been organized in an online NMR / X-ray Structure Pair Data Repository. In addition, several NMR data sets for perdeuterated, methyl-protonated protein samples are included in this repository. As an example of the utility of this repository, these data were used to revisit questions about the precision and accuracy of protein NMR structures first outlined by Levy and coworkers several years ago (Andrec et al., Proteins 2007;69:449-465). These results demonstrate that the agreement between NMR and X-ray crystal structures is improved using modern methods of protein NMR spectroscopy. The NMR / X-ray Structure Pair Data Repository will provide a valuable resource for new computational NMR methods development.
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http://dx.doi.org/10.1002/pro.2774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815321PMC
January 2016

The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS.

Structure 2015 Aug 9;23(8):1382-1393. Epub 2015 Jul 9.

Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA; Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA. Electronic address:

RAS binding is a critical step in the activation of BRAF protein serine/threonine kinase and stimulation of the mitogen-activated protein kinase signaling pathway. Mutations in both RAS and BRAF are associated with many human cancers. Here, we report the solution nuclear magnetic resonance (NMR) and X-ray crystal structures of the RAS-binding domain (RBD) from human BRAF. We further studied the complex between BRAF RBD and the GppNHp bound form of HRAS in solution. Backbone, side-chain, and (19)F NMR chemical shift perturbations reveal unexpected changes distal to the RAS-binding face that extend through the core of the RBD structure. Moreover, backbone amide hydrogen/deuterium exchange NMR data demonstrate conformational ensemble changes in the RBD core structure upon complex formation. These changes in BRAF RBD reveal a basis for allosteric regulation of BRAF structure and function, and suggest a mechanism by which RAS binding can signal the drastic domain rearrangements required for activation of BRAF kinase.
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http://dx.doi.org/10.1016/j.str.2015.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963008PMC
August 2015

The second round of Critical Assessment of Automated Structure Determination of Proteins by NMR: CASD-NMR-2013.

J Biomol NMR 2015 Aug 14;62(4):413-24. Epub 2015 Jun 14.

Department of Biochemistry, School of Biological Sciences, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester, LE1 9HN, UK.

The second round of the community-wide initiative Critical Assessment of automated Structure Determination of Proteins by NMR (CASD-NMR-2013) comprised ten blind target datasets, consisting of unprocessed spectral data, assigned chemical shift lists and unassigned NOESY peak and RDC lists, that were made available in both curated (i.e. manually refined) or un-curated (i.e. automatically generated) form. Ten structure calculation programs, using fully automated protocols only, generated a total of 164 three-dimensional structures (entries) for the ten targets, sometimes using both curated and un-curated lists to generate multiple entries for a single target. The accuracy of the entries could be established by comparing them to the corresponding manually solved structure of each target, which was not available at the time the data were provided. Across the entire data set, 71 % of all entries submitted achieved an accuracy relative to the reference NMR structure better than 1.5 Å. Methods based on NOESY peak lists achieved even better results with up to 100% of the entries within the 1.5 Å threshold for some programs. However, some methods did not converge for some targets using un-curated NOESY peak lists. Over 90% of the entries achieved an accuracy better than the more relaxed threshold of 2.5 Å that was used in the previous CASD-NMR-2010 round. Comparisons between entries generated with un-curated versus curated peaks show only marginal improvements for the latter in those cases where both calculations converged.
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http://dx.doi.org/10.1007/s10858-015-9953-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569658PMC
August 2015

A hybrid NMR/SAXS-based approach for discriminating oligomeric protein interfaces using Rosetta.

Proteins 2015 Feb 19;83(2):309-17. Epub 2014 Dec 19.

Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, 08854.

Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology-based methods, protein-protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet-V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061451PMC
http://dx.doi.org/10.1002/prot.24719DOI Listing
February 2015

Structural and functional characterization of DUF1471 domains of Salmonella proteins SrfN, YdgH/SssB, and YahO.

PLoS One 2014 10;9(7):e101787. Epub 2014 Jul 10.

Fundamental and Computational Sciences Directorate, Pacific Northwest National Laboratory, Richland, Washington, United States of America.

Bacterial species in the Enterobacteriaceae typically contain multiple paralogues of a small domain of unknown function (DUF1471) from a family of conserved proteins also known as YhcN or BhsA/McbA. Proteins containing DUF1471 may have a single or three copies of this domain. Representatives of this family have been demonstrated to play roles in several cellular processes including stress response, biofilm formation, and pathogenesis. We have conducted NMR and X-ray crystallographic studies of four DUF1471 domains from Salmonella representing three different paralogous DUF1471 subfamilies: SrfN, YahO, and SssB/YdgH (two of its three DUF1471 domains: the N-terminal domain I (residues 21-91), and the C-terminal domain III (residues 244-314)). Notably, SrfN has been shown to have a role in intracellular infection by Salmonella Typhimurium. These domains share less than 35% pairwise sequence identity. Structures of all four domains show a mixed α+β fold that is most similar to that of bacterial lipoprotein RcsF. However, all four DUF1471 sequences lack the redox sensitive cysteine residues essential for RcsF activity in a phospho-relay pathway, suggesting that DUF1471 domains perform a different function(s). SrfN forms a dimer in contrast to YahO and SssB domains I and III, which are monomers in solution. A putative binding site for oxyanions such as phosphate and sulfate was identified in SrfN, and an interaction between the SrfN dimer and sulfated polysaccharides was demonstrated, suggesting a direct role for this DUF1471 domain at the host-pathogen interface.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101787PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092069PMC
March 2015

Solution NMR structures of immunoglobulin-like domains 7 and 12 from obscurin-like protein 1 contribute to the structural coverage of the Human Cancer Protein Interaction Network.

J Struct Funct Genomics 2014 Dec 3;15(4):209-14. Epub 2014 Jul 3.

Department of Chemistry, The State University of New York at Buffalo and Northeast Structural Genomics Consortium, Buffalo, NY, 14260, USA.

High-quality solution NMR structures of immunoglobulin-like domains 7 and 12 from human obscurin-like protein 1 were solved. The two domains share 30% sequence identity and their structures are, as expected, rather similar. The new structures contribute to structural coverage of human cancer associated proteins. Mutations of Arg 812 in domain 7 cause the rare 3-M syndrome, and this site is located in a surface area predicted to be involved in protein-protein interactions.
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http://dx.doi.org/10.1007/s10969-014-9185-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4945113PMC
December 2014

Solution NMR structures of homeodomains from human proteins ALX4, ZHX1, and CASP8AP2 contribute to the structural coverage of the Human Cancer Protein Interaction Network.

J Struct Funct Genomics 2014 Dec 19;15(4):201-7. Epub 2014 Jun 19.

Department of Chemistry, The State University of New York at Buffalo and Northeast Structural Genomics Consortium, Buffalo, NY, 14260, USA.

High-quality solution NMR structures of three homeodomains from human proteins ALX4, ZHX1 and CASP8AP2 were solved. These domains were chosen as targets of a biomedical theme project pursued by the Northeast Structural Genomics Consortium. This project focuses on increasing the structural coverage of human proteins associated with cancer.
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http://dx.doi.org/10.1007/s10969-014-9184-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239167PMC
December 2014

Polypeptide backbone, C(β) and methyl group resonance assignments of the 24 kDa plectin repeat domain 6 from human protein plectin.

Biomol NMR Assign 2015 Apr 11;9(1):135-138. Epub 2014 Apr 11.

Department of Chemistry, The State University of New York at Buffalo, and Northeast Structural Genomics Consortium, Buffalo, NY 14260, USA.

The 500 kDa protein plectin is essential for the cytoskeletal organization of most mammalian cells and it is up-regulated in some types of cancer. Here, we report nearly complete sequence-specific polypeptide backbone, (13)C(β) and methyl group resonance assignments for 24 kDa human plectin(4403-4606) containing the C-terminal plectin repeat domain 6.
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http://dx.doi.org/10.1007/s12104-014-9559-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4194182PMC
April 2015

Structure and function of human DnaJ homologue subfamily a member 1 (DNAJA1) and its relationship to pancreatic cancer.

Biochemistry 2014 Mar 19;53(8):1360-72. Epub 2014 Feb 19.

Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.

Pancreatic cancer has a dismal 5 year survival rate of 5.5% that has not been improved over the past 25 years despite an enormous amount of effort. Thus, there is an urgent need to identify truly novel yet druggable protein targets for drug discovery. The human protein DnaJ homologue subfamily A member 1 (DNAJA1) was previously shown to be downregulated 5-fold in pancreatic cancer cells and has been targeted as a biomarker for pancreatic cancer, but little is known about the specific biological function for DNAJA1 or the other members of the DnaJ family encoded in the human genome. Our results suggest the overexpression of DNAJA1 suppresses the stress response capabilities of the oncogenic transcription factor, c-Jun, and results in the diminution of cell survival. DNAJA1 likely activates a DnaK protein by forming a complex that suppresses the JNK pathway, the hyperphosphorylation of c-Jun, and the anti-apoptosis state found in pancreatic cancer cells. A high-quality nuclear magnetic resonance solution structure of the J-domain of DNAJA1 combined with a bioinformatics analysis and a ligand affinity screen identifies a potential DnaK binding site, which is also predicted to overlap with an inhibitory binding site, suggesting DNAJA1 activity is highly regulated.
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http://dx.doi.org/10.1021/bi401329aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985919PMC
March 2014

Allosteric regulation and substrate activation in cytosolic nucleotidase II from Legionella pneumophila.

FEBS J 2014 Mar 17;281(6):1613-1628. Epub 2014 Feb 17.

Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, Karnataka, India.

Unlabelled: Cytosolic nucleotidase II (cN-II) from Legionella pneumophila (Lp) catalyzes the hydrolysis of GMP and dGMP displaying sigmoidal curves, whereas catalysis of IMP hydrolysis displayed a biphasic curve in the initial rate versus substrate concentration plots. Allosteric modulators of mammalian cN-II did not activate LpcN-II although GTP, GDP and the substrate GMP were specific activators. Crystal structures of the tetrameric LpcN-II revealed an activator-binding site at the dimer interface. A double mutation in this allosteric-binding site abolished activation, confirming the structural observations. The substrate GMP acting as an activator, partitioning between the allosteric and active site, is the basis for the sigmoidicity of the initial velocity versus GMP concentration plot. The LpcN-II tetramer showed differences in subunit organization upon activator binding that are absent in the activator-bound human cN-II structure. This is the first observation of a structural change induced by activator binding in cN-II that may be the molecular mechanism for enzyme activation.

Database: The coordinates and structure factors reported in this paper have been submitted to the Protein Data Bank under the accession numbers 2BDE and 4G63. The accession number of GMP complexed LpcN-II is 4OHF.

Structured Digital Abstract: LpcN-II and LpcN-II bind by molecular sieving (View interaction) LpcN-II and LpcN-II bind by x-ray crystallography (View interaction) [Structured digital abstract was added on 5 March 2014 after original online publication].
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http://dx.doi.org/10.1111/febs.12727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982195PMC
March 2014

Structural and biochemical characterization of the bilin lyase CpcS from Thermosynechococcus elongatus.

Biochemistry 2013 Dec 19;52(48):8663-76. Epub 2013 Nov 19.

Department of Biological Sciences, University of New Orleans , New Orleans, LA 70148, United States.

Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded β barrel with two alpha helices and belongs to the lipocalin structural family. TeCpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. TeCpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of TeCpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and TeCpcS was produced.
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http://dx.doi.org/10.1021/bi401192zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3932240PMC
December 2013

DisMeta: a meta server for construct design and optimization.

Methods Mol Biol 2014 ;1091:3-16

Center for Advanced Biotechnology and Medicine, Northeast Structural Genomics Consortium, Rutgers University, Piscataway, NJ, USA.

Intrinsically disordered or unstructured regions in proteins are both common and biologically important, particularly in regulation, signaling, and modulating intermolecular recognition processes. From a practical point of view, however, such disordered regions often can pose significant challenges for crystallization. Disordered regions are also detrimental to NMR spectral quality, complicating the analysis of resonance assignments and three-dimensional protein structures by NMR methods. The DisMeta Server has been used by Northeastern Structural Genomics (NESG) consortium as a primary tool for construct design and optimization in preparing samples for both NMR and crystallization studies. It is a meta-server that generates a consensus analysis of eight different sequence-based disorder predictors to identify regions that are likely to be disordered. DisMeta also identifies predicted secretion signal peptides, transmembrane segments, and low-complexity regions. Identification of disordered regions, by either experimental or computational methods, is an important step in the NESG structure production pipeline, allowing the rational design of protein constructs that have improved expression and solubility, improved crystallization, and better quality NMR spectra.
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http://dx.doi.org/10.1007/978-1-62703-691-7_1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4115584PMC
June 2014

Solution NMR structure of CD1104B from pathogenic Clostridium difficile reveals a distinct α-helical architecture and provides first structural representative of protein domain family PF14203.

J Struct Funct Genomics 2013 Dec 19;14(4):155-60. Epub 2013 Sep 19.

Department of Chemistry, The State University of New York at Buffalo and Northeast Structural Genomics Consortium, Buffalo, NY, 14260, USA.

A high-quality structure of the 68-residue protein CD1104B from Clostridium difficile strain 630 exhibits a distinct all α-helical fold. The structure presented here is the first representative of bacterial protein domain family PF14203 (currently 180 members) of unknown function (DUF4319) and reveals that the side-chains of the only two strictly conserved residues (Glu 8 and Lys 48) form a salt bridge. Moreover, these two residues are located in the vicinity of the largest surface cleft which is predicted to contribute to a surface area involved in protein-protein interactions. This, along with its coding in transposon CTn4, suggests that CD1104B (and very likely all members of Pfam 14203) functions by interacting with other proteins required for the transfer of transposons between different bacterial species.
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http://dx.doi.org/10.1007/s10969-013-9164-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3844015PMC
December 2013

Structure determination and biochemical characterization of a putative HNH endonuclease from Geobacter metallireducens GS-15.

PLoS One 2013 6;8(9):e72114. Epub 2013 Sep 6.

New England Biolabs, Inc. Research Department, Ipswich, Massachusetts, United States of America.

The crystal structure of a putative HNH endonuclease, Gmet_0936 protein from Geobacter metallireducens GS-15, has been determined at 2.6 Å resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel β-sheet that are surrounded by two helices on each face, and reveals a Zn ion bound in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76, which likely plays an important structural role in stabilizing the overall conformation. Structural homologs of Gmet_0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15-20% amino acid sequence identity. An overlay of Gmet_0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon) are conserved. However, Gmet_0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet_0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. Gmet_0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn²⁺-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate appears to be acid and heat-treated DNA with AP sites, suggesting Gmet_0936 may be a DNA repair enzyme.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072114PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765158PMC
June 2014

A bidirectional system for the dynamic small molecule control of intracellular fusion proteins.

ACS Chem Biol 2013 Oct 26;8(10):2293-2300. Epub 2013 Aug 26.

Department of Molecular Cellular and Developmental Biology, Yale University, 219 Prospect Street, New Haven, CT 06511, USA.

Small molecule control of intracellular protein levels allows temporal and dose-dependent regulation of protein function. Recently, we developed a method to degrade proteins fused to a mutant dehalogenase (HaloTag2) using small molecule hydrophobic tags (HyTs). Here, we introduce a complementary method to stabilize the same HaloTag2 fusion proteins, resulting in a unified system allowing bidirectional control of cellular protein levels in a temporal and dose-dependent manner. From a small molecule screen, we identified N-(3,5-dichloro-2-ethoxybenzyl)-2H-tetrazol-5-amine as a nanomolar HALoTag2 Stabilizer (HALTS1) that reduces the Hsp70:HaloTag2 interaction, thereby preventing HaloTag2 ubiquitination. Finally, we demonstrate the utility of the HyT/HALTS system in probing the physiological role of therapeutic targets by modulating HaloTag2-fused oncogenic H-Ras, which resulted in either the cessation (HyT) or acceleration (HALTS) of cellular transformation. In sum, we present a general platform to study protein function, whereby any protein of interest fused to HaloTag2 can be either degraded 10-fold or stabilized 5-fold using two corresponding compounds.
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http://dx.doi.org/10.1021/cb400569kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113957PMC
October 2013

Solution NMR structures provide first structural coverage of the large protein domain family PF08369 and complementary structural coverage of dark operative protochlorophyllide oxidoreductase complexes.

J Struct Funct Genomics 2013 Sep 21;14(3):119-26. Epub 2013 Aug 21.

Department of Chemistry, The State University of New York at Buffalo, Buffalo, NY 14260, USA.

High-quality NMR structures of the C-terminal domain comprising residues 484-537 of the 537-residue protein Bacterial chlorophyll subunit B (BchB) from Chlorobium tepidum and residues 9-61 of 61-residue Asr4154 from Nostoc sp. (strain PCC 7120) exhibit a mixed α/β fold comprised of three α-helices and a small β-sheet packed against second α-helix. These two proteins share 29% sequence similarity and their structures are globally quite similar. The structures of BchB(484-537) and Asr4154(9-61) are the first representative structures for the large protein family (Pfam) PF08369, a family of unknown function currently containing 610 members in bacteria and eukaryotes. Furthermore, BchB(484-537) complements the structural coverage of the dark-operating protochlorophyllide oxidoreductase.
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http://dx.doi.org/10.1007/s10969-013-9159-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982801PMC
September 2013

Crystal structures of malonyl-coenzyme A decarboxylase provide insights into its catalytic mechanism and disease-causing mutations.

Structure 2013 Jul 20;21(7):1182-92. Epub 2013 Jun 20.

Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.

Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design.
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http://dx.doi.org/10.1016/j.str.2013.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701320PMC
July 2013

Two Fe-S clusters catalyze sulfur insertion by radical-SAM methylthiotransferases.

Nat Chem Biol 2013 May 31;9(5):333-8. Epub 2013 Mar 31.

Northeast Structural Genomics Consortium, New York, New York, USA.

How living organisms create carbon-sulfur bonds during the biosynthesis of critical sulfur-containing compounds is still poorly understood. The methylthiotransferases MiaB and RimO catalyze sulfur insertion into tRNAs and ribosomal protein S12, respectively. Both belong to a subgroup of radical-S-adenosylmethionine (radical-SAM) enzymes that bear two [4Fe-4S] clusters. One cluster binds S-adenosylmethionine and generates an Ado• radical via a well-established mechanism. However, the precise role of the second cluster is unclear. For some sulfur-inserting radical-SAM enzymes, this cluster has been proposed to act as a sacrificial source of sulfur for the reaction. In this paper, we report parallel enzymological, spectroscopic and crystallographic investigations of RimO and MiaB, which provide what is to our knowledge the first evidence that these enzymes are true catalysts and support a new sulfation mechanism involving activation of an exogenous sulfur cosubstrate at an exchangeable coordination site on the second cluster, which remains intact during the reaction.
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http://dx.doi.org/10.1038/nchembio.1229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118475PMC
May 2013

Structures of apo- and ssDNA-bound YdbC from Lactococcus lactis uncover the function of protein domain family DUF2128 and expand the single-stranded DNA-binding domain proteome.

Nucleic Acids Res 2013 Feb 8;41(4):2756-68. Epub 2013 Jan 8.

Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, and the Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.

Single-stranded DNA (ssDNA) binding proteins are important in basal metabolic pathways for gene transcription, recombination, DNA repair and replication in all domains of life. Their main cellular role is to stabilize melted duplex DNA and protect genomic DNA from degradation. We have uncovered the molecular function of protein domain family domain of unknown function DUF2128 (PF09901) as a novel ssDNA binding domain. This bacterial domain strongly associates into a dimer and presents a highly positively charged surface that is consistent with its function in non-specific ssDNA binding. Lactococcus lactis YdbC is a representative of DUF2128. The solution NMR structures of the 20 kDa apo-YdbC dimer and YdbC:dT(19)G(1) complex were determined. The ssDNA-binding energetics to YdbC were characterized by isothermal titration calorimetry. YdbC shows comparable nanomolar affinities for pyrimidine and mixed oligonucleotides, and the affinity is sufficiently strong to disrupt duplex DNA. In addition, YdbC binds with lower affinity to ssRNA, making it a versatile nucleic acid-binding domain. The DUF2128 family is related to the eukaryotic nuclear protein positive cofactor 4 (PC4) family and to the PUR family both by fold similarity and molecular function.
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http://dx.doi.org/10.1093/nar/gks1348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575825PMC
February 2013

Solution NMR structure of the helicase associated domain BVU_0683(627-691) from Bacteroides vulgatus provides first structural coverage for protein domain family PF03457 and indicates domain binding to DNA.

J Struct Funct Genomics 2013 Mar 16;14(1):19-24. Epub 2012 Nov 16.

Department of Chemistry, The State University of New York at Buffalo, Buffalo, NY 14260, USA.

A high-quality NMR structure of the helicase associated (HA) domain comprising residues 627-691 of the 753-residue protein BVU_0683 from Bacteroides vulgatus exhibits an all α-helical fold. The structure presented here is the first representative for the large protein domain family PF03457 (currently 742 members) of HA domains. Comparison with structurally similar proteins supports the hypothesis that HA domains bind to DNA and that binding specificity varies greatly within the family of HA domains constituting PF03457.
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http://dx.doi.org/10.1007/s10969-012-9148-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3637686PMC
March 2013

Principles for designing ideal protein structures.

Nature 2012 Nov;491(7423):222-7

University of Washington, Department of Biochemistry and Howard Hughes Medical Institute, Seattle, Washington 98195, USA.

Unlike random heteropolymers, natural proteins fold into unique ordered structures. Understanding how these are encoded in amino-acid sequences is complicated by energetically unfavourable non-ideal features--for example kinked α-helices, bulged β-strands, strained loops and buried polar groups--that arise in proteins from evolutionary selection for biological function or from neutral drift. Here we describe an approach to designing ideal protein structures stabilized by completely consistent local and non-local interactions. The approach is based on a set of rules relating secondary structure patterns to protein tertiary motifs, which make possible the design of funnel-shaped protein folding energy landscapes leading into the target folded state. Guided by these rules, we designed sequences predicted to fold into ideal protein structures consisting of α-helices, β-strands and minimal loops. Designs for five different topologies were found to be monomeric and very stable and to adopt structures in solution nearly identical to the computational models. These results illuminate how the folding funnels of natural proteins arise and provide the foundation for engineering a new generation of functional proteins free from natural evolution.
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http://dx.doi.org/10.1038/nature11600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3705962PMC
November 2012

Structure of a specialized acyl carrier protein essential for lipid A biosynthesis with very long-chain fatty acids in open and closed conformations.

Biochemistry 2012 Sep 7;51(37):7239-49. Epub 2012 Sep 7.

Department of Chemistry and Biochemistry, Northeast Structural Genomics Consortium, Miami University, Oxford, Ohio 45056, United States.

The solution nuclear magnetic resonance (NMR) structures and backbone (15)N dynamics of the specialized acyl carrier protein (ACP), RpAcpXL, from Rhodopseudomonas palustris, in both the apo form and holo form modified by covalent attachment of 4'-phosphopantetheine at S37, are virtually identical, monomeric, and correspond to the closed conformation. The structures have an extra α-helix compared to the archetypical ACP from Escherichia coli, which has four helices, resulting in a larger opening to the hydrophobic cavity. Chemical shift differences between apo- and holo-RpAcpXL indicated some differences in the hinge region between α2 and α3 and in the hydrophobic cavity environment, but corresponding changes in nuclear Overhauser effect cross-peak patterns were not detected. In contrast to the NMR structures, apo-RpAcpXL was observed in an open conformation in crystals that diffracted to 2.0 Å resolution, which resulted from movement of α3. On the basis of the crystal structure, the predicted biological assembly is a homodimer. Although the possible biological significance of dimerization is unknown, there is potential that the resulting large shared hydrophobic cavity could accommodate the very long-chain fatty acid (28-30 carbons) that this specialized ACP is known to synthesize and transfer to lipid A. These structures are the first representatives of the AcpXL family and the first to indicate that dimerization may be important for the function of these specialized ACPs.
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http://dx.doi.org/10.1021/bi300546bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104962PMC
September 2012

Solution NMR and X-ray crystal structures of Pseudomonas syringae Pspto_3016 from protein domain family PF04237 (DUF419) adopt a "double wing" DNA binding motif.

J Struct Funct Genomics 2012 Sep 3;13(3):155-62. Epub 2012 Aug 3.

Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.

The protein Pspto_3016 is a 117-residue member of the protein domain family PF04237 (DUF419), which is to date a functionally uncharacterized family of proteins. In this report, we describe the structure of Pspto_3016 from Pseudomonas syringae solved by both solution NMR and X-ray crystallography at 2.5 Å resolution. In both cases, the structure of Pspto_3016 adopts a "double wing" α/β sandwich fold similar to that of protein YjbR from Escherichia coli and to the C-terminal DNA binding domain of the MotA transcription factor (MotCF) from T4 bacteriophage, along with other uncharacterized proteins. Pspto_3016 was selected by the Protein Structure Initiative of the National Institutes of Health and the Northeast Structural Genomics Consortium (NESG ID PsR293).
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http://dx.doi.org/10.1007/s10969-012-9140-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697073PMC
September 2012

Crystal structure of a catalytically active GG(D/E)EF diguanylate cyclase domain from Marinobacter aquaeolei with bound c-di-GMP product.

J Struct Funct Genomics 2012 Sep 29;13(3):177-83. Epub 2012 Jul 29.

Department of Biological Sciences, The Northeast Structural Genomics Consortium Columbia University, New York, NY, 10032, USA.

Recent studies of signal transduction in bacteria have revealed a unique second messenger, bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), which regulates transitions between motile states and sessile states, such as biofilms. C-di-GMP is synthesized from two GTP molecules by diguanylate cyclases (DGC). The catalytic activity of DGCs depends on a conserved GG(D/E)EF domain, usually part of a larger multi-domain protein organization. The domains other than the GG(D/E)EF domain often control DGC activation. This paper presents the 1.83 Å crystal structure of an isolated catalytically competent GG(D/E)EF domain from the A1U3W3_MARAV protein from Marinobacter aquaeolei. Co-crystallization with GTP resulted in enzymatic synthesis of c-di-GMP. Comparison with previously solved DGC structures shows a similar orientation of c-di-GMP bound to an allosteric regulatory site mediating feedback inhibition of the enzyme. Biosynthesis of c-di-GMP in the crystallization reaction establishes that the enzymatic activity of this DGC domain does not require interaction with regulatory domains.
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http://dx.doi.org/10.1007/s10969-012-9136-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683829PMC
September 2012

Three structural representatives of the PF06855 protein domain family from Staphyloccocus aureus and Bacillus subtilis have SAM domain-like folds and different functions.

J Struct Funct Genomics 2012 Sep 29;13(3):163-70. Epub 2012 Jul 29.

Center for Advanced Biotechnology and Medicine, The State University of New Jersey, Piscataway, 08854, USA.

Protein domain family PF06855 (DUF1250) is a family of small domains of unknown function found only in bacteria, and mostly in the order Bacillales and Lactobacillales. Here we describe the solution NMR or X-ray crystal structures of three representatives of this domain family, MW0776 and MW1311 from Staphyloccocus aureus and yozE from Bacillus subtilis. All three proteins adopt a four-helix motif similar to sterile alpha motif (SAM) domains. Phylogenetic analysis classifies MW1311 and yozE as functionally equivalent proteins of the UPF0346 family of unknown function, but excludes MW0776, which likely has a different biological function. Our structural characterization of the three domains supports this separation of function. The structures of MW0776, MW1311, and yozE constitute the first structural representatives from this protein domain family.
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http://dx.doi.org/10.1007/s10969-012-9134-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4075964PMC
September 2012

Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples.

Proc Natl Acad Sci U S A 2012 Jul 25;109(27):10873-8. Epub 2012 Jun 25.

Biomolecular NMR and Munich Center for Integrated Protein Science, Department Chemie, Technische Universität München, 85747 Garching, Germany.

We have developed an approach for determining NMR structures of proteins over 20 kDa that utilizes sparse distance restraints obtained using transverse relaxation optimized spectroscopy experiments on perdeuterated samples to guide RASREC Rosetta NMR structure calculations. The method was tested on 11 proteins ranging from 15 to 40 kDa, seven of which were previously unsolved. The RASREC Rosetta models were in good agreement with models obtained using traditional NMR methods with larger restraint sets. In five cases X-ray structures were determined or were available, allowing comparison of the accuracy of the Rosetta models and conventional NMR models. In all five cases, the Rosetta models were more similar to the X-ray structures over both the backbone and side-chain conformations than the "best effort" structures determined by conventional methods. The incorporation of sparse distance restraints into RASREC Rosetta allows routine determination of high-quality solution NMR structures for proteins up to 40 kDa, and should be broadly useful in structural biology.
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http://dx.doi.org/10.1073/pnas.1203013109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3390869PMC
July 2012

NMR structure of lipoprotein YxeF from Bacillus subtilis reveals a calycin fold and distant homology with the lipocalin Blc from Escherichia coli.

PLoS One 2012 5;7(6):e37404. Epub 2012 Jun 5.

Department of Chemistry, State University of New York at Buffalo, Buffalo, New York, United States of America.

The soluble monomeric domain of lipoprotein YxeF from the Gram positive bacterium B. subtilis was selected by the Northeast Structural Genomics Consortium (NESG) as a target of a biomedical theme project focusing on the structure determination of the soluble domains of bacterial lipoproteins. The solution NMR structure of YxeF reveals a calycin fold and distant homology with the lipocalin Blc from the Gram-negative bacterium E.coli. In particular, the characteristic β-barrel, which is open to the solvent at one end, is extremely well conserved in YxeF with respect to Blc. The identification of YxeF as the first lipocalin homologue occurring in a Gram-positive bacterium suggests that lipocalins emerged before the evolutionary divergence of Gram positive and Gram negative bacteria. Since YxeF is devoid of the α-helix that packs in all lipocalins with known structure against the β-barrel to form a second hydrophobic core, we propose to introduce a new lipocalin sub-family named 'slim lipocalins', with YxeF and the other members of Pfam family PF11631 to which YxeF belongs constituting the first representatives. The results presented here exemplify the impact of structural genomics to enhance our understanding of biology and to generate new biological hypotheses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037404PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367933PMC
October 2012