Publications by authors named "Alexander Eletsky"

34 Publications

Skp1 Dimerization Conceals Its F-Box Protein Binding Site.

Biochemistry 2020 04 13;59(15):1527-1536. Epub 2020 Apr 13.

Skp1 is an adapter that links F-box proteins to cullin-1 in the Skp1/cullin-1/F-box (SCF) protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent protein degradation. Skp1 from the amoebozoan forms a stable homodimer with a of 2.5 μM as determined by sedimentation velocity studies yet is monomeric in crystal complexes with F-box proteins. To investigate the molecular basis for the difference, we determined the solution NMR structure of a doubly truncated Skp1 homodimer (Skp1ΔΔ). The solution structure of the Skp1ΔΔ dimer reveals a 2-fold symmetry with an interface that buries ∼750 Å of predominantly hydrophobic surface. The dimer interface overlaps with subsite 1 of the F-box interaction area, explaining why only the Skp1 monomer binds F-box proteins (FBPs). To confirm the model, Rosetta was used to predict amino acid substitutions that might disrupt the dimer interface, and the F97E substitution was chosen to potentially minimize interference with F-box interactions. A nearly full-length version of Skp1 with this substitution (Skp1ΔF97E) behaved as a stable monomer at concentrations of ≤500 μM and actively bound a model FBP, mammalian Fbs1, which suggests that the dimeric state is not required for Skp1 to carry out a basic biochemical function. Finally, Skp1ΔF97E is expected to serve as a monomer model for high-resolution NMR studies previously hindered by dimerization.
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http://dx.doi.org/10.1021/acs.biochem.0c00094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521599PMC
April 2020

NMR Resonance Assignment Methodology: Characterizing Large Sparsely Labeled Glycoproteins.

J Mol Biol 2019 05 26;431(12):2369-2382. Epub 2019 Apr 26.

Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA. Electronic address:

Characterization of proteins using NMR methods begins with assignment of resonances to specific residues. This is usually accomplished using sequential connectivities between nuclear pairs in proteins uniformly labeled with NMR active isotopes. This becomes impractical for larger proteins, and especially for proteins that are best expressed in mammalian cells, including glycoproteins. Here an alternate protocol for the assignment of NMR resonances of sparsely labeled proteins, namely, the ones labeled with a single amino acid type, or a limited subset of types, isotopically enriched with N or C, is described. The protocol is based on comparison of data collected using extensions of simple two-dimensional NMR experiments (correlated chemical shifts, nuclear Overhauser effects, residual dipolar couplings) to predictions from molecular dynamics trajectories that begin with known protein structures. Optimal pairing of predicted and experimental values is facilitated by a software package that employs a genetic algorithm, ASSIGN_SLP_MD. The approach is applied to the 36-kDa luminal domain of the sialyltransferase, rST6Gal1, in which all phenylalanines are labeled with N, and the results are validated by elimination of resonances via single-point mutations of selected phenylalanines to tyrosines. Assignment allows the use of previously published paramagnetic relaxation enhancements to evaluate placement of a substrate analog in the active site of this protein. The protocol will open the way to structural characterization of the many glycosylated and other proteins that are best expressed in mammalian cells.
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http://dx.doi.org/10.1016/j.jmb.2019.04.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554063PMC
May 2019

The operon protects from copper toxicity: CopL is an extracellular membrane-associated copper-binding protein.

J Biol Chem 2019 03 17;294(11):4027-4044. Epub 2019 Jan 17.

From the Department of Biochemistry and Microbiology, Rutgers, the State University of New Jersey, New Brunswick, New Jersey 08901,

As complications associated with antibiotic resistance have intensified, copper (Cu) is attracting attention as an antimicrobial agent. Recent studies have shown that copper surfaces decrease microbial burden, and host macrophages use Cu to increase bacterial killing. Not surprisingly, microbes have evolved mechanisms to tightly control intracellular Cu pools and protect against Cu toxicity. Here, we identified two genes ( and ) encoded within the arginine-catabolic mobile element (ACME) that we hypothesized function in Cu homeostasis. Supporting this hypothesis, mutational inactivation of or increased copper sensitivity. We found that are co-transcribed and that their transcription is increased during copper stress and in a strain in which , encoding a Cu-responsive transcriptional repressor, was mutated. Moreover, displayed genetic synergy with suggesting that CopB functions in Cu export. We further observed that CopL functions independently of CopB or CopA in Cu toxicity protection and that CopL from the clone USA300 is a membrane-bound and surface-exposed lipoprotein that binds up to four Cu ions. Solution NMR structures of the homologous CopL, together with phylogenetic analysis and chemical-shift perturbation experiments, identified conserved residues potentially involved in Cu coordination. The solution NMR structure also revealed a novel Cu-binding architecture. Of note, a CopL variant with defective Cu binding did not protect against Cu toxicity Taken together, these findings indicate that the ACME-encoded CopB and CopL proteins are additional factors utilized by the highly successful USA300 clone to suppress copper toxicity.
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http://dx.doi.org/10.1074/jbc.RA118.004723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422080PMC
March 2019

Paramagnetic Tag for Glycosylation Sites in Glycoproteins: Structural Constraints on Heparan Sulfate Binding to Robo1.

ACS Chem Biol 2018 09 16;13(9):2560-2567. Epub 2018 Aug 16.

Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, and Bijvoet Center for Biomolecular Research , Utrecht University , Utrecht , The Netherlands.

An enzyme- and click chemistry-mediated methodology for the site-specific nitroxide spin labeling of glycoproteins has been developed and applied. The procedure relies on the presence of single N-glycosylation sites that are present natively in proteins or that can be engineered into glycoproteins by mutational elimination of all but one glycosylation site. Recombinantly expressing glycoproteins in HEK293S (GnT1-) cells results in N-glycans with high-mannose structures that can be processed to leave a single GlcNAc residue. This can in turn be modified by enzymatic addition of a GalNAz residue that is subject to reaction with an alkyne-carrying TEMPO moiety using copper(I)-catalyzed click chemistry. To illustrate the procedure, we have made an application to a two-domain construct of Robo1, a protein that carries a single N-glycosylation site in its N-terminal domains. The construct has also been labeled with N at amide nitrogens of lysine residues to provide a set of sites that are used to derive an effective location of the paramagnetic nitroxide moiety of the TEMPO group. This, in turn, allowed measurements of paramagnetic perturbations to the spectra of a new high affinity heparan sulfate ligand. Calculation of distance constraints from these data facilitated determination of an atomic level model for the docked complex.
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http://dx.doi.org/10.1021/acschembio.8b00511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161356PMC
September 2018

Glycosylation Promotes the Random Coil to Helix Transition in a Region of a Protist Skp1 Associated with F-Box Binding.

Biochemistry 2018 02 28;57(5):511-515. Epub 2017 Dec 28.

Department of Biochemistry & Molecular Biology, ‡Complex Carbohydrate Research Center, and §Center for Tropical and Emerging Global Diseases, University of Georgia , Athens, Georgia 30602, United States.

Cullin-ring-ligases mediate protein polyubiquitination, a signal for degradation in the 26S proteasome. The CRL1 class consists of Skp1/cullin-1/F-box protein/Rbx1 (SCF) complexes that cyclically associate with ubiquitin-E2 to build the polyubiquitin chain. Within the SCF complex, the 162-amino acid DdSkp1 from Dictyostelium bridges cullin-1 with an F-box protein (FBP), the specificity factor for substrate selection. The hydroxylation-dependent glycosylation of Pro143 of DdSkp1 by a pentasaccharide forms the basis of a novel O-sensing mechanism in the social amoeba Dictyostelium and other protists. Previous evidence indicated that glycosylation promotes increased α-helical content correlating with enhanced interaction with three F-box proteins. To localize these differences, we used nuclear magnetic resonance (NMR) methods to compare nonglycosylated DdSkp1 and a glycoform with a single GlcNAc sugar (Gn-DdSkp1). We report NMR assignments of backbone H, N, C, and CO nuclei as well as side-chain C and methyl C/H nuclei of Ile(δ1), Leu, and Val in both unmodified DdSkp1 and Gn-DdSkp1. The random coil index and N{H} HNOE indicate that the C-terminal region, which forms a helix-loop-helix motif centered on Pro143 at the crystallographically defined binding interface with F-box domains, remains dynamic in both DdSkp1 and Gn-DdSkp1. Chemical shifts indicate that the variation of conformation in Gn-DdSkp1, relative to DdSkp1, is limited to this region and characterized by increased helical fold. Extension of the glycan chain results in further changes, also limited to this region. Thus, glycosylation may control F-box protein interactions via a local effect on DdSkp1 conformation, by a mechanism that may be general to many unicellular eukaryotes.
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http://dx.doi.org/10.1021/acs.biochem.7b01033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282176PMC
February 2018

Accurate de novo design of hyperstable constrained peptides.

Nature 2016 Oct 14;538(7625):329-335. Epub 2016 Sep 14.

Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.

Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 18-47 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or N-C backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs.
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http://dx.doi.org/10.1038/nature19791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161715PMC
October 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

Solution NMR Experiment for Measurement of (15)N-(1)H Residual Dipolar Couplings in Large Proteins and Supramolecular Complexes.

J Am Chem Soc 2015 Sep 27;137(35):11242-5. Epub 2015 Aug 27.

Department of Chemistry and ‡Department of Biological Sciences, State University of New York at Buffalo , Buffalo, New York 14260, United States.

NMR residual dipolar couplings (RDCs) are exquisite probes of protein structure and dynamics. A new solution NMR experiment named 2D SE2 J-TROSY is presented to measure N-H RDCs for proteins and supramolecular complexes in excess of 200 kDa. This enables validation and refinement of their X-ray crystal and solution NMR structures and the characterization of structural and dynamic changes occurring upon complex formation. Accurate N-H RDCs were measured at 750 MHz (1)H resonance frequency for 11-mer 93 kDa (2)H,(15)N-labeled Trp RNA-binding attenuator protein tumbling with a correlation time τc of 120 ns. This is about twice as long as that for the most slowly tumbling system, for which N-H RDCs could be measured, so far, and corresponds to molecular weights of ∼200 kDa at 25 °C. Furthermore, due to the robustness of SE2 J-TROSY with respect to residual (1)H density from exchangeable protons, increased sensitivity at (1)H resonance frequencies around 1 GHz promises to enable N-H RDC measurement for even larger systems.
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http://dx.doi.org/10.1021/jacs.5b07010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905564PMC
September 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 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

Identification of low-molecular-weight compounds inhibiting growth of corynebacteria: potential lead compounds for antibiotics.

ChemMedChem 2014 Feb 8;9(2):282-5. Epub 2014 Jan 8.

Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304 (USA).

The bacterial genus Corynebacteria contains several pathogenic species that cause diseases such as diphtheria in humans and "cheesy gland" in goats and sheep. Thus, identifying new therapeutic targets to treat Corynebacteria infections is both medically and economically important. CG2496, a functionally uncharacterized protein from Corynebacterium glutamicum, was evaluated using an NMR ligand-affinity screen. A total of 11 compounds from a library of 460 biologically active compounds were shown to selectively bind CG2496 in a highly conserved region of the protein. The best binder was identified to be methiothepin (KD =54 ± 19 µM), an FDA-approved serotonin receptor antagonist. Methiothepin was also shown to inhibit the growth of C. glutamicum, but not bacteria that lack CG2496 homologs. Our results suggest that CG2496 is a novel therapeutic target and methiothepin is a potential lead compound or structural scaffold for developing new antibiotics specifically targeting Corynebacteria.
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http://dx.doi.org/10.1002/cmdc.201300386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977743PMC
February 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

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

Highly precise measurement of kinetic isotope effects using 1H-detected 2D [13C,1H]-HSQC NMR spectroscopy.

J Am Chem Soc 2012 Dec 11;134(51):20589-92. Epub 2012 Dec 11.

Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000, USA.

A new method is presented for measuring kinetic isotope effects (KIEs) by (1)H-detected 2D [(13)C,(1)H]-heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The high accuracy of this approach was exemplified for the reaction catalyzed by glucose-6-phosphate dehydrogenase by comparing the 1-(13)C KIE with the published value obtained using isotope ratio mass spectrometry. High precision was demonstrated for the reaction catalyzed by 1-deoxy-D-xylulose-5-phosphate reductoisomerase from Mycobacterium tuberculosis. 2-, 3-, and 4-(13)C KIEs were found to be 1.0031(4), 1.0303(12), and 1.0148(2), respectively. These KIEs provide evidence for a cleanly rate-limiting retroaldol step during isomerization. The high intrinsic sensitivity and signal dispersion of 2D [(13)C,(1)H]-HSQC offer new avenues to study challenging systems where low substrate concentration and/or signal overlap impedes 1D (13)C NMR data acquisition. Moreover, this approach can take advantage of highest-field spectrometers, which are commonly equipped for (1)H detection with cryogenic probes.
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http://dx.doi.org/10.1021/ja310353cDOI Listing
December 2012

Solution NMR structure of yeast succinate dehydrogenase flavinylation factor Sdh5 reveals a putative Sdh1 binding site.

Biochemistry 2012 Oct 19;51(43):8475-7. Epub 2012 Oct 19.

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

The yeast mitochondrial protein Sdh5 is required for the covalent attachment of flavin adenine dinucleotide (FAD) to protein Sdh1, a subunit of the heterotetrameric enzyme succinate dehydrogenase. The NMR structure of Sdh5 represents the first eukaryotic structure of Pfam family PF03937 and reveals a conserved surface region, which likely represents a putative Sdh1-Sdh5 interaction interface. Point mutations in this region result in the loss of covalent flavinylation of Sdh1. Moreover, chemical shift perturbation measurements showed that Sdh5 does not bind FAD in vitro, indicating that it is not a simple cofactor transporter in vivo.
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http://dx.doi.org/10.1021/bi301171uDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3667956PMC
October 2012

Blind testing of routine, fully automated determination of protein structures from NMR data.

Structure 2012 Feb;20(2):227-36

Magnetic Resonance Center, University of Florence, 50019 Sesto Fiorentino, Italy.

The protocols currently used for protein structure determination by nuclear magnetic resonance (NMR) depend on the determination of a large number of upper distance limits for proton-proton pairs. Typically, this task is performed manually by an experienced researcher rather than automatically by using a specific computer program. To assess whether it is indeed possible to generate in a fully automated manner NMR structures adequate for deposition in the Protein Data Bank, we gathered 10 experimental data sets with unassigned nuclear Overhauser effect spectroscopy (NOESY) peak lists for various proteins of unknown structure, computed structures for each of them using different, fully automatic programs, and compared the results to each other and to the manually solved reference structures that were not available at the time the data were provided. This constitutes a stringent "blind" assessment similar to the CASP and CAPRI initiatives. This study demonstrates the feasibility of routine, fully automated protein structure determination by NMR.
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http://dx.doi.org/10.1016/j.str.2012.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609704PMC
February 2012

Solution NMR structures reveal unique homodimer formation by a winged helix-turn-helix motif and provide first structures for protein domain family PF10771.

J Struct Funct Genomics 2012 Mar 6;13(1):1-7. Epub 2012 Jan 6.

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

High-quality NMR structures of the homo-dimeric proteins Bvu3908 (69-residues in monomeric unit) from Bacteroides vulgatus and Bt2368 (74-residues) from Bacteroides thetaiotaomicron reveal the presence of winged helix-turn-helix (wHTH) motifs mediating tight complex formation. Such homo-dimer formation by winged HTH motifs is otherwise found only in two DNA-binding proteins with known structure: the C-terminal wHTH domain of transcriptional activator FadR from E. coli and protein TubR from B. thurigensis, which is involved in plasmid DNA segregation. However, the relative orientation of the wHTH motifs is different and residues involved in DNA-binding are not conserved in Bvu3908 and Bt2368. Hence, the proteins of the present study are not very likely to bind DNA, but are likely to exhibit a function that has thus far not been ascribed to homo-dimers formed by winged HTH motifs. The structures of Bvu3908 and Bt2368 are the first atomic resolution structures for PFAM family PF10771, a family of unknown function (DUF2582) currently containing 128 members.
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http://dx.doi.org/10.1007/s10969-011-9121-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654790PMC
March 2012

Solution NMR structures reveal a distinct architecture and provide first structures for protein domain family PF04536.

J Struct Funct Genomics 2012 Mar 24;13(1):9-14. Epub 2011 Dec 24.

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

The protein family (Pfam) PF04536 is a broadly conserved domain family of unknown function (DUF477), with more than 1,350 members in prokaryotic and eukaryotic proteins. High-quality NMR structures of the N-terminal domain comprising residues 41-180 of the 684-residue protein CG2496 from Corynebacterium glutamicum and the N-terminal domain comprising residues 35-182 of the 435-residue protein PG0361 from Porphyromonas gingivalis both exhibit an α/β fold comprised of a four-stranded β-sheet, three α-helices packed against one side of the sheet, and a fourth α-helix attached to the other side. In spite of low sequence similarity (18%) assessed by structure-based sequence alignment, the two structures are globally quite similar. However, moderate structural differences are observed for the relative orientation of two of the four helices. Comparison with known protein structures reveals that the α/β architecture of CG2496(41-180) and PG0361(35-182) has previously not been characterized. Moreover, calculation of surface charge potential and identification of surface clefts indicate that the two domains very likely have different functions.
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http://dx.doi.org/10.1007/s10969-011-9122-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609422PMC
March 2012

Solution NMR structure of MED25(391-543) comprising the activator-interacting domain (ACID) of human mediator subunit 25.

J Struct Funct Genomics 2011 Sep 23;12(3):159-66. Epub 2011 Jul 23.

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

The solution NMR structure of protein MED25(391-543), comprising the activator interacting domain (ACID) of subunit 25 of the human mediator, is presented along with the measurement of polypeptide backbone heteronuclear 15N-{1H} NOEs to identify fast internal motional modes. This domain interacts with the acidic transactivation domains of Herpes simplex type 1 (HSV-1) protein VP16 and the Varicella-zoster virus (VZV) major transactivator protein IE62, which initiate transcription of viral genes. The structure is similar to the β-barrel domains of the human protein Ku and the SPOC domain of human protein SHARP, and provides a starting point to understand the structural biology of initiation of HSV-1 and VZV gene activation. Homology models built for the two ACID domains of the prostate tumor overexpressed (PTOV1) protein using the structure of MED25(391-543) as a template suggest that differential biological activities of the ACID domains in MED25 and PTOV1 arise from modulation of quite similar protein-protein interactions by variable residues grouped around highly conserved charged surface areas.
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http://dx.doi.org/10.1007/s10969-011-9115-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609412PMC
September 2011

NMR structure determination for larger proteins using backbone-only data.

Science 2010 Feb 4;327(5968):1014-8. Epub 2010 Feb 4.

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

Conventional protein structure determination from nuclear magnetic resonance data relies heavily on side-chain proton-to-proton distances. The necessary side-chain resonance assignment, however, is labor intensive and prone to error. Here we show that structures can be accurately determined without nuclear magnetic resonance (NMR) information on the side chains for proteins up to 25 kilodaltons by incorporating backbone chemical shifts, residual dipolar couplings, and amide proton distances into the Rosetta protein structure modeling methodology. These data, which are too sparse for conventional methods, serve only to guide conformational search toward the lowest-energy conformations in the folding landscape; the details of the computed models are determined by the physical chemistry implicit in the Rosetta all-atom energy function. The new method is not hindered by the deuteration required to suppress nuclear relaxation processes for proteins greater than 15 kilodaltons and should enable routine NMR structure determination for larger proteins.
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http://dx.doi.org/10.1126/science.1183649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909653PMC
February 2010

Analysis of the varicella-zoster virus IE62 N-terminal acidic transactivating domain and its interaction with the human mediator complex.

J Virol 2009 Jun 8;83(12):6300-5. Epub 2009 Apr 8.

Departments of Microbiology and Immunology, University at Buffalo SUNY, Buffalo, New York 14214, USA.

The varicella-zoster virus major transactivator, IE62, contains a potent N-terminal acidic transcriptional activation domain (TAD). Our experiments revealed that the minimal IE62 TAD encompasses amino acids (aa) 19 to 67. We showed that the minimal TAD interacts with the human Mediator complex. Site-specific mutations revealed residues throughout the minimal TAD that are important for both activation and Mediator interaction. The TAD interacts directly with aa 402 to 590 of the MED25 subunit, and site-specific TAD mutations abolished this interaction. Two-dimensional nuclear magnetic resonance spectroscopy revealed that the TAD is intrinsically unstructured. Our studies suggest that transactivation may involve the TAD adopting a defined structure upon binding MED25.
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http://dx.doi.org/10.1128/JVI.00054-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2687382PMC
June 2009

NMR and X-RAY structures of human E2-like ubiquitin-fold modifier conjugating enzyme 1 (UFC1) reveal structural and functional conservation in the metazoan UFM1-UBA5-UFC1 ubiquination pathway.

J Struct Funct Genomics 2009 Apr 20;10(2):127-36. Epub 2008 Dec 20.

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

For cell regulation, E2-like ubiquitin-fold modifier conjugating enzyme 1 (Ufc1) is involved in the transfer of ubiquitin-fold modifier 1 (Ufm1), a ubiquitin like protein which is activated by E1-like enzyme Uba5, to various target proteins. Thereby, Ufc1 participates in the very recently discovered Ufm1-Uba5-Ufc1 ubiquination pathway which is found in metazoan organisms. The structure of human Ufc1 was solved by using both NMR spectroscopy and X-ray crystallography. The complementary insights obtained with the two techniques provided a unique basis for understanding the function of Ufc1 at atomic resolution. The Ufc1 structure consists of the catalytic core domain conserved in all E2-like enzymes and an additional N-terminal helix. The active site Cys(116), which forms a thio-ester bond with Ufm1, is located in a flexible loop that is highly solvent accessible. Based on the Ufc1 and Ufm1 NMR structures, a model could be derived for the Ufc1-Ufm1 complex in which the C-terminal Gly(83) of Ufm1 may well form the expected thio-ester with Cys(116), suggesting that Ufm1-Ufc1 functions as described for other E1-E2-E3 machineries. alpha-helix 1 of Ufc1 adopts different conformations in the crystal and in solution, suggesting that this helix plays a key role to mediate specificity.
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http://dx.doi.org/10.1007/s10969-008-9054-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2850604PMC
April 2009

Consistent blind protein structure generation from NMR chemical shift data.

Proc Natl Acad Sci U S A 2008 Mar 7;105(12):4685-90. Epub 2008 Mar 7.

Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Protein NMR chemical shifts are highly sensitive to local structure. A robust protocol is described that exploits this relation for de novo protein structure generation, using as input experimental parameters the (13)C(alpha), (13)C(beta), (13)C', (15)N, (1)H(alpha) and (1)H(N) NMR chemical shifts. These shifts are generally available at the early stage of the traditional NMR structure determination process, before the collection and analysis of structural restraints. The chemical shift based structure determination protocol uses an empirically optimized procedure to select protein fragments from the Protein Data Bank, in conjunction with the standard ROSETTA Monte Carlo assembly and relaxation methods. Evaluation of 16 proteins, varying in size from 56 to 129 residues, yielded full-atom models that have 0.7-1.8 A root mean square deviations for the backbone atoms relative to the experimentally determined x-ray or NMR structures. The strategy also has been successfully applied in a blind manner to nine protein targets with molecular masses up to 15.4 kDa, whose conventional NMR structure determination was conducted in parallel by the Northeast Structural Genomics Consortium. This protocol potentially provides a new direction for high-throughput NMR structure determination.
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http://dx.doi.org/10.1073/pnas.0800256105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2290745PMC
March 2008

Probing structure and functional dynamics of (large) proteins with aromatic rings: L-GFT-TROSY (4,3)D HCCH NMR spectroscopy.

J Am Chem Soc 2005 Oct;127(42):14578-9

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

NMR assignment of aromatic rings in proteins is a prerequisite for obtaining high-quality solution structures of proteins and for studying the dynamics and folding of their molecular cores. Here we present sensitive PFG-PEP L-GFT-(TROSY) (4,3)D HCCH NMR for identification of aromatic spin systems based on four-dimensional (4D) spectral information which can be rapidly obtained with high digital resolution. The G-matrix Fourier Transform (GFT) experiment relies on newly introduced longitudinal relaxation (L-)optimization for aromatic protons and is optimally suited for both sensitivity and sampling limited data collection, making it particularly attractive for NMR-based structural genomics. Applications are presented for 21 and 13 kDa proteins HR41 and MaR11, targets of the Northeast Structural Genomics Consortium for which data collection is, respectively, sensitivity and sampling limited. Complete assignment of aromatic rings enabled high-quality NMR structure determination, and nearly complete analysis of aromatic proton line widths allowed one to assess the flipping of most rings in HR41. Specifically, the ring of Tyr90 flips very slowly on the seconds time scale, thereby proving the absence of fast larger-amplitude motional modes which could allow the ring to flip. This indicates remarkable rigidity of the substructure in which the ring is embedded. Tyr90 is conserved among ubiquitin-conjugating enzymes E2, to which HR41 belongs, and is located in spatial proximity to the interface between E2 and ubiquitin protein ligase E3. Hence, the conformational rigidity and/or the slow motional mode probed by the ring might be of functional importance.
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http://dx.doi.org/10.1021/ja054895xDOI Listing
October 2005

Investigation of ligand binding and protein dynamics in Bacillus subtilis chorismate mutase by transverse relaxation optimized spectroscopy-nuclear magnetic resonance.

Biochemistry 2005 May;44(18):6788-99

Department of Chemistry and Applied Bioscience, Swiss Federal Institute of Technology, ETH Hönggerberg, 8093 Zürich, Switzerland.

The structural and dynamical consequences of ligand binding to a monofunctional chorismate mutase from Bacillus subtilis have been investigated by solution NMR spectroscopy. TROSY methods were employed to assign 98% of the backbone (1)H(N), (1)H(alpha), (15)N, (13)C', and (13)C(alpha) resonances as well as 86% of the side chain (13)C resonances of the 44 kDa trimeric enzyme at 20 degrees C. This information was used to map chemical shift perturbations and changes in intramolecular mobility caused by binding of prephenate or a transition state analogue to the X-ray structure. Model-free interpretation of backbone dynamics for the free enzyme and its complexes based on (15)N relaxation data measured at 600 and 900 MHz showed significant structural consolidation of the protein in the presence of a bound ligand. In agreement with earlier structural and biochemical studies, substantial ordering of 10 otherwise highly flexible residues at the C-terminus is particularly notable. The observed changes suggest direct contact between this protein segment and the bound ligand, providing support for the proposal that the C-terminus can serve as a lid for the active site, limiting diffusion into and out of the pocket and possibly imposing conformational control over substrate once bound. Other regions of the protein that experience substantial ligand-induced changes also border the active site or lie along the subunit interfaces, indicating that the enzyme adapts dynamically to ligands by a sort of induced fit mechanism. It is believed that the mutase-catalyzed chorismate-to-prephenate rearrangement is partially encounter controlled, and backbone motions on the millisecond time scale, as seen here, may contribute to the reaction barrier.
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http://dx.doi.org/10.1021/bi0474259DOI Listing
May 2005

Resonance assignment of proteins with high shift degeneracy based on 5D spectral information encoded in G2FT NMR experiments.

J Am Chem Soc 2005 Apr;127(13):4554-5

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

A suite of novel (5,3)D G2FT triple resonance NMR experiments encoding highly resolved 5D spectral information is presented for sequential resonance assignment of proteins exhibiting high chemical shift degeneracy. Efficient resonance assignment is achieved by separate joint sampling of (i) chemical shifts which solely serve to provide increased resolution and (ii) shifts which also provide sequential connectivities. In these G2FT experiments, two G-matrix transformations are employed. Peaks are resolved along a first GFT dimension at both Omega(15N) + Omega(13C') and Omega(15N) - Omega(13C'), or at Omega(15N) + Omega(13Calpha) and Omega(15N) - Omega(13Calpha), to break backbone 15N,1HN chemical shift degeneracy. Sequential connectivities are established along a second GFT dimension by measuring intraresidue and sequential correlations at 2Omega(13Calpha), Omega(13Calpha + 13Cbeta), and Omega(13Calpha - 13Cbeta), or at Omega(13Calpha + 1Halpha) and Omega(13Calpha - 1Halpha), to resolve 13Calpha/beta,1Halpha chemical shift degeneracy. It is demonstrated that longitudinal proton relaxation optimization of out-and-back implementations suitable for deuterated proteins and nonlinear data sampling combined with maximum entropy reconstruction further accelerate G2FT NMR data acquisition speed. As a result, the spectral information can be obtained within hours, so that (5,3)D G2FT experiments are viable options for high-throughput structure determination in structural genomics. Applications are presented for 17 kDa alpha-helical protein YqbG and 13.5 kDa protein rps24e, targets of the Northeast Structural Genomics consortium, as well as for 9 kDa protein Z-domain. The high resolving power of the G2FT NMR experiments makes them attractive choices to study alpha-helical globular/membrane or (partially) unfolded proteins, thus promising to pave the way for NMR-based structural genomics of membrane proteins.
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http://dx.doi.org/10.1021/ja042562eDOI Listing
April 2005

A novel strategy for the assignment of side-chain resonances in completely deuterated large proteins using 13C spectroscopy.

J Biomol NMR 2003 Jun;26(2):167-79

Laboratory of Physical Chemistry, Swiss Federal Institute of Technology, ETH-Hönggerberg, CH-8093 Zürich, Switzerland.

The assignment of the aliphatic (13)C resonances of trimeric Bacillus Subtilis chorismate mutase, a protein with a molecular mass of 44 kDa, consisting of three 127-residue monomers is presented by use of two-dimensional (2D) (13)C-start and (13)C-observe NMR experiments. These experiments start with (13)C excitation and end with (13)C observation while relying on the long transverse relaxation times of (13)C spins in uniformly deuterated and (13)C,(15)N-labeled large proteins. Gains in sensitivity are achieved by the use of a paramagnetic relaxation enhancement agent to reduce (13)C T(1) relaxation times with little effect on (13)C T(2) relaxation times. Such 2D (13)C-only NMR experiments circumvent problems associated with the application of conventional experiments for side-chain assignment to proteins of larger sizes, for instance, the absence or low concentration of the side-chain (1)H spins, the transfer of the side-chain spin polarization to the (1)H(N) spins for signal acquisition, or the necessity of a quantitative reprotonation of the methyl moieties in the otherwise fully deuterated side-chains. We demonstrate that having obtained a nearly complete assignment of the side-chain aliphatic (13)C resonances, the side-chain (1)H chemical shifts can be assigned in a semiautomatic fashion using 3D (15)N-resolved and (13)C-resolved NOESY experiments measured with a randomly partially protonated protein sample. We also discuss perspectives for structure determination of larger proteins by using novel strategies which are based on the (1)H,(1)H NOEs in combination with multiple residual dipolar couplings between adjacent (13)C spins determined with 2D (13)C-only experiments.
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http://dx.doi.org/10.1023/a:1023572320699DOI Listing
June 2003