Assignment validation software suite for the evaluation and presentation of protein resonance assignment data

Moseley, Hunter N. B.; Sahota, Gurmukh; Montelione, Gaetano T.

doi:10.1023/b:jnmr.0000015420.44364.06

Cited by 96 publications

(125 citation statements)

References 21 publications

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“…Histidine tautomeric states were elucidated by two-dimensional 1 H-15 N heteronuclear multiple-quantum coherence spectroscopy (15). Resonance assignments were validated using the Assignment Validation Suite software package (16) and deposited in the BioMagResDB (accession number 16272).…”

Section: Methodsmentioning

confidence: 99%

Structural Basis of O6-Alkylguanine Recognition by a Bacterial Alkyltransferase-like DNA Repair Protein

Aramini

Tubbs

Kanugula

et al. 2010

Journal of Biological Chemistry

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Alkyltransferase-like proteins (ATLs) are a novel class of DNA repair proteins related to O 6 -alkylguanine-DNA alkyltransferases (AGTs) that tightly bind alkylated DNA and shunt the damaged DNA into the nucleotide excision repair pathway. Here, we present the first structure of a bacterial ATL, from Vibrio parahaemolyticus (vpAtl). We demonstrate that vpAtl adopts an AGT-like fold and that the protein is capable of tightly binding to O 6 -methylguanine-containing DNA and disrupting its repair by human AGT, a hallmark of ATLs. Mutation of highly conserved residues Tyr 23 and Arg 37 demonstrate their critical roles in a conserved mechanism of ATL binding to alkylated DNA. NMR relaxation data reveal a role for conformational plasticity in the guanine-lesion recognition cavity. Our results provide further evidence for the conserved role of ATLs in this primordial mechanism of DNA repair. O6 -Alkylguanine-DNA alkyltransferases (AGTs) 3 are a large family (Pfam, PF01035; EC 2.1.1.63) of alkyl damage-response proteins that reverse endogenous and exogenous alkylation at the O 6 position of guanines, cytotoxic lesions that otherwise cause G:C to A:T mutations in DNA (1). Human AGT, also called MGMT, interferes with alkylating chemotherapies making it a target for anticancer drug design (1, 2). AGTs are ubiquitous suicide enzymes that mediate the irreversible transfer of the alkyl group to a reactive cysteine within a highly conserved PCHRV active site sequence motif by a direct reversal mechanism, featuring sequence-independent minor-groove binding to a helix-turn-helix motif and flipping of the damaged nucleotide (3, 4).Alkyltransferase-like proteins (ATLs), thus far identified in prokaryotes and lower eukaryotes, constitute a new subclass with sequence similarity to AGTs but lacking the critical cysteine alkyl receptor, which is most often replaced by a tryptophan (5, 6). ATLs tightly bind a wide range of O 6 -alkylguanine adducts and block the repair of O 6 -mG by human AGT but exhibit no alkyltransferase, glycosylase, or endonuclease activities (7-9). The recent first structural study of an ATL (10), from the fission yeast Schizosaccharomyces pombe (spAtl1) which lacks an AGT, provides strong evidence for a novel mechanism of DNA repair in which an ATL binds alkylated DNA in a manner analogous to AGTs, and the resulting nonenzymatic ATL⅐DNA complex triggers the NER pathway (10, 11).Here, we present the solution NMR structure of the 100-residue ATL from Vibrio parahaemolyticus AQ3810 (SwissProt entry A6B4U8_VIBPA; Northeast Structural Genomics code, VpR247; hereafter referred to as vpAtl), whose structure was solved as part of the Northeast Structural Genomics consortium of the National Institutes of Health, NIGMS, Protein Structure Initiative. The vpAtl protein shares 47% sequence identity with spAtl1 and features a PWFRV active site sequence motif (Fig. 1A). We demonstrate that the structure of vpAtl is highly analogous to the AGT fold and that this bacterial protein is capable of tightly binding to O 6 -mGcontaini...

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Section: Methodsmentioning

confidence: 99%

Structural Basis of O6-Alkylguanine Recognition by a Bacterial Alkyltransferase-like DNA Repair Protein

Aramini

Tubbs

Kanugula

et al. 2010

Journal of Biological Chemistry

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“…Backbone dihedral angle restraints were obtained by using the TALOS program (34) with backbone chemical shifts that were calibrated for isotope and TROSY effects (35). Predictions of and dihedral angles from the program were used in the structure calculations as input restraints with Ϯ30°error tolerances.…”

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confidence: 99%

Structure of outer membrane protein G by solution NMR spectroscopy

Liang

Tamm

2007

Proc. Natl. Acad. Sci. U.S.A.

143

138

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The bacterial outer membrane protein G (OmpG), a monomeric pH-gated porin, was overexpressed in Escherichia coli and refolded in ␤-octyl glucoside micelles. After transfer into dodecylphosphocholine micelles, the solution structure of OmpG was determined by solution NMR spectroscopy at pH 6.3. Complete backbone assignments were obtained for 234 of 280 residues based on CA, CB, and CO connection pathways determined from a series of TROSY-based 3D experiments at 800 MHz. The global fold of the 14-stranded ␤-barrel was determined based on 133 long-range NOEs observed between neighboring strands and local chemical shift and NOE information. The structure of the barrel is very similar to previous crystal structures, but the loops of the solution structure are quite flexible.porin ͉ membrane protein folding ͉ dodecylphosphocholine micelle M embrane proteins (MP) continue to be prime drug targets because they function as receptors for cellular communication and gatekeepers for ion and substrate transport across cell membranes. Despite the tremendous progress that has been made in the past decade in solving an increasing number of MP structures (1), the total number of structure determinations of MPs still lags far behind the corresponding number for soluble proteins. Most MP structures solved to date have been and probably will continue to be determined by x-ray crystallography. However, some MPs resist crystallization, and some functional aspects of MP structure as well as their dynamics are better studied in solution. Solution NMR spectroscopy is the ideal tool to study the structural dynamics of proteins and recently has been successfully applied to such studies of very large protein complexes (2). Recent advances in instrumentation and pulse program implementation have proven solution NMR to also be a valuable method to determine the structures and dynamic aspects of polytopic integral MPs (3-9). To study MPs by solution NMR, they are usually reconstituted into micelles formed by nondenaturing detergents that are thought to mimic the natural membrane environment quite well, while retaining the overall size of the protein/micelle complex still manageable by solution NMR techniques (10). Various detergents and detergent/lipid combinations have been reported in solution NMR structural studies of MPs with variable degrees of success (11-13). Despite many recent efforts and successes in this area, it is clear that MP sample preparation remains one of the most critical steps in obtaining spectra of a quality that is suitable for a structure determination. In general, ␣-helical MPs have to be natively expressed and extracted from the cell membranes, whereas ␤-barrel MPs can often be produced in insoluble inclusion bodies and then refolded in micellar systems that are suitable for solution NMR (3,4,6).The outer membrane protein G (OmpG) is an integral MP that resides in the outer membrane of Gram-negative bacteria. It functions as a monomeric porin and facilitates the uptake of large oligosaccharides (14). Because the prim...

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“…NMR based structure determination frequently uses a crude initial experimental structure to resolve ambiguities in assignment of NOE peaks before going on to produce high resolution structures. A correct computational model could serve a similar purpose [39][40][41]. Backbone folds also can be used in combination with paramagnetic perturbations and RDCs to produce assignment of backbone resonances in the absence of a complete set of triple resonance experiments [7].…”

Section: Discussionmentioning

confidence: 99%

Rapid classification of protein structure models using unassigned backbone RDCs and probability density profile analysis (PDPA)

Bansal

Miao

Adams

et al. 2008

Journal of Magnetic Resonance

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A method of identifying the best structural model for a protein of unknown structure from a list of structural candidates using unassigned 15 N-1 H residual dipolar coupling (RDC) data and probability density profile analysis (PDPA) is described. Ten candidate structures have been obtained for the structural genomics target protein PF2048.1 using ROBETTA. 15 N-1 H residual dipolar couplings have been measured from NMR spectra of the protein in two alignment media and these data have been analyzed using PDPA to rank the models in terms of their ability to represent the actual structure.A number of advantages in using this method to characterize a protein structure become apparent. RDCs can easily and rapidly be acquired, and without the need for assignment, the cost and duration of data acquisition is greatly reduced. The approach is quite robust with respect to imprecise and missing data. In the case of PF2048.1, a 79 residue protein, only 58 and 55 of the total RDC data were observed. The method can accelerate structure determination at higher resolution using traditional NMR spectroscopy by providing a starting point for the addition of NOEs and other NMR structural data.

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Assignment validation software suite for the evaluation and presentation of protein resonance assignment data

Cited by 96 publications

References 21 publications

Structural Basis of O6-Alkylguanine Recognition by a Bacterial Alkyltransferase-like DNA Repair Protein

Structural Basis of O6-Alkylguanine Recognition by a Bacterial Alkyltransferase-like DNA Repair Protein

Structure of outer membrane protein G by solution NMR spectroscopy

Rapid classification of protein structure models using unassigned backbone RDCs and probability density profile analysis (PDPA)

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