ArsD is a metallochaperone that delivers As(III) to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773.  Conserved ArsD cysteine residues (Cys12, Cys13 and Cys18) construct the As(III) binding site of the protein, however a global structural understanding of this arsenic binding remains unclear.  We have obtained NMR assignments for ArsD as a starting point for probing structural changes on the protein that occur in response to metalloid binding and upon formation of a complex with ArsA.  The predicted solution structure of ArsD is in agreement with recently published crystallographic structural results

He, Yanan

Rosen, Barry P.

Skalicky, Jack

Stemmler, Timothy L.

Ye, Jun

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Wayne State UniversityDigitalCommons@WayneStateBiochemistry and Molecular Biology FacultyPublications Department of Biochemistry and Molecular Biology11-10-2010Resonance Assignments and Secondary StructurePredictions of the As(III) Metallochaperone ArsDin SolutionJun YeWayne State UniversityYanan HeWayne State UniversityJack SkalickyUniversity of UtahBarry P. RosenFlorida International UniversityTimothy L. StemmlerWayne State University, tstemmle@med.wayne.eduThis Article is brought to you for free and open access by the Department of Biochemistry and Molecular Biology at DigitalCommons@WayneState. Ithas been accepted for inclusion in Biochemistry and Molecular Biology Faculty Publications by an authorized administrator ofDigitalCommons@WayneState.Recommended CitationYe J, He Y et al (2011) Resonance assignments and secondary structure prediction of the As(III) metallochaperone ArsD in solution.Biomol NMR Assign 5(1): 109-112 doi:10.1007/s12104-010-9279-9Available at: http://digitalcommons.wayne.edu/med_biochem/12                         This article is the author’s final version after peer-review. A publisher version (Springer Verlag) of this article previously appeared in BIOMOLECULAR NMR ASSIGNMENTS (November 2010). Available online at: http://www.springerlink.com/content/x755666345978657/   Secondary structure prediction of the As(III) metallochaperone ArsD in solution |  Jun Ye, et. al DIGITALCOMMONS@WSU  |  2010 2   Resonance assignments and secondary structure prediction of the As(III) metallochaperone ArsD in solution  Jun Ye1, Yanan He1, Jack Skalicky2, Barry P. Rosen3 & Timothy L. Stemmler1, § 1 Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA 2 Department of Biochemistry, University of Utah, Salt Lake City, UT 84132 USA 3 Department of Cellular Biology and Pharmacology, Florida International University, Herbert Wertheim College of Medicine, Miami, FL 33199  § Corresponding Author: tstemmle@med.wayne.edu;  Phone: 313 577 5712;  Fax: 313 577 2765  Abstract ArsD is a metallochaperone that delivers As(III) to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773.  Conserved ArsD cysteine residues (Cys12, Cys13 and Cys18) construct the As(III) binding site of the protein, however a global structural understanding of this arsenic binding remains unclear.  We have obtained NMR assignments for ArsD as a starting point for probing structural changes on the protein that occur in response to metalloid binding and upon formation of a complex with ArsA.  The predicted solution structure of ArsD is in agreement with recently published crystallographic structural results.   Keywords NMR assignments, arsenic, ARS Operon, metallochaperone Secondary structure prediction of the As(III) metallochaperone ArsD in solution |  Jun Ye, et. al DIGITALCOMMONS@WSU  |  2010 3Biological context  Arsenic is one of the most toxic elements found in the environment and ranks first on the Environmental Protection Agency Superfund list of Hazardous Substances (http://www.atsdr.cdc.gov/cercla/07list.html).  Since arsenic is toxic to most life forms, it is no surprise that nearly every organism, from bacteria to archaea to eukaryotes, including humans, have evolved detoxification systems for the metalloid (Bhattacharjee and Rosen, 2007).  In Escherichia coli, the arsRDABC operon of plasmid R773 confers resistance to arsenite and antimonite, where ArsAB is an ATPases that pumps trivalent metalloids (As(III) and Sb(III)) out of the cell.  ArsD has recently been demonstrated to be a metallochaperone for As(III), ferrying the metalloid through the cytosol to the catalytic subunit of the efflux pump, the ArsA ATPase (Lin, Walmsley and Rosen, 2006).  In various ars operons, the arsD gene is almost always adjacent to the arsA gene, suggesting a related function.  Interaction with ArsD increases the affinity of ArsA for As(III), which in turn increases the detoxification efficiency for the system (Lin, Yang and Rosen, 2007).  Direct transfer of As(III) from ArsD to ArsA occurs in the presence of chelators, consistent with the channeling of the metalloid between protein partners;  transfer is dependent on the presence of ATP (Yang, Rawat, Stemmler and Rosen, 2010).  These studies indicate a direct interaction between ArsD and ArsA during transfer of As(III) from one protein to the other.  In this study, we have undertaken NMR characterization of ArsD to provide a basis for understanding the structural aspects of how the protein functions.  ArsD is a homodimer of two 120-residue subunits in which residues Cys12, Cys13 and Cys18 form the As(III) binding site required for arsenic metallochaperone activity (Lin, Yang and Rosen, 2007).  Each subunit has eight cysteine residues, including three vicinal pairs, Cys12-13, Cys112-113 and Cys119-120.  Although all three vicinal pairs bind As(III), only Cys12, Cys13 and Cys18 are conserved, and these three are required for chaperone activity.  The crystal structure of apo ArsD was recently reported (Ye, Ajees, Yang and Rosen, 2010).  The overall structure of the ArsD monomer has a thioredoxin fold, with a core of four beta-strands flanked by four α-helices (PDB code 3MWH). The region of ArsD with the cysteines that form the arsenic binding site was not visible in that structure, but a structure of an oxidized form of an ArsD homologue, showing the location of the cysteines in disulfide bonds, has been reported (PDB code 3KTB).   In this report, we have initiated determination of the solution structure of ArsD for comparison with the static structure and to allow identification of amino acid-specific chemical environmental changes in ArsD coupled with binding of arsenite and antimonite or during the interaction with ArsA.  Therefore, the work outlined in this report provides the basis for Secondary structure prediction of the As(III) DIGITALCOMMONS@WSU  |  20104future experiments directed at understanding the mechanism by which ArsD transfers As(III) to ArsA and hence mediates arsenic toxicity in  Dispersion in the high-resolution protein is well folded, with the number of peaks consistent with the protein existing as a symmetric homodimer (Fig. 1).  Assignments for the 118 amino acid ArsDmajority of native protein residues.  In the absence of As(III), amide chemical shifts for the three conserved cysteines (Cys12, Cys13 and Cysthese residues exist in an unstructured environment.  Chemical shift analysis of the backbone atom resonances (Wishart, Sykes and Richards, 1991)the protein in solution (Fig. 2).  Prediction analysis suggests the protein is flanked bterminal β-strand.  The active site residues Cysregion between β-strand 1 and α-helix 1.   In addition, the protein contains an extensive Cunstructured tail ranging from residues  Figure 1. Assigned 1H,15N TROSY HSQC spectrum of ArsD.  Data were collected on the National Magnet Laboratory’s Varian INOVA 720 MHz spectrometer at 293 K. metallochaperone ArsD in solution |  Jun Ye, et. al E. coli. 15N TROSY HSQC spectrum (Pervushin, 2000) construct were completed for the 18) occur in the unfolded region of the spectrum, indicating  was applied to predict the secondary structure for 12, Cys13 and Cys18 all exist in the unstructured 100 to 118.   indicates the y a 7 residue N--terminal   Secondary structure prediction of the As(III) DIGITALCOMMONS@WSU  |  20105A comparison of predicted secondary structural elements for ArsD in solution with thstructure results indicate a reasonable agreement between the structures (Fig. 2), with a few differences.  The solution structure is less ordered than the crystal structure, especially in the length and location of α-helix 2 and Cys13 and Cys18) exist in an unstructured region between indicated for the predicted solution structure.  It is only when the cysteine residues are artifactuallyoxidized to form disulfide bonds that they could be resolved by crystallography.  These results suggest this region of apo ArsD is normally very flexible and folds around As(III), allowing the three cysteine residues to form the binding site.  In a previomodeled and this metallated protein was docked 2010).  These modeling experiments offer testable predictions for which the solution characterization of ArsD outlined above will be informative.  Therefore, these NMR data provide the basis for future experiments directed at monitoring structural changes coupled with binding of As(III) and/or ArsA.  Using the crystal structure as the global structural template, we will be able to map binding surfaces between proteins and monitor structural changes during As(III) bidelivery in the future using NMR spectroscopy.  These results will provide significant insight in the molecular mechanism of  ArsD metallochaperone function. Figure 2.  Summary of proposed secondary structural elements for ArsD in solution (mistructural characterization obtained from crystallographic analysis (bottom) NMRView (Johnson, 2004).  Single letter residue designations are provided (top) with predicted secondary structural elements displayed in red (helix), blue (strand) or grey (unfolded). metallochaperone ArsD in solution |  Jun Ye, et. al β-strand 3.  By both methods, the conserved cysteines (Cysβ-strand 1 and αus study, ArsD with As(III) bound was in silico with ArsA (Ye, Ajees, Yang and Rosen,  from CSI analysis using  e crystal 12, -helix 1, as earlier  nding and  ddle) compared with  Secondary structure prediction of the As(III) metallochaperone ArsD in solution |  Jun Ye, et. al DIGITALCOMMONS@WSU  |  2010 6Methods and experiments  Labeling, expression and purification of  ArsD An ArsD construct containing residues 1 to 118 with the C-terminal cysteine pair (Cys119,Cys120) replaced by a six histidine tag (sequence: MKTLMVFDPA MCCSTGVCGT DVDQALVDFS TDVQWLKQCG VQIERFNLAQ QPMSFVQNEK VKAFIEASGA EGLPLLLLDG ETVMAGRYPK RAELARWFGI PLDKVGLAPS GCCGGNTSHH HHHH) for a total size of 124 residues and a mass of 13,543 Daltons was expressed in E. coli strain BL21(DE3).  Uniformly labeled 15N, 15N/13C and 2H/15N/13C protein samples were prepared by growing cells on M9 medium containing 15NH4Cl, 13C glucose and/or 100% D2O (Cambridge Isotope Labs, Andover, MA).  Expression and purification of the His6-tagged ArsD was as described previously (Lin, Yang and Rosen, 2007).  Eluted proteins were concentrated, quickly frozen and stored at –80 °C until use. Protein concentrations were between 0.8 to 1.3 mM as determined by UV-vis spectroscopy at 280 nm (using A280 of 1.0 cm-1 = 75 µM).  For NMR experiments, proteins were exchanged into a buffer containing 20 mM sodium phosphate, 100 mM NaCl, 5 mM DTT, 90% H2O/10% D2O (pH 6.0) using Micro Bio-Spin columns (Bio-Rad, Hercules, CA); under similar buffer conditions the protein is a stable homodimer (Lin, Yang and Rosen, 2007).  NMR samples were cycled through multiple vacuum/Ar purge cycles using a Schlenk line.  Samples were then sealed under argon directly within the NMR tube.  Nuclear magnetic resonance spectroscopy (NMR) NMR spectra were acquired at 293 K on a Varian INOVA 720 MHz spectrometer (National High Field Magnet Laboratory, Tallahassee FL) and on an in house Varian INOVA 600 MHz spectrometer.  The 720 MHz spectrometer was equipped with a triple resonance gradient room temperature probe while the 600 MHz spectrometer utilized a Varian triple resonance gradient cold probe.  Backbone assignments were made using the following experiments: 15N-HSQC, HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB and CBCA(CO)NH (Cavanagh, 1996).  Partial side chain atom assignments were made using a 15N TOCSY and a CCONH calibrated to account for the deuterium labeled aliphatic carbons. Spectra were analyzed according to established protocols on a Macintosh G5 PC using the processing programs NMRPipe (Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax, 1995) and Sparky (Goddard and Kneller, 2001).  Chemical shift analysis for secondary structure prediction from the NMR data was performed using CSI provided by the Sykes laboratory utilizing backbone and Cb residues only (Wishart, Sykes and Richards, 1991).  Secondary structure prediction of the As(III) metallochaperone ArsD in solution |  Jun Ye, et. al DIGITALCOMMONS@WSU  |  2010 7Assignments and data deposition Residue assignments for the 248 residue symmetric ArsD homodimer are provided in the 15N HSQC spectrum (Fig. 1).  Amide backbone assignments were completed for 109 of the 112 non-proline residues, minus the protein’s N-terminus, 6 prolines and the histidine tag residues.  Side chain assignments were achieved for 106 of the 118 residues in the ArsD monomeric primary structure.  Chemical shift assignments are deposited in the Biological Magnetic Resonance Bank (http://www.bmrb.wisc.edu/) under the accession code BMRB-17231.  Acknowledgements   We thank the National High Magnetic Field Laboratory (NHMFL) in Tallahassee, FL. for 720 MHz NMR time as part of their user’s program.  The INOVA 600 MHz spectrometer with cold probe was purchased through NIH support (1S10RR16626-01).  The work was supported by NIH grants AI043428 (BPR) and DK068139 (TLS). References Bhattacharjee, H., Rosen, B. P., Arsenic metabolism in prokaryotic and eukaryotic microbes. In: D. H. S. Nies, Simon (Ed.), Molecular microbiology of heavy metals, Vol. 6. Springer-Verlag, Heidelberg/New York, 2007, pp. 371-406. Cavanagh, J., Fairbrother,W.J., Palmer III,A.G., Skelton,N.J., (1996). Protein NMR Spectroscopy: Principles and Practice. Academic Press, San Diego. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., Bax, A., (1995). NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 6, 277-93. Goddard, T. D., Kneller, D. G., SPARKY 3. University of California, San Francisco, 2001. Johnson, B. A., (2004). Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol Biol. 278, 313-52. Lin, Y. F., Walmsley, A. R., Rosen, B. P., (2006). An arsenic metallochaperone for an arsenic detoxification pump. Proc Natl Acad Sci U S A. 103, 15617-22. Lin, Y. F., Yang, J., Rosen, B. P., (2007). ArsD residues Cys12, Cys13, and Cys18 form an As(III)-binding site required for arsenic metallochaperone activity. J Biol Chem. 282, 16783-91. Pervushin, K., Braun,D., Fernandez,C., Wuthrich,K., (2000). [15N,1H]/[13C,1H]-TROSY for simultaneous detection of backbone 15N-1H, aromatic 13C-1H and side-chain 15N-1H2 correlations in large proteins. J. Biomol NMR. 17, 195-202. Wishart, D. S., Sykes, B. D., Richards, F. M., (1991). Relationship between Nuclear Magnetic Resonance Chemical Shift and Protein Secondary Structure. J. Mol. Bio. 222, 311-333. Secondary structure prediction of the As(III) metallochaperone ArsD in solution |  Jun Ye, et. al DIGITALCOMMONS@WSU  |  2010 8Yang, J., Rawat, S., Stemmler, T. L., Rosen, B. P., (2010). Arsenic binding and transfer by the ArsD As(III) metallochaperone. Biochemistry. in press. Ye, J., Ajees, A. A., Yang, J., Rosen, B. P., (2010). The 1.4 A crystal structure of the ArsD arsenic metallochaperone provides insights into its interaction with the ArsA ATPase. Biochemistry. 49, 5206-12.   

Resonance Assignments and Secondary Structure Predictions of the As(III) Metallochaperone ArsD in Solution

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Resonance Assignments and Secondary Structure Predictions of the As(III) Metallochaperone ArsD in Solution

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