Mapping the DNA gyrase:ParE toxin interface using chemical crosslinking

Mapping the DNA gyrase:Par E toxin interface using chemical crosslinkingJessica R. Ames*, Dr. Meena Muthu, Dr. Christina R. BourneInterdisciplinary Research Collaborative Undergraduate Student Research SymposiumOctober 28, 2016Rose-Hulman Institute of Technology, Department of Chemical Engineering University of Oklahoma, Department of Chemistry and Biochemistry

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Project Goal and SignificanceEstablish a crosslinked structure of gyrase, DNA, and toxinFurther our knowledge on the conformational changes of gyraseDevelop new inhibition of an established antibiotic targetPotential alternative for fluoroquinolone-resistant infections2

Center for Disease Dynamics, Economics & Policy Resistance Map, Antibiotic Resistance of E. coli in United States

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Gyrase BasicsA2B2 heterotetramerType II topoisomeraseInduces negative supercoilingTolerates mutations in the active siteGives rise to quinolone resistance

PDB ID: 4plb, S. aureus gyrase. Papillon, et al. Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase. Colored by Ames, J. R. via UCSF Chimera (2016).3

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Gyrase Basics-Two Proposed StructuresCryo-EM Map

SAXS envelope

Papillon, et al. Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase. Baker, Weigand, Maar-Mathias, Mondragon Solution structures of DNA-bound gyrase Nucl. Acids Res. (2011) 39:7554

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Fusion Protein ExpressionAmes, J.R. (2016)5

T7 Promotor

GyrBGyrA4 Amino Acid Linker

6-His Tag

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Research Question #1Can we obtain the E. coli GyrBA fusion protein in sufficient quantity and purity for experiments?

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Gyrase PurificationAffinity ChromatographySize Exclusion ChromatographyAmes, J. R. (2016)7

161310719

M710131619250 kDa130 kDa

28 kDa

M510127250 kDa130 kDa510127

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Research Question #2How do different concentrations of glutaraldehyde affect protein-protein crosslinking in the GyrBA fusion protein?8

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Crosslinking BasicsGlutaraldehyde crosslinks lysine residuesExpect a dimer of MW=386 kDa ProtocolGyrBA fusion protein (and toxin)Incubate at 37C Add glutaraldehyde

PDB ID: 4plb, colored by Ames, J. R. (2016)9

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Gyrase Protein-Protein CrosslinkingVarying concentrations of glutaraldehydeUncrosslinked v. Crosslinked samples10

250 kDa130 kDa

386 kDa193 kDaM12Ames, J. R. (2016)460250171117

4% stacking, 6% resolving polyacrylamide gel3%-15% gradient gel

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Research Question #3How does toxin affect crosslinking?11

PDB ID: 3kxe colored by Ames, J. R. (2016)

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Digested Crosslinked Gyrase with and without ToxinWith ToxinWithout Toxin12

UnCLCLMCLMAmes, J. R. (2016)75 kDa25 kDa35 kDa48 kDa63 kDa75 kDa25 kDa35 kDa48 kDa63 kDa

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Final Comparisons13

M123MCrosslinked-++Toxin--+

Ames, J. R. (2016)75 kDa25 kDa35 kDa48 kDa63 kDa75 kDa25 kDa35 kDa48 kDa63 kDa

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ConclusionsThe E. coli GyrBA fusion protein can be purified with a yield of approximately 3 mg per liter of cultureGlutaraldehyde completely crosslinks GyrBA fusion at a concentration of 0.05% in both the presence and absence of ADPNPThe addition of toxin appears to protect GyrBA fusion from crosslinkingFuture directions for this project include mass spectrometry analysis of trypsinized peptides and EM evaluation of the crosslinked conformations of gyrase.

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ReferencesPapillon, J., Menetret, J., Batisse, C., Helye, R., Schultz, P., Potier, N., & Lamour, V. (2013, June 26). Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase.Nucleic Acids Research,41(16), 7815-7827. http://dx.doi.org/10.1093/nar/gkt560 Figure S5Baker, Weigand, Maar-Mathias, Mondragon Solution structures of DNA-bound gyrase Nucl. Acids Res. (2011) 39:755Kumar, R., Riley, J. E., Parry, D., Bates, A. D., & Nagaraja, V. (2012, September 18). Binding of two DNA molecules by type II topoisomerases for decatenation.Nucleic Acids Research,40(21), 10904-10915.

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AcknowledgementsResearch reported in this work was supported by a National Science Foundation Research Experience for Undergraduates award (DBI-1359457) and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103640. We appreciate the assistance of Dr. Phil Bourne and the OU Protein Production Core facility, funded by the NIGMS of the NIH (Award Number P20GM103640). We would also like recognize John C. White for purification of Gyrase A and B subunits and the At-1 toxin.I would also like to thank Drs. Broughton and Brandt for directing the IRC, along with Wabash Valley Local Section of the ACS for supporting this initiative.

Questions?

Gyrase Basics-Proposed Catalytic Cycle

Assemble functional unitBind DNAWrap DNA around GyrA and CTDBind ATP, cleave DNALigation happens between or during steps 5 and 6Papillon, et al. Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase18

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