Constraining Conformation D.S. Magruder1, L. Li2, J. Unruh2 & K. Si2 1Rhodes College: 2000 N. Parkway, Memphis, TN 38112 2Stowers Institue for Medical Research: 1000 E. 50th Street, Kansas City, MO 64110 Predicting Prions Methodology

Prions’ conformational dexterity makes their structure elusive. Crystallization [i] [ii] cannot constrain prions to a uniform state and predictive software cannot N N N N N N 1,5,9 handle the search space complexity . Pivotal in both memory and incurable N Prion domain

(variable) rz

diseases, discovering prions’ mechanism of action is paramount. While failing rz

rz rz

rz rz rz

Orb2 Orb2

Orb2 Orb2 independently, hybridizing biological and in silico assays may reveal structural Orb2 Orb2 Orb2A TEV clevage site insight of prions. Orb2, a mnemonic prion, forms fibers in vitro; the repeating Quencher RRM rz Quencher 11-13,16 C terminus cTEV cTEV cTEV

segment’s dimensions are known (Fig. 1) . Orb2’s C terminus (Orb2rz) is Orb2 nTEV nTEV nTEV GFP less dynamic, non-prion-like and contains two conserved domains; the ZZ domain reTEV GFP 1,5,9,11-13,16 structure of an Orb2rz human homolog has been solved . Thus Linker 5 C template-based modeling can accurately predict Orb2rz . 5. Test for ber formation

4. Optimize prion domain to t Repeating unit Repeating unit within ber segment Application of split TEV to Orb2 may restrict its conformation, Fig. 1: Electron micrograph of Orb2A oligomers [iii]

Prion domain Prion domain Prion domain Prion domain Prion domain Prion domain rz rz rz rz allowing in silico analysis of oligomers. Split TEV protease protocol requires rz rz rz rz

(asterisks) and amyloid fibers (arrow). Repeating (variable) (variable) (variable) (variable) (variable) (variable)

Orb2 Orb2 segment dimensions are 17 x 58 nm. Scale bar: Orb2 Orb2 Orb2 Orb2 fusion of N/C terminus halves of TEV protease (nTEV and cTEV protease 1. Predict structures respectively) to proteins with suspected protein-protein interactions (PPIs)15. 0.2 µm. (Hervás et al., unpublished) of fusion proteins Upon a PPI, TEV protease is reconstituted (reTEV) and reTEV protease’s 1.5 p = 0.0006 2. Dock fusion proteins 2 to reconstitute TEV

catalytic function activates a designated signal (Fig. 5B). Fusion of split TEV R = 0.37 N rz rz

Prion domain Prion domain Prion domain Prion domain Prion domain Prion domain rz rz rz rz rz rz Orb2

slope = 0.43 (variable) (variable) (variable) (variable) (variable) Orb2 (variable) Orb2 protease to Orb2’s C termini, during traditional memory paradigms in Orb2 Orb2 Orb2

1.0 rz Drosophila melanogaster, demonstrates fusion does not impede either protein’s N N 3. Orient docked Orb2rz

Orb2 to t within ber 16 function (Fig. 2) . Conjoined, the concurrency and independence of reTEV Repeating unit 58 x 17 nm

protease and Orb2 activity imply Orb2rzs are within vicinity of one another rz rz

Prion domain Prion domain Prion domain Prion domain Prion domain Prion domain Orb2 rz rz rz rz rz rz

Orb2 rz rz rz rz rz

0.5 nTEV Fiber (variable) (variable) (variable) (variable) (variable) (variable) Orb2 Orb2 Orb2 thus restricting Orb2’s conformational space. Orb2 Orb2 Orb2 Orb2 Orb2 Normalized GFP Signal

cTEV N nTEV reTEV Hence I modeled the fusion proteins, docked them, and filtered by reTEV reTEV protease occurrence for a pair of Orb2rzs4,5,8. Using Orb2rzs’ orientation, we 0.0

0.0 0.2 0.4 0.6 0.8 1.0 rz rz rz

Prion domain Prion domain Prion domain Prion domain Prion domain Prion domain rz rz rz rz Memory Index rz rz Orb2 Orb2

configured the docked Orb2rzs to fit within the repeat segment (Fig. 5C). Orb2 (variable) (variable) (variable) (variable) (variable) (variable) Orb2 Ongoing work aims at optimizing the prion-like domain to fit within the Orb2 Orb2 Orb2 Fig. 2: Correlation of GFP signal unsuppressed via cTEV remaining space and subsequently testing optimized models against TEV protease cleaving to the Memory Index of flies. themselves to evaluate fiber forming potential. These models will then be Thus also a correlation of reTEV protease formation Fiber

Prion domain Prion domain Prion domain Prion domain rz rz and Orb2 oligomerization. Courtesy of Liying Li. rz rz rz

analyzed as candidate structures for the fiber conformation of Orb2 to later be (variable) (variable) (variable) (variable) Prion domain Orb2 Orb2 Orb2 Orb2 (variable)

rz

rz Prion domain

tested via biological assays. Orb2 Orb2 (variable)

[i] [ii] [iii]

Fig. 5: [i] Model of Orb2A. The blue cylinder represents homology modeling of Orb2’s RNA Recognition Motif (RRM) and ZZ domain (ZZ) and is thus labeled Orb2rz. [ii] Orb2 oligomerization and split TEV prtoease. When Orb2 monomers interact, the prion domain undergoes a * conformational change conducive for oligomerization. When split TEV protease is attached, this allows for reTEV protease formation. reTEV protease can then cleave a designed signal, such as the linker connecting a suppressor (Quencher) to GFP allowing for the PPI to be quantified (see Fig. 2). [iii] (1) Prediction of the Orb2 + split TEV protease fusion proteins. Only Orb2rz + split TEV protease are predicted via homology modeling5. The N terminus is included to represent the full fusion proteins, and our prediction goal. (2) The fusion proteins (Orb2rz + split TEV protease) are docked such that reTEV protease is produced8. (3) The docked structure is oriented to fit within the dimensions of the repeating segment of an Orb2 fiber. (4) Prion domain predictions. Orb2A’s remaining amino acids are predicted such that the structure is optimized to fit within the repeating segment’s dimensions. (5) Optimized structures are tested against each other for docking potential. Only those that allow of potential fiber formation are candidate models to be confirmed via biological assays. Fig. 3: [i] Crystal structure of Tobacco Etch Virus Nuclear Inclusion-a 3C endopeptidase (TEV Protease) with in red2, nTEV protease in purple, and cTEV protease in green. [ii] Split TEV protease shown as reTEV protease with same coloring scheme as in [i]. [iii] Orb2A C terminus’ RNA Recognition Motif (RRM) and ZZ-like Domain (ZZ) (Orb2rz). ZZ left of asterisk, RRM right thereof. Acknowledgements For their contributions, time and insight: Liying Li, Thomas Shafee, Kelley Lawrence, Malcolm Cook, Mark Miller, Michael Wehr, Christopher Seidel, Thom Vreven, David Waugh, Jeffery Gray, Brian Slaughter, Blake Ebner, Paulo Leal, Kerson Haung, Andras Aszódi, Angle Herrez, & Mark D’Agostino. In remembrance of Thomas E Creighton. TEV: A Tool for Taming Variability References

[1] Afroz, T., L. Skrisovska, E. Belloc, J. Guillen-Boixet, R. Mendez, and F. H.- T. Allain. "A Fly Trap Mechanism Provides Sequence-specific RNA Recognition by CPEB Proteins." Genes & Development 28.13 (2014): 1498-514. Web. TEV protease demonstrates profound specificity attributed to its highly conserved [2] Arad, Dorit, Racheli Kreisberg, and Michael Shokhen. "Structural and Mechanistic Aspects of 3C from the Picornavirus Family." Journal of Chemical Information and Modeling J. Chem. Inf. Model. 33.3 (1993): 345-49. 2-4,7,10,14,15,17 [3] Cleland, W.w., P. Frey, and J. Gerlt. "Low-Barrier Hydrogen Bonds and Enzymatic Catalysis." Archives of Biochemistry and Biophysics 382.1 (2000): 1-5. Web. structure; its catalytic chemistry is mostly elucidated . Previous work [4] Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/ [5] Kelley, Lawrence A., Stefans Mezulis, Christopher M. Yates, Mark N. Wass, and Michael J E Sternberg. "The Phyre2 Web Portal for Protein Modeling, Prediction and Analysis." Nature Protocols Nat Protoc 10.6 (2015): 845-58. suggests that more than 1Å deviation at catalytic residues renders TEV protease [6] Majumdar, Amitabha, Wanda Colón Cesario, Erica White-Grindley, Huoqing Jiang, Fengzhen Ren, Mohammed “Repon” Khan, Liying Li, Edward Man-Lik Choi, Kasthuri Kannan, Fengli Guo, Jay Unruh, Brian Slaughter, and Kausik Si. 2-4,7,10,14,15,17 "Critical Role of Amyloid-like Oligomers of Drosophila Orb2 in the Persistence of Memory." Cell 148.3 (2012): 515-29. Web. inactive (Fig. 4). Thus viability of catalytically active reTEV protease [7] Phan, J., A. Zdanov, A. Evdokimov, J. Tropea, H. Peters, R. Kapust, M. Li, A. Wlodawer, and D. Waugh. "Structural Basis for the Specificity of Tobacco Etch Virus Protease." Journal of Biological Chemistry 277.52 (2002): readily restricts reTEV protease’s structure to that of TEV protease. A five amino 50564-0572. Web. [8] Pierce BG, Wiehe K, Hwang H, Kim BH, Vreven T, Weng Z. (2014) ZDOCK Server: Interactive Docking Prediction of Protein-Protein Complexes and Symmetric Multimers. Bioinformatics 30(12): 1771-3. acid linker attaching split TEV protease to the structurally conserved domains of [9] Saunders, Alison, and Daniel Cox. "Finding the Structure of CPEB." UC Davis Physics REU: REU 09. UC Davis, 18 Aug. 2009. Web. 12 June 2015. [10] Shafee, Thomas, Pietro Gatti-Lafranconi, Ralph Minter, and Florian Hollfelder. "Handicap-Recover Evolution Leads to a Chemically Versatile, Nucleophile-Permissive Protease." ChemBioChem (2015): n. pag. Web. the relatively large Orb2 protein, at minimum, restricts Orb2rz to a finite set of [11] Si, Kausik, Maurizio Giustetto, Amit Etkin, Ruby Hsu, Agnieszka M. Janisiewicz, Maria Conchetta Miniaci, Joung-Hun Kim, Huixiang Zhu, and Eric R. Kandel. "A Neuronal Isoform of CPEB Regulates Local Protein Synthesis and Stabilizes Fig. 4: Distances between TEV protease’s Synapse-Specific Long-Term Facilitation in Aplysia." Cell 115.7 (2003): 893-904. Web. orientations conducive for reTEV protease. Consequentially, knowing the [12] Si, Kausik, Susan Lindquist, and Eric R. Kandel. "A Neuronal Isoform of the Aplysia CPEB Has Prion-Like Properties." Cell 115.7 (2003): 879-91. Web. catalytic triad (His46, Asp81, Cys151). Catalytic [13] Si, Kausik, Yun-Beom Choi, Erica White-Grindley, Amitabha Majumdar, and Eric R. Kandel. "Aplysia CPEB Can Form Prion-like Multimers in Sensory Neurons That Contribute to Long-Term Facilitation." Cell 140.3 (2010): 421-35. structure and orientation of Orbrzs restricts the rest of Orb2 to the remaining triad may be a misnomer, as the 3C protease [14] Tyndall, Joel D. A., Tessa Nall, and David P. Fairlie. "Proteases Universally Recognize ß- Strands in Their Active Sites." ChemInform 36.21 (2005): n. pag. Web. [15] Wehr, M.C., L. Reinecke, A. Botvinnik, and M.J. Rossner. "Analysis of Transient Phosphorylation-dependent Protein-protein Interactions in Living Mammalian Cells Using Split-TEV." BMC Biotechnology 8.1 (2008): 55.. space within the dimensions of a fiber’s repeating segment. “triad” has been shown to be inefficient, [16] White-Grindley, E., L. Li, R. Mohammad Khan, F. Ren, A. Saraf, L. Florens, and K. Si. "Contribution of Orb2A Stability in Regulated Amyloid-Like Oligomerization of Drosophila Orb2." PLoS Biol PLoS Biology 12.2 (2014): n. pag. Web. suggesting involvement of other residues2. [17] Yin, Jiang, Chunying Niu, Maia M. Cherney, Jianmin Zhang, Carly Huitema, Lindsay D. Eltis, John C. Vederas, and Michael N.g. James. "A Mechanistic View of Inhibition and Peptide Hydrolysis in the of the SARS-CoV 3C-like Peptidase." Journal of Molecular Biology 371.4 (2007): 1060-074. Web.