The designability of protein switches by chemical rescue of structure: mechanisms of inactivation and reactivation
Open accessAbstract
The ability to selectively activate function of particular proteins via pharmacological agents is a longstanding goal in chemical biology. Recently, we reported an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. However, rationally identifying analogous de novo binding sites in other enzymes represents a key challenge for extending this approach to introduce allosteric control into other enzymes. Here we show that mutation sites leading to protein inactivation via tryptophan-to-glycine substitution and allowing (partial) reactivation by the subsequent addition of indole are remarkably frequent. Through a suite of methods including a cell-based reporter assay, computational structure prediction and energetic analysis, fluorescence studies, enzymology, pulse proteolysis, X-ray crystallography, and hydrogen-deuterium mass spectrometry, we find that these switchable proteins are most commonly modulated indirectly, through control of protein stability. Addition of indole in these cases rescues activity not by reverting a discrete conformational change, as we had observed in the sole previously reported example, but rather rescues activity by restoring protein stability. This important finding will dramatically impact the design of future switches and sensors built by this approach, since evaluating stability differences associated with cavity-forming mutations is a far more tractable task than predicting allosteric conformational changes. By analogy to natural signaling systems, the insights from this study further raise the exciting prospect of modulating stability to design optimal recognition properties into future de novo switches and sensors built through chemical rescue of structure.
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Protein structure and function are most frequently modulated indirectly through control of protein stability.
“A suite of computational and experimental methods, collectively led to the insight that protein structure and function were most frequently modulated indirectly through control of protein stability (Xia et al., 2013).”
From Allostery in Its Many Disguises: From Theory to Applications (2019) · cites this paper as Xia et al., 2013
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NIH Image to ImageJ: 25 years of image analysis
Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012) · Nature Methods · doi:10.1038/nmeth.2089 · Cited by 64,638 · see citing works
Engineering and characterization of a superfolder green fluorescent protein
Pédelacq, J. D., Cabantous, S., Tran, T. H., Terwilliger, T. C., & Waldo, G. S. (2005) · Nature Biotechnology · doi:10.1038/nbt1172 · Cited by 2,295 · see citing works
Leaver-Fay (2011) · doi:10.1016/b978-0-12-381270-4.00019-6 · Cited by 1,904 · see citing works
Hydrogen exchange and structural dynamics of proteins and nucleic acids
Englander, S. W., & Kallenbach, N. R. (1983) · Quarterly Reviews of Biophysics · doi:10.1017/s0033583500005217 · Cited by 1,359 · see citing works
Alleviating Cancer Drug Toxicity by Inhibiting a Bacterial Enzyme
Wallace, B. D., Wang, H., Lane, K. T., Scott, J. E., Orans, J., Koo, J. S., Venkatesh, M., Jobin, C., Yeh, L.-A., Mani, S., et al. (2010) · Science · doi:10.1126/science.1191175 · Cited by 1,080 · see citing works
Zhang, Z., & Smith, D. L. (1993) · Protein Science · doi:10.1002/pro.5560020404 · Cited by 1,017 · see citing works
Wright, P. E., & Dyson, H. J. (2009) · Current Opinion in Structural Biology · doi:10.1016/j.sbi.2008.12.003 · Cited by 1,012 · see citing works
Sauer-Eriksson, A. E., Baase, W. A., Zhang, X. ‐., Heinz, D. W., Blaber, M., Baldwin, E. P., & Matthews, B. W. (1992) · Science · doi:10.1126/science.1553543 · Cited by 920 · see citing works
Banaszynski, L. A., Chen, L.-C., Maynard-Smith, L. A., Ooi, A. G. L., & Wandless, T. J. (2006) · Cell · doi:10.1016/j.cell.2006.07.025 · Cited by 902 · see citing works
Controlling Signal Transduction with Synthetic Ligands
Spencer, D. M., Wandless, T. J., Schreiber, S. L., & Crabtree, G. R. (1993) · Science · doi:10.1126/science.7694365 · Cited by 845 · see citing works
Intrinsically disordered proteins: regulation and disease
Babu, M. M., van der Lee, R., de Groot, N. S., & Gsponer, J. A. (2011) · Current Opinion in Structural Biology · doi:10.1016/j.sbi.2011.03.011 · Cited by 597 · see citing works
Structure prediction for CASP8 with all‐atom refinement using Rosetta
Raman, S., Vernon, R. M., Thompson, J., Tyka, M. D., Sadreyev, R. I., Pei, J., Kim, D. E., Kellogg, E. H., DiMaio, F., Lange, O. F., et al. (2009) · Proteins Structure Function and Bioinformatics · doi:10.1002/prot.22540 · Cited by 511 · see citing works
Surface Sites for Engineering Allosteric Control in Proteins
Lee, J., Natarajan, M., Nashine, V. C., Socolich, M., Vo, T., Russ, W. P., Benkovic, S. J., & Ranganathan, R. (2008) · Science · doi:10.1126/science.1159052 · Cited by 345 · see citing works
High-resolution structure prediction and the crystallographic phase problem
Qian, B., Raman, S., Das, R., Bradley, P. H., McCoy, A. J., Read, R. J., & Baker, D. A. (2007) · Nature · doi:10.1038/nature06249 · Cited by 317 · see citing works
Reprogramming Control of an Allosteric Signaling Switch Through Modular Recombination
Dueber, J. E., Yeh, B. J., Chak, K., et al. (2003) · Science · doi:10.1126/science.1085945 · Cited by 298 · see citing works
A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene
Sauer-Eriksson, A. E., Baase, W. A., Wozniak, J. A., & Matthews, B. W. (1992) · Nature · doi:10.1038/355371a0 · Cited by 266 · see citing works
Huang, Y., & Liu, Z. (2009) · Journal of Molecular Biology · doi:10.1016/j.jmb.2009.09.010 · Cited by 266 · see citing works
Exchange of hydrogen atoms in insulin with deuterium atoms in aqueous solutions
Hvidt, A., & Linderström-Lang, K. (1954) · Biochimica et Biophysica Acta · doi:10.1016/0006-3002(54)90241-3 · Cited by 244 · see citing works
The structural basis for spectral variations in green fluorescent protein
Palm, G. J., Zdanov, A. S., Gaitanaris, G., Stauber, R. H., Pavlakis, G. N., & Wlodawer, A. (1997) · Nature Structural & Molecular Biology · doi:10.1038/nsb0597-361 · Cited by 243 · see citing works
Xu, J., Baase, W. A., Baldwin, E. P., & Matthews, B. W. (1998) · Protein Science · doi:10.1002/pro.5560070117 · Cited by 229 · see citing works
Allosite: a method for predicting allosteric sites
Huang, W., Lu, S., Huang, Z., Liu, X., Mou, L., Luo, Y., Zhao, Y., Liu, Y., Chen, Z., Hou, T., et al. (2013) · Bioinformatics · doi:10.1093/bioinformatics/btt399 · Cited by 227 · see citing works
Guntas, G., Mansell, T. J., Kim, J. R., & Ostermeier, M. (2005) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0502673102 · Cited by 215 · see citing works
Engineered allosteric activation of kinases in living cells
Karginov, A. V., Ding, F., Kota, P. B., Dokholyan, N. V., & Hahn, K. M. (2010) · Nature Biotechnology · doi:10.1038/nbt.1639 · Cited by 203 · see citing works
Pan, H. C., Lee, J. C., & Hilser, V. J. (2000) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.220240297 · Cited by 201 · see citing works
Protein dynamics viewed by hydrogen exchange
Skinner, J. J., Lim, W. K., Bédard, S., Black, B. E., & Englander, S. W. (2012) · Protein Science · doi:10.1002/pro.2081 · Cited by 189 · see citing works
Directed evolution of ligand dependence: small-molecule-activated protein splicing.
Buskirk, A. R., Ong, Y., Gartner, Z. J., & Liu, D. R. (2004) · Proceedings of the National Academy of Sciences of the United States of America · doi:10.1073/pnas.0402762101 · Cited by 169 · see citing works
Protein Conformational Switches: From Nature to Design
Ha, J., & Loh, S. N. (2012) · Chemistry - A European Journal · doi:10.1002/chem.201200348 · Cited by 165 · see citing works
Green‐fluorescent protein mutants with altered fluorescence excitation spectra
Ehrig, T., O’Kane, D. J., & Prendergast, F. G. (1995) · FEBS Letters · doi:10.1016/0014-5793(95)00557-p · Cited by 160 · see citing works
Engineering Biosensors with Extended, Narrowed, or Arbitrarily Edited Dynamic Range
Vallée‐Bélisle, A., Ricci, F., & Plaxco, K. W. (2011) · Journal of the American Chemical Society · doi:10.1021/ja209850j · Cited by 155 · see citing works
Bell, C. E., Frescura, P., Hochschild, A., & Lewis, M. (2000) · Cell · doi:10.1016/s0092-8674(00)80891-0 · Cited by 151 · see citing works
Protein hydrogen exchange: Testing current models
Skinner, J. J., Lim, W. K., Bédard, S., et al. (2012) · Protein Science · doi:10.1002/pro.2082 · Cited by 149 · see citing works
The Database of Macromolecular Motions: new features added at the decade mark
Flores, S. C. (2005) · Nucleic Acids Research · doi:10.1093/nar/gkj046 · Cited by 146 · see citing works
Coupling of receptor conformation and ligand orientation determine graded activity
Bruning, J. B., Parent, A. A., Gil, G., Zhao, M., Nowak, J., Pace, M. C., Smith, C. L., Afonine, P. V., Adams, P. D., Katzenellenbogen, J. A., et al. (2010) · Nature Chemical Biology · doi:10.1038/nchembio.451 · Cited by 142 · see citing works
Revisited gene regulation in bacteriophage λ
Dodd, I. B., Shearwin, K. E., & Egan, J. B. (2005) · Current Opinion in Genetics & Development · doi:10.1016/j.gde.2005.02.001 · Cited by 140 · see citing works
The role of protein conformational fluctuations in allostery, function, and evolution
Wrabl, J. O., Gu, J., Liu, T., Schrank, T. P., Whitten, S. T., & Hilser, V. J. (2011) · Biophysical Chemistry · doi:10.1016/j.bpc.2011.05.020 · Cited by 134 · see citing works
Rational design of a ligand-controlled protein conformational switch
Dağliyan, O., Shirvanyants, D. G., Karginov, A. V., Ding, F., Fee, L. R., Chandrasekaran, S. N., Freisinger, C. M., Smolen, G. A., Anna Huttenlocher, A., Hahn, K. M., et al. (2013) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1218319110 · Cited by 128 · see citing works
Agonism/antagonism switching in allosteric ensembles
Motlagh, H. N., & Hilser, V. J. (2012) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1120519109 · Cited by 102 · see citing works
SPACER: server for predicting allosteric communication and effects of regulation
Goncearenco, A., Mitternacht, S., Yong, T., Eisenhaber, B., Eisenhaber, F., & Berezovsky, I. N. (2013) · Nucleic Acids Research · doi:10.1093/nar/gkt460 · Cited by 102 · see citing works
Limitations of Induced Folding in Molecular Recognition by Intrinsically Disordered Proteins
Házy, E., & Tompa, P. (2009) · ChemPhysChem · doi:10.1002/cphc.200900205 · Cited by 98 · see citing works
Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt
Pratt, M. R., Schwartz, E. C., & Muir, T. W. (2007) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0700816104 · Cited by 89 · see citing works
Structural Dynamics, Intrinsic Disorder, and Allostery in Nuclear Receptors as Transcription Factors
Hilser, V. J., & Thompson, E. B. (2011) · Journal of Biological Chemistry · doi:10.1074/jbc.r111.278929 · Cited by 82 · see citing works
Dixit, A., & Verkhivker, G. M. (2011) · PLoS Computational Biology · doi:10.1371/journal.pcbi.1002179 · Cited by 81 · see citing works
A review about nothing: Are apolar cavities in proteins really empty?
Matthews, B. W., & Liu, L. (2009) · Protein Science · doi:10.1002/pro.61 · Cited by 81 · see citing works
Laine, É., Gonçalves, C., Karst, J. C., Lesnard, A., Rault, S., Tang, W., Malliavin, T. E., Ladant, D., & Blondel, A. (2010) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0914611107 · Cited by 75 · see citing works
Intrinsically disordered proteins may escape unwanted interactions via functional misfolding
Uversky, V. N. (2011) · Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics · doi:10.1016/j.bbapap.2011.03.010 · Cited by 65 · see citing works
Structure-Based Predictive Models for Allosteric Hot Spots
Demerdash, O., Daily, M. D., & Mitchell, J. C. (2009) · PLoS Computational Biology · doi:10.1371/journal.pcbi.1000531 · Cited by 64 · see citing works
Rapid optimization of gene dosage in E. coli using DIAL strains
Kittleson, J. T., Cheung, S. F., & Anderson, J. (2011) · Journal of Biological Engineering · doi:10.1186/1754-1611-5-10 · Cited by 64 · see citing works
Self-citationRacemic crystallography—Easy crystals and easy structures: What's not to like?
Matthews, B. W. (2009) · Protein Science · doi:10.1002/pro.125 · Cited by 63 · see citing works
Computation of Conformational Coupling in Allosteric Proteins
Kidd, B. A., Baker, D. A., & Thomas, W. E. (2009) · PLoS Computational Biology · doi:10.1371/journal.pcbi.1000484 · Cited by 63 · see citing works
Engineered Allosteric Activation of Kinases in Living Cells
Karginov, A. V., Ding, F., Kota, P. B., et al. (2011) · Biophysical Journal · doi:10.1016/j.bpj.2010.12.3017 · Cited by 59 · see citing works
Designing Allosteric Control into Enzymes by Chemical Rescue of Structure
Deckert, K., Budiardjo, S. J., Brunner, L. C., et al. (2012) · Journal of the American Chemical Society · doi:10.1021/ja301409g · Cited by 57 · see citing works
Interplay between allostery and intrinsic disorder in an ensemble
Motlagh, H. N., Li, J., Thompson, E. B., & Hilser, V. J. (2012) · Biochemical Society Transactions · doi:10.1042/bst20120163 · Cited by 55 · see citing works
Converting a protein into a switch for biosensing and functional regulation
Stratton, M. M., & Loh, S. N. (2010) · Protein Science · doi:10.1002/pro.541 · Cited by 50 · see citing works
Rapid and Tunable Control of Protein Stability in Caenorhabditis elegans Using a Small Molecule
Cho, U. H., Zimmerman, S. M., Chen, L., Owen, E., Kim, J. V., Kim, S. K., & Wandless, T. J. (2013) · PLoS ONE · doi:10.1371/journal.pone.0072393 · Cited by 49 · see citing works
Amemiya, T., Koike, R., Fuchigami, S., et al. (2011) · Journal of Molecular Biology · doi:10.1016/j.jmb.2011.02.058 · Cited by 46 · see citing works
A cavity‐forming mutation in insulin induces segmental unfolding of a surrounding α‐helix
Xu, B., Hua, Q. X., Nakagawa, S. H., et al. (2002) · Protein Science · doi:10.1110/ps.32102 · Cited by 37 · see citing works
An Engineered Calmodulin‐Based Allosteric Switch for Peptide Biosensing
Meister, G. E., & Joshi, N. S. (2013) · ChemBioChem · doi:10.1002/cbic.201300168 · Cited by 34 · see citing works
Protein Switch Engineering by Domain Insertion
Kanwar, M., Wright, R. C., Date, A., et al. (2013) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/b978-0-12-394292-0.00017-5 · Cited by 34 · see citing works
A cavity-forming mutation in insulin induces segmental unfolding of a surrounding alpha-helix
Xu, B. (2002) · Protein Science · doi:10.1110/ps.ps.32102 · Cited by 23 · see citing works
Choi, J. H., San, A., & Ostermeier, M. (2013) · Protein Science · doi:10.1002/pro.2234 · Cited by 16 · see citing works
Predicting the structure of protein cavities created by mutation
Machicado, C., Bueno, M., & Sancho, J. (2002) · Protein Engineering Design and Selection · doi:10.1093/protein/15.8.669 · Cited by 15 · see citing works
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- [18]Leaver-Fay A Tyka M Lewis SM Lange OF Thompson J Jacak R Kaufman K Renfrew PD Smith CA Sheffler W Davis IW Cooper S Treuille A Mandell DJ Richter F Ban YE Fleishman SJ Corn JE Kim DE Lyskov S Berrondo M Mentzer S Popovic Z Havranek JJ Karanicolas J Das R Meiler J Kortemme T Gray JJ Kuhlman B Baker D Bradley P Methods Enzymol 2011 487 545 21187238 PMC4083816
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Allostery in Its Many Disguises: From Theory to Applications
Wodak, S. J., Paci, E., Dokholyan, N. V., Berezovsky, I. N., Horovitz, A., Li, J., Hilser, V. J., Bahar, I., Karanicolas, J., Stock, G., et al. (2019) · Structure · doi:10.1016/j.str.2019.01.003 · Cited by 404 · see citing works
Self-citationKeppel, T. R., Sarpong, K., Murray, E. M., Monsey, J., Zhu, J., & Bose, R. (2017) · Journal of Biological Chemistry · doi:10.1074/jbc.m116.747485 · Cited by 29 · see citing works
Self-citationFull and partial agonism of a designed enzyme switch
Budiardjo, S. J., Licknack, T. J., Cory, M. B., Kapros, D., Roy, A., Lovell, S., Douglas, J., & Karanicolas, J. (2016) · ACS Synthetic Biology · doi:10.1021/acssynbio.6b00097 · Cited by 13 · see citing works
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- Authors
- Xia, Y., DiPrimio, N., Keppel, T. R., Vo, B., Fraser, K., Battaile, K. P., Egan, C., Bystroff, C., Lovell, S., Weis, D. D., Anderson, J. C., & Karanicolas, J.
- Affiliations
- University of Kansas (US)
- University of California, Berkeley (US)
- Hauptman-Woodward Medical Research Institute (US)
- Rensselaer Polytechnic Institute (US)
- Corresponding author
- Karanicolas, John
- ORCID
- Venue
- Journal of the American Chemical Society
- Publisher
- American Chemical Society (ACS)
- Published
- 2013-12-06
- Volume
- 135
- Issue
- 50
- Pages
- 18840-18849
- ISSN
- 0002-7863 (ISSN-L)
- 1520-5126
- PMC
- PMC3919134
- OpenAlex
- W2132125753
- Cited by
- 33
- References
- 61
- Citations by year
- 2026: 2 · 2025: 1 · 2024: 1 · 2023: 2 · 2022: 3 · 2021: 2 · 2020: 1 · 2019: 6 · 2018: 4 · 2017: 3 · 2016: 4 · 2015: 3 · 2014: 1
- FWCI
- 1.19
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Journal of the American Chemical Society (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- KU ScholarWorks (University of Kansas) (acceptedVersion, other-oa)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Institute of General Medical Sciences (R01GM099827, P20GM103420)
- National Center for Research Resources (P20RR017708)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
- Keywords
- protein switches, indole, protein stability, allosteric modulation, enzyme inactivation, protein reactivation, protein designability
- MeSH terms
- Electrophoresis, Polyacrylamide Gel, Indoles, Mutation, Protein Conformation, Proteins, Genes, Reporter, Green Fluorescent Proteins
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