Rational design of a ligand-controlled protein conformational switch

Design of a regulatable multistate protein is a challenge for protein engineering. Here we design a protein with a unique topology, called uniRapR, whose conformation is controlled by the binding of a small molecule. We confirm switching and control ability of uniRapR in silico, in vitro, and in vivo. As a proof of concept, uniRapR is used as an artificial regulatory domain to control activity of kinases. By activating Src kinase using uniRapR in single cells and whole organism, we observe two unique phenotypes consistent with its role in metastasis. Activation of Src kinase leads to rapid induction of protrusion with polarized spreading in HeLa cells, and morphological changes with loss of cell–cell contacts in the epidermal tissue of zebrafish. The rational creation of uniRapR exemplifies the strength of computational protein design, and offers a powerful means for targeted activation of many pathways to study signaling in living organisms.

[1]  Shuangye Yin,et al.  Eris: an automated estimator of protein stability , 2007, Nature Methods.

[2]  Stephen L. Mayo,et al.  An efficient algorithm for multistate protein design based on FASTER , 2010, J. Comput. Chem..

[3]  W. P. Russ,et al.  Surface Sites for Engineering Allosteric Control in Proteins , 2008, Science.

[4]  Sheila M. Thomas,et al.  Cellular functions regulated by Src family kinases. , 1997, Annual review of cell and developmental biology.

[5]  Brian Kuhlman,et al.  Design of protein conformational switches. , 2006, Current opinion in structural biology.

[6]  N. Grishin.  Fold change in evolution of protein structures. , 2001, Journal of structural biology.

[7]  H. Stanley,et al.  Discrete molecular dynamics studies of the folding of a protein-like model. , 1998, Folding & design.

[8]  David Baker,et al.  Computation of Conformational Coupling in Allosteric Proteins , 2009, PLoS Comput. Biol..

[9]  Menachem Fromer,et al.  Dead‐end elimination for multistate protein design , 2007, J. Comput. Chem..

[10]  D. Baker,et al.  Design of a Novel Globular Protein Fold with Atomic-Level Accuracy , 2003, Science.

[11]  K. Hahn,et al.  Light regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP. , 2011, Journal of the American Chemical Society.

[12]  M. Ostermeier.  Designing switchable enzymes. , 2009, Current opinion in structural biology.

[13]  S. L. Mayo,et al.  De novo protein design: fully automated sequence selection. , 1997, Science.

[14]  Daniel W. Kulp,et al.  Design of a switchable eliminase , 2011, Proceedings of the National Academy of Sciences.

[15]  KumarShankar,et al.  The weighted histogram analysis method for free-energy calculations on biomolecules. I , 1992 .

[16]  Pradeep Kota,et al.  Engineered allosteric activation of kinases in living cells , 2010, Nature Biotechnology.

[17]  Jan E. Schnitzer,et al.  Role of GTP Hydrolysis in Fission of Caveolae Directly from Plasma Membranes , 1996, Science.

[18]  H. Ding,et al.  Antifungal rapamycin analogues with reduced immunosuppressive activity. , 2000, Bioorganic & medicinal chemistry letters.

[19]  Corey W. Liu,et al.  Characterization of the FKBP.rapamycin.FRB ternary complex. , 2005, Journal of the American Chemical Society.

[20]  Stuart L. Schreiber,et al.  Structure of the FKBP12-Rapamycin Complex Interacting with Binding Domain of Human FRAP , 1996, Science.

[21]  B. Kuhlman,et al.  A genetically-encoded photoactivatable Rac controls the motility of living cells , 2009, Nature.

[22]  P. Harbury,et al.  Automated design of specificity in molecular recognition , 2003, Nature Structural Biology.

[23]  Kevin M. D'Auria,et al.  Structural and dynamic determinants of protein-peptide recognition. , 2011, Structure.

[24]  M. Merkx,et al.  Engineering Protein Switches: Sensors, Regulators, and Spare Parts for Biology and Biotechnology , 2011, Chembiochem : a European journal of chemical biology.

[25]  Tobias Meyer,et al.  An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways , 2005, Nature Methods.

[26]  Boris N. Kholodenko,et al.  Signalling ballet in space and time , 2010, Nature Reviews Molecular Cell Biology.

[27]  A. Moreno,et al.  Src Utilizes Cas to Block Gap Junctional Communication Mediated by Connexin43* , 2007, Journal of Biological Chemistry.

[28]  W. Lim,et al.  Reprogramming Control of an Allosteric Signaling Switch Through Modular Recombination , 2003, Science.

[29]  Feng Ding,et al.  Rapid Flexible Docking Using a Stochastic Rotamer Library of Ligands , 2010, J. Chem. Inf. Model..

[30]  C. Walsh,et al.  Evaluation of the catalytic mechanism of recombinant human Csk (C-terminal Src kinase) using nucleotide analogs and viscosity effects. , 1994, The Journal of biological chemistry.

[31]  Kevin H. Gardner,et al.  Structural Basis of a Phototropin Light Switch , 2003, Science.

[32]  Birte Höcker,et al.  Computational design of ligand binding is not a solved problem , 2009, Proceedings of the National Academy of Sciences.

[33]  S. Cutler,et al.  Identification and Mechanism of ABA Receptor Antagonism , 2010, Nature Structural &Molecular Biology.

[34]  L. Regan,et al.  Characterization of a helical protein designed from first principles. , 1988, Science.

[35]  P. Oeller,et al.  Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. , 1997, Science.

[36]  J. Parsons,et al.  Src family kinases, key regulators of signal transduction , 2004, Oncogene.

[37]  Xinming Cai,et al.  Structural Basis for the Autoinhibition of Focal Adhesion Kinase , 2007, Cell.

[38]  Feng Ding,et al.  Multiscale modeling of nucleosome dynamics. , 2007, Biophysical journal.

[39]  R. Swendsen,et al.  THE weighted histogram analysis method for free‐energy calculations on biomolecules. I. The method , 1992 .

[40]  F. Ding,et al.  Ab initio folding of proteins with all-atom discrete molecular dynamics. , 2008, Structure.