title: Allosteric activation of apicomplexan calcium-dependent protein kinases.
abstract: Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7-kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.
authors: Whitehead Institute for Biomedical Research, Cambridge, MA 02142;
authors: Jessica R
authors: JR
authors: Ingram
authors: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors: Kevin E
authors: KE
authors: Knockenhauer
authors: Whitehead Institute for Biomedical Research, Cambridge, MA 02142;
authors: Benedikt M
authors: BM
authors: Markus
authors: Whitehead Institute for Biomedical Research, Cambridge, MA 02142;
authors: Joseph
authors: J
authors: Mandelbaum
authors: D. E. Shaw Research, New York, NY 10036;
authors: Alexander
authors: A
authors: Ramek
authors: D. E. Shaw Research, New York, NY 10036;
authors: Yibing
authors: Y
authors: Shan
authors: D. E. Shaw Research, New York, NY 10036; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032.
authors: David E
authors: DE
authors: Shaw
authors: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors: Thomas U
authors: TU
authors: Schwartz
authors: Whitehead Institute for Biomedical Research, Cambridge, MA 02142; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors: Hidde L
authors: HL
authors: Ploegh
authors: Whitehead Institute for Biomedical Research, Cambridge, MA 02142; lourido@wi.mit.edu.
authors: Sebastian
authors: http://orcid.org/0000-0002-5237-1095
authors: S
authors: Lourido
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D000913:Antibodies, Protozoan / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002161:Camelids, New World; D002478:Cells, Cultured; D018360:Crystallography, X-Ray; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D056004:Molecular Dynamics Simulation; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics; D017871:Calcium-Calmodulin-Dependent Protein Kinases
chemical_list: D000913:Antibodies, Protozoan; D002118:Calcium; D007143:Immunoglobulin Heavy Chains; D011494:Protein Kinases; D015800:Protozoan Proteins; D061905:Single-Domain Antibodies; D017871:Calcium-Calmodulin-Dependent Protein Kinases
keywords: VHH; calcium-dependent protein kinase; kinase activation