title: Stability of the Plasmodium falciparum AMA1-RON2 Complex Is Governed by the Domain II (DII) Loop.
abstract: Plasmodium falciparum is an obligate intracellular protozoan parasite that employs a highly sophisticated mechanism to access the protective environment of the host cells. Key to this mechanism is the formation of an electron dense ring at the parasite-host cell interface called the Moving Junction (MJ) through which the parasite invades. The MJ incorporates two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, the latter one being targeted to the host cell membrane during invasion. Crystal structures of AMA1 have shown that a partially mobile loop, termed the DII loop, forms part of a deep groove in domain I and overlaps with the RON2 binding site. To investigate the mechanism by which the DII loop influences RON2 binding, we measured the kinetics of association and dissociation and binding equilibria of a PfRON2sp1 peptide with both PfAMA1 and an engineered form of PfAMA1 where the flexible region of the DII loop was replaced by a short Gly-Ser linker (ΔDII-PfAMA1). The reactions were tracked by fluorescence anisotropy as a function of temperature and concentration and globally fitted to acquire the rate constants and corresponding thermodynamic profiles. Our results indicate that both PfAMA1 constructs bound to the PfRON2sp1 peptide with the formation of one intermediate in a sequential reversible reaction: A↔B↔C. Consistent with Isothermal Titration Calorimetry measurements, final complex formation was enthalpically driven and slightly entropically unfavorable. Importantly, our experimental data shows that the DII loop lengthened the complex half-life time by 18-fold (900 s and 48 s at 25°C for Pf and ΔDII-Pf complex, respectively). The longer half-life of the Pf complex appeared to be driven by a slower dissociation process. These data highlight a new influential role for the DII loop in kinetically locking the functional binary complex to enable host cell invasion.
authors.affiliation: Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, CNRS, UMR 7275, 660, route des Lucioles, Sophia Antipolis, 06560, Valbonne, France.
authors.forename: Roberto F
authors.initials: RF
authors.lastname: Delgadillo
authors.affiliation: Department of Biochemistry & Microbiology, University of Victoria, PO Box 3055 STN CSC, Victoria, BC, V8W 3P6, Canada.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Parker
authors.affiliation: UMR 5235 CNRS, Université de Montpellier, 34095, Montpellier, France.
authors.forename: Maryse
authors.initials: M
authors.lastname: Lebrun
authors.affiliation: Department of Biochemistry & Microbiology, University of Victoria, PO Box 3055 STN CSC, Victoria, BC, V8W 3P6, Canada.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Boulanger
authors.affiliation: Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, CNRS, UMR 7275, 660, route des Lucioles, Sophia Antipolis, 06560, Valbonne, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Douguet
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D016880:Anisotropy; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry; D006207:Half-Life; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
chemical_list: D000953:Antigens, Protozoan; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D010455:Peptides; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface; C090077:apical membrane antigen I, Plasmodium