RESUMEN
We have used magnetic resonance to map the interaction surface of an integral membrane protein for its regulatory target, an integral membrane enzyme. Phospholamban (PLN) regulates cardiac contractility via its modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. Impairment of this regulatory process causes heart failure. To map the molecular details of the PLN/SERCA interaction, we have functionally reconstituted SERCA with labeled PLN in dodecylphosphocholine micelles for high-resolution NMR spectroscopy and in both micelles and lipid bilayers for EPR spectroscopy. Differential perturbations in NMR linewidths and chemical shifts, measured as a function of position in the PLN sequence, provide a vivid picture of extensive SERCA contacts in both cytoplasmic and transmembrane domains of PLN and provide structural insight into previously reported functional mutagenesis data. NMR and EPR data show clear and complementary evidence for a dynamic (micros-to-ms) equilibrium between two conformational states in the cytoplasmic domain of PLN. These results support the hypothesis that SERCA attracts the cytoplasmic domain of PLN away from the lipid surface, shifting the preexisting equilibrium of PLN conformers toward a structure that is poised to interact with the regulatory target. EPR shows that this conformational switch behaves similarly in micelles and lipid membranes. Based on structural and dynamics data, we propose a model in which PLN undergoes allosteric activation upon encountering SERCA.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Contracción Miocárdica/fisiología , Animales , Micelas , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estructura Terciaria de Proteína , Conejos , ATPasas Transportadoras de Calcio del Retículo SarcoplásmicoRESUMEN
We have purified from potato tubers, the lectin STA devoid of chitinase activity and two chitinases devoid of lectin activity. Both enzymes are 16 kDa glycoproteins, and probably belong to a new family of plant chitinases. The respective antifungal properties of lectin and chitinases were studied by following their effects against early developmental stages of Fusarium oxysporum, a fungal potato pathogen. Here we demonstrate that: (1) lectin does not inhibit mycelial growth but irreversibly inhibits conidia germination and alters the germ tubes; and (2) chitinases block mycelial growth as well as conidia germination and lyse germ tubes.
