RESUMO
The (pro)renin receptor (PRR) is an important component of the renin-angiotensin system (RAS), which regulates blood pressure and cardiovascular function. The integral membrane protein PRR contains a large extracellular domain (â¼310 amino acids), a single transmembrane domain (â¼20 amino acids) and an intracellular domain (â¼19 amino acids). Although short, the intracellular (IC) domain of the PRR has functionally important roles in a number of signal transduction pathways activated by (pro)renin binding. Meanwhile, together with the transmembrane domain and a small portion of the extracellular domain (â¼30 amino acids), the IC domain is also involved in assembly of V(0) portion of the vacuolar proton-translocating ATPase (V-ATPase). To better understand structural and multifunctional roles of the PRR-IC, we report the crystal structure of the PRR-IC domain as maltose-binding protein (MBP) fusion proteins at 2.0Å (maltose-free) and 2.15Å (maltose-bound). In the two separate crystal forms having significantly different unit-cell dimensions and molecular packing, MBP-PRR-IC fusion protein was found to be a dimer, which is different with the natural monomer of native MBP. The PRR-IC domain appears as a relatively flexible loop and is responsible for the dimerization of MBP fusion protein. Residues in the PRR-IC domain, particularly two tyrosines, dominate the intermonomer interactions, suggesting a role for the PRR-IC domain in protein oligomerization.
Assuntos
Proteínas Ligantes de Maltose/química , Maltose/química , Receptores de Superfície Celular/química , Proteínas Recombinantes de Fusão/química , ATPases Vacuolares Próton-Translocadoras/química , Cristalografia por Raios X , Humanos , Proteínas Ligantes de Maltose/genética , Multimerização Proteica , Estrutura Terciária de Proteína , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Tirosina/química , Tirosina/genética , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
PorB class II from Neisseria meningitidis is a pore-forming, outer-membrane protein that can translocate to the host-cell membrane during Neisserial infections. This report describes development of tethered bilayer lipid membrane (tBLM) system to measure PorB conductance properties. The tBLM was fabricated by depositing a self-assembled monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) tethering lipid on a gold electrode and then using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes to deposit the upper tBLM leaflet. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to monitor tBLM formation and subsequent PorB incorporation. The highly insulating tBLM exhibited a membrane resistance and capacitance of 2.5MOmegacm(2) and 0.7 microF/cm(2), respectively. PorB was incorporated into the tBLM in an active conformation, as evidenced by its mediation of ion passage and the decrease in membrane impedance. After PorB incorporation, the membrane resistance decreased to 0.6MOmegacm(2). As expected, the PorB channel was found to be non-selective, allowing the transport of both cations and anions. Cyclic voltammetry indicated that ferricyanide ions can also pass through the pores. The PorB-containing biomimetic interface developed in this study could potentially be used to screen for compounds that modulate PorB activity.