Pore dynamics in PIP aquaporin closed state

GERARDO ZERBETTO DE PALMA; AGUSTINA CANESSA FORTUNA; ALIPERTI CAR, LUCIO; ARMENTIA, LUCIANO; ARI ZEIDA; DARIO ESTRIN; KARINA ALLEVA

La Plata

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

PIP are membrane proteins from the aquaporin family that facilitates watertransport across plasma membrane in plants. The permeability of these channelsare determined by the pore inner structure and regulatory elements in the proteinstructure. Previous reports pointed to a conserved histidine residue located inloopD as a key element of the gating mechanism, trapping loopD in a closedconformation when protonated at low pH (Tournaire-Roux et al., Nature 2003,Törnroth-Horsefield et al., Nature 2006). Additionally, by means of structural dataobtained from the X-ray crystallography of a PIP2, it was suggested that pore lininghydrophobic residues in loopD are also involved in the gating mechanism. Thesehydrophobic residues are proposed to be inserted into a cavity near the entranceof the pore when the PIP channel is in a closed conformation, modifying the innerstructure of the pore.In this work we combine functional assays and classical molecular dynamicssimulations of PIPs to elucidate the relationship between a pore lining hydrophobicresidue in loopD (Leu 206 or Leu 197, in BvPIP2;2 or SoPIP2:1 respectively) and thepore inner structure in its vicinity.Experiments expressing wild type or L206A BvPIP2;2 in Xenopus oocytes showedthat mutant PIP exhibit less transport inhibition when tested at high intracellularproton concentration. Molecular dynamics simulations of SoPIP2;1 starting fromcrystal structure and BvPIP2;2 homology models (both in a closed conformation)show a larger pore radius and lower water chemical potential across the porecoordinate near the mutated residue. Intrinsic permeability and hydrogen bondsbetween water molecules crossing the pore and pore lining residues were alsoanalyzed from simulations. Our results suggest that, when in a closed state, AlaPIP mutants have a less hindered water transit across the pore, providingexperimental evidence about the fine details of pore closure promoted after loopDconformational change.