Crystal structure of the aquaglyceroporin PfAQP from the malarial parasite Plasmodium falciparum

Abstract

The 2.05-A resolution structure of the aquaglyceroporin from the malarial parasite Plasmodium falciparum (PfAQP), a protein important in the parasite's life cycle, has been solved. The structure provides key evidence for the basis of water versus glycerol selectivity in aquaporin family members. Unlike its closest homolog of known structure, GlpF, the channel conducts both glycerol and water at high rates, framing the question of what determines high water conductance in aquaporin channels. The universally conserved arginine in the selectivity filter is constrained by only two hydrogen bonds in GlpF, whereas there are three in all water-selective aquaporins and in PfAQP. The decreased cost of dehydrating the triply-satisfied arginine cation may provide the basis for high water conductance. The two Asn-Pro-Ala (NPA) regions of PfAQP, which bear rare substitutions to Asn-Leu-Ala (NLA) and Asn-Pro-Ser (NPS), participate in preserving the orientation of the selectivity filter asparagines in the center of the channel.

Figure 1

Channel architecture. Ribbon representations of the tetramer and monomer view of PfAQP. N- and C-terminal pseudo two-fold related portions of the channel are gold and maroon, respectively. (a) The PfAQP monomer constituting the asymmetric unit as viewed parallel to the membrane. Helices are labeled M1–M8. The pseudo two-fold axis protrudes from the page at the center of the membrane where helices M3 and M7 meet. The conduction pathway of glycerol is shown by sphere representations of the molecules observed in the crystal structure. (b) PfAQP tetramer viewed normal to the membrane down the crystallographic four-fold symmetry axis. Glycerol molecules contained in each conduction pore are shown in CPK representation. (c) Side view of the tetramer. Helices from two adjacent monomers are gray. Tyr44 residues from all four monomers are shown in stick representation. F_o – F_c density around the four-fold axis is contoured at 3 σ and shown in orange mesh. The solid diagonal line represents the length of the density in the figure. All structural renderings were done with PyMOL (http://www.pymol.org).

Figure 2

Details of the PfAQP conduction channel. (a) Plot of channel radius versus position along the conduction pore for the water-specific AQP1 and the aquaglyceroporins AQPM, GlpF and PfAQP. The regions of the channel comprising the extracellular vestibule, intracellular vestibule, selectivity filter (SF) and NPA motif region are labeled below. All calculations of pore dimensions were done with the program Hole2 (ref. 26). (b) Schematic of protein interactions with channel contents. Generated with ChemDraw Ultra (CambridgeSoft). (c) PfAQP is represented by a surface representation of two opened halves that contribute the predominantly hydrophobic (right) or hydrophilic (left) interactions to the conduction channel. The blue surface indicates residues that are identical to GlpF. Nonidentical residues are gray. Stick representations of the three glycerol molecules in the conduction channel are shown. The boundaries of the conduction channel are roughly defined by dashed lines.

Figure 3

Selectivity in aquaporin channels. Ribbon representations of PfAQP, GlpF and AQP1. Residues comprising the selectivity filter of each channel are shown in stick representation. Glycerols are drawn as yellow sticks and waters as red spheres. The hydrogen bond network around each arginine residue is shown with measurements in angstroms. Helices M1 and M4 have been removed for easier viewing of the selectivity filter region.

Figure 4

Water and glycerol conduction assay. Water (left) and glycerol (right) flux measurements in liposomes with PfAQP. Control measurements were made in empty liposomes containing no protein. Rates were determined from a fitting of a single exponential curve to the data ± s.d. from seven replicate measurements. The inset in the glycerol conduction plot is an expansion of the initial kinetics for glycerol conduction.

Figure 5

Alterations to the conserved NPA motif in PfAQP. (a) Ribbon representation of PfAQP showing the NLA and NPS regions of PfAQP in stick representation. Hydrogen bonds are indicated with dashed lines. (b) Structural alignment of PfAQP (gold) and AQP1 (silver). For both proteins, helices are shown in ribbon representation and residues around the Leu71 of PfAQP and corresponding proline in AQP1 are shown in stick representation.