Cooperativity in Plant Plasma Membrane Intrinsic Proteins (PIPs): Mechanism of Increased Water Transport in Maize PIP1 Channels in Hetero-tetramers

Abstract

Plant aquaporins (AQPs) play vital roles in several physiological processes. Plasma membrane intrinsic proteins (PIPs) belong to the subfamily of plant AQPs. They are further subdivided into two closely related subgroups PIP1s and PIP2s. While PIP2 members are efficient water channels, PIP1s from some plant species have been shown to be functionally inactive. Aquaporins form tetramers under physiological conditions. PIP2s can enhance the water transport of PIP1s when they form hetero-tetramers. However, the role of monomer-monomer interface and the significance of specific residues in enhancing the water permeation of PIP1s have not been investigated at atomic level. We have performed all-atom molecular dynamics (MD) simulations of homo-tetramers and four different hetero-tetramers containing ZmPIP1;2 and ZmPIP2;5 from Zea mays. ZmPIP1;2 in a tetramer assembly will have two interfaces, one formed by transmembrane segments TM4 and TM5 and the other formed by TM1 and TM2. We have analyzed channel radius profiles, water transport and potential of mean force profiles of ZmPIP1;2 monomers. Results of MD simulations clearly revealed the influence of TM4-TM5 interface in modulating the water transport of ZmPIP1;2. MD simulations indicate the importance of I93 residue from the TM2 segment of ZmPIP2;5 for the increased water transport in ZmPIP1;2.

Figure 1

ZmPIP1;2 homo-tetramer showing two different monomer-monomer interfaces formed by TM1-TM2 (orange) and TM4-TM5 (blue) segments. The monomer under consideration is shown in opaque and the other three monomers are displayed as transparent. The convention for naming the interfaces is explained in the main text. Unless otherwise stated, the molecular plots in this figure and subsequent figures were generated using VMD software.

Figure 2

Temporal channel radius profiles of (A) ZmPIP1;2 homo-tetramer and (B) ZmPIP1;2 monomer from Type-I category in the 3:1 hetero-tetramer system. The point ‘0’ along the channel axis represents the NPA region. The ar/R selectivity filter is located at approximately 10 Ǻ. Each point along the channel axis is the average calculated from 2 ns bin for all the monomers considered. For example, for the homo-tetramer system, all four monomers from the two independent simulations (total 8 monomers) were considered and a point along the channel axis in this 2D-profile is an average over 8 monomers in a 2 ns bin at that time.

Figure 3

Temporal channel radius profiles of ZmPIP1;2 monomers (A) Type II, (B) Type III and (C) Type IV categories from hetero-tetramers. The number of monomers used to generate the 2D channel radius profiles for each category is 4, 4 and 6 respectively for Type II, Type III and Type IV from two independent simulations. For all other details, see caption of Fig. 2 and Table 2.

Figure 4

Temporal channel radius profiles of ZmPIP1;2 monomers consolidated for (A) Type I/Type III and (B) Type II/Type IV categories from different hetero-tetramer systems. A total of 6 and 10 monomers were considered for Type I/Type III and Type II/Type IV respectively from two independent simulations. (C) Average PMF profiles calculated for ZmPIP1;2 monomers from homo-tetrameric system, ZmPIP1;2 monomers belonging to Type I/Type III and Type II/Type IV groups. The regions corresponding to the aromatic/arginine selectivity filter (SF) and the conserved NPA motif (NPA) are indicated.

Figure 5

(A) Pairwise sequence alignment of TM1 and TM2 helical segments along with the linker region are shown for ZmPIP1;2 and ZmPIP2;5. Residues that are at the interface and are different between the two sequences are marked. The transmembrane helical regions are indicated. The seven marked positions are mutated in the 2:2(d) heteromer system and the mutant oligomer was simulated to understand the influence of the interfacial residues. Molecular plots showing the interfaces formed by TM4-TM5 of ZmPIP1;2 with TM1-TM2 of (B) ZmPIP1;2 and (C) ZmPIP2;5 monomers. The helices of TM1 and TM2 are shown in green and pink for ZmPIP1;2 and ZmPIP2;5 respectively. Residues occurring at the monomer-monomer interface that are different between ZmPIP1;2 and ZmPIP2;5 are labeled and are shown in space-filling representation.

Figure 6

Initial configuration of two mutant systems. (A) One of the ZmPIP2;5 monomer (solid pink) in the 2:2(d) hetero-tetramer was mutated at 7 positions to mimic ZmPIP1;2 so that the adjacent ZmPIP1;2 monomer will have Type III-like interface. The mutated helix in ZmPIP2;5 is shown in solid green. (B) ZmPIP1;2 homo-tetramer with a single mutation of A103I at the TM2 position in one ZmPIP1;2 (solid green) is shown. All the wild-type monomers are shown transparent in green (ZmPIP1;2) or pink (ZmPIP2;5). The red and blue dots show the expected water permeation behavior of the corresponding monomers, with the red dot (Type I/Type III interface) indicating low expected permeation and the blue (Type II/Type IV interface) indicating high expected permeation.

Figure 7

(A) The residue I93 from TM2 of ZmPIP2;5 at the monomer-monomer interface exhibit enhanced interactions with several bulky residues (I190, I194 and F220) from TM4 of ZmPIP1;2 in hetero-tetramers belonging to Type II or Type IV category. Interacting residues are labeled and are displayed in stick representation. Only two of the interacting monomers in the tetrameric system are shown. (B) Average PMF profiles calculated for monomers of ZmPIP1;2 homo-tetramers, ZmPIP1;2 monomers of Type I category from the A103I mutant homo-tetramers and ZmPIP1;2 monomer which acquired Type II-like interface in the mutant homo-tetramer. The regions corresponding to the aromatic/arginine selectivity filter (SF) and the conserved NPA motif (NPA) are indicated in the figure. Temporal channel radius profiles of (C) Type I ZmPIP1;2 monomers and (D) ZmPIP1;2 monomer with Type II-like interface from two independent simulations of the mutant homo-tetramer systems.

Figure 8

First eigenvectors, shown as arrows, depicting the direction of motion of TM5 (blue) for ZmPIP1;2 monomers from homo-tetramers viewed (A) parallel to the membrane and (B) down from the extracellular side. The direction of first principal component shown for TM5 (red) from ZmPIP1;2 monomers belonging to Type II/Type IV category viewed (C) parallel to the membrane and (D) down from the extracellular side.