Role of C-terminal Domain and Transmembrane Helices 5 and 6 in Function and Quaternary Structure of Major Intrinsic Proteins. ANALYSIS OF AQUAPORIN/GLYCEROL FACILITATOR CHIMERIC PROTEINS (original) (raw)
Related papers
Water and glycerol permeation through the glycerol channel GlpF and the aquaporin family
Journal of Synchrotron Radiation, 2004
The 2.2 Å resolution crystal structure of GlpF, an E.coli aquaporin that facilitates the flow of glycerol, water and other small solutes, provides much insight into the molecular function and selectivity of aquaporins. Using GlpF and its atomic structure as a paradigm for the ten highly conserved human aquaporins, site-directed mutagenesis has been used to mutate residues that are possibly integral to the structure and function of different aquaporins. X-ray crystallography and other biophysical and molecular simulation methods allows for assessment of these changes at the structural and functional level. Initial attempts to convert the glycerol specific properties of GlpF towards a water specific aquaporin resulted in the shifting of GlpF channel properties towards that of the water aquaporins. This result reveals the great possibility of emulating and deciphering the function of other aquaporins with GlpF via mutagenesis and investigation of structure and function.
Oligomerization of water and solute channels of the major intrinsic protein (MIP) family
Kidney International, 2001
(250 to 290 amino acids), and the high conservation intrinsic protein (MIP) family. Water and small solute fluxes throughout the MIP family may indicate a common fold: through cell membranes are ensured in many tissues by seleca NH 2 cytosolic portion followed by a hydrophobic stretch tive pores that belong to the major intrinsic protein family of six transmembrane helices ). Among highly (MIP). This family includes the water channels or aquaporins conserved amino acids in the family, two repetitions of (AQP) and the neutral solute facilitators such as the glycerol facilitator (GlpF). We have compared the characteristics of Asp, Pro, Ala residues (NPA box) localized in the B and representatives of each subfamily. Following solubilization in E loops draw the sequence signature of the family (Fig. the nondenaturing detergents n-octyl-glucoside (OG) and Tri-1B). The folding of these two loops in the membrane ton X-100 (T-X100), AQPs remain in their native homotetrabilayer is proposed to be responsible of the pore formameric state, while GlpF always behaves as a monomer. Solute facilitators are fully solubilized by the detergent N-lauroyl sartion and solute movement across the membrane [2, 3]. cosine (NLS), while AQPs are not. Analyses of mutants and Interestingly, AQPs are widely distributed in bacteria, chimeras demonstrate a close correlation between the water plants, and animals, while GlpFs have been characterized transport function and the resistance to NLS solubilization. only within microorganisms such as bacteria or yeast. In Thus, AQPs and solute facilitators exhibit different behaviors mammals, ten MIPs have been cloned and functionally in mild detergents; this could reflect differences in quaternary organization within the membranes. We propose that the oligocharacterized. Some of them, such as AQP1, AQP3, and merization state or the strength of self-association is part of the AQP4, are widely distributed in the body [4]. In contrast, mechanisms used by MIP proteins to ensure solute selectivity.
Structural Determinants of Oligomerization of the Aquaporin-4 Channel
Journal of Biological Chemistry, 2016
The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family.
Aquaporins: More Than Functional Monomers in a Tetrameric Arrangement
Cells
Aquaporins (AQPs) function as tetrameric structures in which each monomer has its own permeable pathway. The combination of structural biology, molecular dynamics simulations, and experimental approaches has contributed to improve our knowledge of how protein conformational changes can challenge its transport capacity, rapidly altering the membrane permeability. This review is focused on evidence that highlights the functional relationship between the monomers and the tetramer. In this sense, we address AQP permeation capacity as well as regulatory mechanisms that affect the monomer, the tetramer, or tetramers combined in complex structures. We therefore explore: (i) water permeation and recent evidence on ion permeation, including the permeation pathway controversy—each monomer versus the central pore of the tetramer—and (ii) regulatory mechanisms that cannot be attributed to independent monomers. In particular, we discuss channel gating and AQPs that sense membrane tension. For th...
The 3.7 Å projection map of the glycerol facilitator GlpF: a variant of the aquaporin tetramer
EMBO reports, 2000
GlpF, the glycerol facilitator protein of Escherichia coli, is an archetypal member of the aquaporin superfamily. To assess its structure, recombinant histidine-tagged protein was overexpressed, solubilized in octylglucoside and purified to homogeneity. Negative stain electron microscopy of solubilized GlpF protein revealed a tetrameric structure of ∼80 Å side length. Scanning transmission electron microscopy yielded a mass of 170 kDa, corroborating the tetrameric nature of GlpF. Reconstitution of GlpF in the presence of lipids produced highly ordered two-dimensional crystals, which diffracted electrons to 3.6 Å resolution. Cryoelectron microscopy provided a 3.7 Å projection map exhibiting a unit cell comprised of two tetramers. In projection, GlpF is similar to AQP1, the erythrocyte water channel. However, the major density minimum within each monomer is distinctly larger in GlpF than in AQP1.
Selectivity and conductance among the glycerol and water conducting aquaporin family of channels
FEBS Letters, 2003
The atomic structures of a transmembrane water plus glycerol conducting channel (GlpF), and now of aquaporin Z (AqpZ) from the same species, Escherichia coli, bring the total to three atomic resolution structures in the aquaporin (AQP) family. Members of the AQP family each assemble as tetramers of four channels. Common helical axes support a wider channel in the glycerol plus water channel paradigm, GlpF. Water molecules form a single hydrogen bonded ¢le throughout the 28 A î long channel in AqpZ. The basis for absolute exclusion of proton or hydronium ion conductance through the line of water is explored using simulations. (R.M. Stroud).
Frontiers in Physiology, 2020
Xenopus oocytes expressing human aquaporin-7 (AQP7) exhibit greater osmotic water permeability and 3 H-glycerol uptake vs. those expressing the bacterial glycerol facilitator GlpF. AQP7-expressing oocytes exposed to increasing extracellular [glycerol] under isosmolal conditions exhibit increasing swelling rates, whereas GlpF-expressing oocytes do not swell at all. To provide a structural basis for these observed physiological differences, we performed X-ray crystallographic structure determination of AQP7 and molecular-dynamics simulations on AQP7 and GlpF. The structure reveals AQP7 tetramers containing two monomers with 3 glycerols, and two monomers with 2 glycerols in the pore. In contrast to GlpF, no glycerol is bound at the AQP7 selectivity filter (SF), comprising residues F74, G222, Y223, and R229. The AQP7 SF is resolved in its closed state because F74 blocks the passage of small solutes. Molecular dynamics simulations demonstrate that F74 undergoes large and rapid conformational changes, allowing glycerol molecules to permeate without orientational restriction. The more rigid GlpF imposes orientational constraints on glycerol molecules passing through the SF. Moreover, GlpF-W48 (analogous to AQP7-F74) undergoes rare but long-lasting conformational changes that block the pore to H 2 O and glycerol.
The Hidden Intricacies of Aquaporins: Remarkable Details in a Common Structural Scaffold
Small
Evolution turned aquaporins (AQPs) into the most efficient facilitators of passive water flow through cell membranes at no expense of solute discrimination. In spite of a plethora of solved AQP structures, many structural details remain hidden. Here, by combining extensive sequence-and structural-based analysis of a unique set of 20 non-redundant high-resolution structures and molecular dynamics simulations of 4 representatives, we identify key aspects of AQP stability, gating, selectivity, pore geometry and oligomerization, with a potential impact on channel functionality. We challenge the general view of AQPs possessing a continuous open water pore and depict that AQPs selectivity is not exclusively shaped by pore lining residues but also by the relative arrangement of transmembrane helices. Moreover, our analysis reveals that hydrophobic interactions constitute the main determinant of protein thermal stability. Finally, we establish a novel numbering scheme of the conserved AQP scaffold facilitating direct comparison and prediction of potential structural effects of e.g. disease-causing mutations. Additionally, our results pave the way for the design of optimized AQP water channels to be utilized in biotechnological applications. 1. Introduction Aquaporins (AQPs), part of a larger family of major intrinsic proteins, are one of the best studied protein families with currently 20 non-redundant high-resolution structures (≤3,70 Å) solved. Since their first discovery in 1992 by Peter Agre and coworkers (1, 2), thirteen different types of aquaporins (AQP0-12) were discovered in mammals (3). The narrow AQP pores combine enormous permeability, conducting water in a single-file manner close to the diffusion limit of water in bulk, with exceptional selectivity (4). A subset of AQPs, the aquaglyceroporins (AQGPs), paralogs of AQPs, are also able to conduct glycerol and other small neutral solutes (5, 6). Bacteria also express members of AQPs and AQGPs, generally functioning with one copy of each paralog and, interestingly, some lacking both. Unicellular eukaryotes and fungi follow a similar pattern, with a clear division between AQPs or AQGPs and a heterogeneous distribution in the number of copies of each paralog in the different genera (7). So far, no archaea has been found that possesses both paralogs concurrently. Plants exhibit the highest AQP diversity, with five main subfamilies (plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26 like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and X intrinsic proteins (XIPs)), which are each further divided into subgroups (7). Furthermore, in primitive plant species, two additional subfamilies, GLPF-like intrinsic proteins (GIPs) and hybrid intrinsic proteins (HIPs), have been found (8). However, the full diversity of AQ(G)Ps is still not represented by the numerous high-resolution structures, as exemplified by only three plant aquaporin structures and none of the unorthodox AQ(G)Ps (represented by AQP11 and 12 in mammals).
Proceedings of the National Academy of Sciences, 2010
Aquaporins are homotetrameric channel proteins, which allow the diffusion of water and small solutes across biological membranes. According to their transport function, aquaporins can be divided into "orthodox aquaporins", which allow the flux of water molecules only, and "aquaglyceroporins", which facilitate the diffusion of glycerol and other small solutes in addition to water. The contribution of individual residues in the pore to the selectivity of orthodox aquaporins and aquaglyceroporins is not yet fully understood. To gain insights into aquaporin selectivity, we focused on the sequence variation and electrostatics of their channels. The continuum Poisson-Boltzmann electrostatic potential along the channel was calculated and compared for ten three-dimensionalstructures which are representatives of different aquaporin subfamilies, and a panel of functionally characterized mutants, for which high-accuracy three-dimensional-models could be derived. Interestingly, specific electrostatic profiles associated with the main selectivity to water or glycerol could be identified. In particular: (i) orthodox aquaporins showed a distinctive electrostatic potential maximum at the periplasmic side of the channel around the aromatic/Arg (ar/R) constriction site; (ii) aquaporin-0 (AQP0), a mammalian aquaporin with considerably low water permeability, had an additional deep minimum at the cytoplasmic side; (iii) aquaglyceroporins showed a rather flat potential all along the channel; and (iv) the bifunctional protozoan PfAQP had an unusual all negative profile. Evaluation of electrostatics of the mutants, along with a thorough sequence analysis of the aquaporin pore-lining residues, illuminated the contribution of specific residues to the electrostatics of the channels and possibly to their selectivity. channel proteins | electrostatic potentials | pore analysis | comparative analysis
Biophysical Journal, 2013
Aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). The high-resolution X-ray structure of the human aquaporin 5 (HsAQP5) shows that HsAQP5, as all the other known aquaporins, exhibits tetrameric structure. By means of molecular dynamics simulations we analyzed the role of spontaneous fluctuations on the structural behavior of the human AQP5. We found that different conformations within the tetramer lead to a distribution of monomeric channel structures, which can be characterized as open or closed. The switch between the two states of a channel is a tap-like mechanism at the cytoplasmic end which regulates the water passage through the pore. The channel is closed by a translation of the His67 residue inside the pore. Moreover, water permeation rate calculations revealed that the selectivity filter, located at the other end of the channel, regulates the flow rate of water molecules when the channel is open, by locally modifying the orientation of His173. Furthermore, the calculated permeation rates of a fully open channel are in good agreement with the reported experimental value.