Functional reconstitution and characterization of AqpZ, the E. coli water channel protein 1 1 Edited by W. Baumeister (original) (raw)

The Hidden Intricacies of Aquaporins: Remarkable Details in a Common Structural Scaffold

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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).

Reconstitution of water channel function and 2D-crystallization of human aquaporin 8

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2012

Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H 2 O 2 in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of three human aquaporins (HsAQP1, HsAQP4, and HsAQP5) have been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP1, HsAQP4 and HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent-lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3 Å.

Architecture and Selectivity in Aquaporins: 2.5 A X-ray Structure of Aquaporin Z

Aquaporins are a family of water and small molecule channels found in organisms ranging from bacteria to animals. One of these channels, the E. coli protein aquaporin Z (AqpZ), has been shown to selectively conduct only water at high rates. We have expressed, purified, crystallized, and solved the X-ray structure of AqpZ. The 2.5 Å resolution structure of AqpZ suggests aquaporin selectivity results both from a steric mechanism due to pore size and from specific amino acid substitutions that regulate the preference for a hydrophobic or hydrophilic substrate. This structure provides direct evidence on the molecular mechanisms of specificity between water and glycerol in this family of channels from a single species. It is to our knowledge the first atomic resolution structure of a recombinant aquaporin and so provides a platform for combined genetic, mutational, functional, and structural determinations of the mechanisms of aquaporins and, more generally, the assembly of multimeric membrane proteins.

The atomic-level mechanism underlying the functionality of aquaporin-0

So far, more than 82,000 protein structures have been reported in the Protein Data Bank, but the driving force and structures that allow for protein functions have not been elucidated at the atomic level for even one protein. We have been able to clarify that the inter-subunit hydrophobic interaction driving the electrostatic opening of the pore in aquaporin 0 (AQP0). Aquaporins are membrane channels for water and small non-ionic solutes found in animals, plants, and microbes. The structures of aquaporins have high homology and consist of homotetramers, each monomer of which has one pore for a water channel. Each pore has two narrow portions: one is the narrowest constriction region consisting of aromatic residues and an arginine (ar/R), and another is two asparagine-proline-alanine (NPA) homolog portions. Here we show that an inter-subunit hydrophobic interaction in AQP0 drives a stick portion consisting of four amino acids toward the pore and the tip of the stick portion, consisti...

The importance of aquaporin water channel protein structures

The EMBO Journal, 2000

The history of the water channel and recent structural and functional analyses of aquaporins are reviewed. These ubiquitous channels are important for bacteria, plants and animals, exhibit a pronounced sequence homology and share functional as well as structural similarities. Aquaporins allow water or small specific solutes to pass unhindered, but block the passage of ions to prevent dissipation of the transmembrane potential. Besides advances in structure determination, recent experiments suggest that many of these channels are regulated by pH variations, phosphorylation and binding of auxiliary proteins.

Molecular Cloning and Characterization of AqpZ, a Water Channel from Escherichia coli

Journal of Biological Chemistry, 1995

The aquaporin family of molecular water channels is widely expressed throughout the plant and animal kingdoms. No bacterial aquaporins are known; however, sequence-related bacterial genes have been identified that encode glycerol facilitators (glpF). By homology cloning, a novel aquaporin-related DNA (aqpZ) was identified that contained no surface N-glycosylation consensus. The aqpZ RNA was not identified in mammalian mRNA by Northern analysis and exhibited bacterial codon usage preferences. Southern analysis failed to demonstrate aqpZ in mammalian genomic DNA, whereas a strongly reactive DNA was present in chromosomal DNA from Escherichia coli and other bacterial species and did not correspond to glpF. The aqpZ DNA isolated from E. coli contained a 693-base pair open reading frame encoding a polypeptide 28 -38% identical to known aquaporins. When compared with other aquaporins, aqpZ encodes a 10-residue insert preceding exofacial loop C, truncated NH 2 and COOH termini, and no cysteines at known mercury-sensitive sites. Expression of aqpZ cRNA conferred Xenopus oocytes with a 15-fold increase in osmotic water permeability, which was maximal after 5 days of expression, was not inhibited with HgCl 2 , exhibited a low activation energy (E a ‫؍‬ 3.8 kcal/mol), and failed to transport nonionic solutes such as urea and glycerol. In contrast, oocytes expressing glpF transported glycerol but exhibited limited osmotic water permeability. Phylogenetic comparison of aquaporins and homologs revealed a large separation between aqpZ and glpF, consistent with an ancient gene divergence.

Aquaporins: A Multidisciplinary Perspective on The Water Channel Proteins

Acta Medica, 2020

Aquaporins are unique water channel proteins located at cell membranes that possess high water permeability and high solute rejection. Their primary function is to maintain the osmotic balance of the cells via regulating the water transport. However, their discovery had also provided the scientists to understand the pathophysiology of some diseases. In fact, aquaporins are shown to be strongly related to cancer by taking part in several tumor-related processes such as cell migration, cell proliferation and cell adhesion. Other than their functions in human body, recently, aquaporins have started to be used in engineering biomimetic membranes, for different applications such as desalination. This review investigates the properties and functions of the aquaporins in a multidisciplinary point of view and demonstrates the recent developments in aquaporin-based research.

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 channel architecture of aquaporin 0 at a 2.2-A resolution

Proceedings of the National Academy of Sciences, 2004

We determined the x-ray structure of bovine aquaporin 0 (AQP0) to a resolution of 2.2 Å. The structure of this eukaryotic, integral membrane protein suggests that the selectivity of AQP0 for water transport is based on the identity and location of signature amino acid residues that are hallmarks of the water-selective arm of the AQP family of proteins. Furthermore, the channel lumen is narrowed only by two, quasi-2-fold related tyrosine side chains that might account for reduced water conductance relative to other AQPs. The channel is functionally open to the passage of water because there are eight discreet water molecules within the channel. Comparison of this structure with the recent electron-diffraction structure of the junctional form of sheep AQP0 at pH 6.0 that was interpreted as closed shows no global change in the structure of AQP0 and only small changes in side-chain positions. We observed no structural change to the channel or the molecule as a whole at pH 10, which could be interpreted as the postulated pH-gating mechanism of AQP0-mediated water transport at pH >6.5. Contrary to the electron-diffraction structure, the comparison shows no evidence of channel gating induced by association of the extracellular domains of AQP0 at pH 6.0. Our structure aids the analysis of the interaction of the extracellular domains and the possibility of a cell-cell adhesion role for AQP0. In addition, our structure illustrates the basis for formation of certain types of cataracts that are the result of mutations.