Aquaporins are membrane channels that facilitate the flow of water across biological membranes. Two conserved regions are central for selective function: the dual asparagine-proline-alanine (NPA) ...aquaporin signature motif and the aromatic and arginine selectivity filter (SF). Here, we present the crystal structure of a yeast aquaporin at 0.88 angstrom resolution. We visualize the H-bond donor interactions of the NPA motifs asparagine residues to passing water molecules; observe a polarized water-water H-bond configuration within the channel; assign the tautomeric states of the SF histidine and arginine residues; and observe four SF water positions too closely spaced to be simultaneously occupied. Strongly correlated movements break the connectivity of SF waters to other water molecules within the channel and prevent proton transport via a Grotthuss mechanism.
Human aquaporin 2 (AQP2) is a water channel found in the kidney collecting duct, where it plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between ...intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin and defective trafficking results in nephrogenic diabetes insipidus (NDI). Here we present the X-ray structure of human AQP2 at 2.75 Å resolution. The C terminus of AQP2 displays multiple conformations with the C-terminal α-helix of one protomer interacting with the cytoplasmic surface of a symmetry-related AQP2 molecule, suggesting potential protein–protein interactions involved in cellular sorting of AQP2. Two Cd ²⁺-ion binding sites are observed within the AQP2 tetramer, inducing a rearrangement of loop D, which facilitates this interaction. The locations of several NDI-causing mutations can be observed in the AQP2 structure, primarily situated within transmembrane domains and the majority of which cause misfolding and ER retention. These observations provide a framework for understanding why mutations in AQP2 cause NDI as well as structural insights into AQP2 interactions that may govern its trafficking.
Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N ...terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 A resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Atomic-resolution X-ray crystallography, functional analyses, and molecular dynamics simulations suggest a novel mechanism for the regulation of water flux through the yeast Aqy1 water channel.
...Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from
Pichia pastoris
at 1.15 Å resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of
P. pastoris
. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.
All living organisms must regulate precisely the flow of water into and out of cells in order to maintain cell shape and integrity. Proteins of one family, the aquaporins, are found in virtually every living organism and play a major role in maintaining water homeostasis by acting as regulated water channels. Here we describe the first crystal structure of a yeast aquaporin, Aqy1, at 1.15 Å resolution, which represents the highest resolution structural data obtained to date for a membrane protein. Using this structural information, we address an outstanding biological question surrounding yeast aquaporins: what is the functional role of the amino-terminal extension that is characteristic of yeast aquaporins? Our structural data show that the amino terminus of Aqy1 fulfills a novel gate-like function by folding to form a cytoplasmic helical bundle with a tyrosine residue entering the water channel and occluding the cytoplasmic entrance. Molecular dynamics simulations and functional studies in combination with site-directed mutagenesis suggest that water flow is regulated through a combination of mechanosensitive gating and post-translational modifications such as phosphorylation. Our study therefore provides insight into a unique mechanism for the regulation of water flux in yeast.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cytosolic thymidine kinase 1, TK1, is a well known cell-cycle-regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs such as ...3′-azido-3′-deoxythymidine (AZT). We have now determined the structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in complex with the feedback inhibitor dTTP. The TK1s have a tetrameric structure in which each subunit contains an α/β-domain that is similar to ATPase domains of members of the RecA structural family and a domain containing a structural zinc. The zinc ion connects β-structures at the root of a β-ribbon that forms a stem that widens to a lasso-type loop. The thymidine of dTTP is hydrogen-bonded to main-chain atoms predominantly coming from the lasso loop. This binding is in contrast to other deoxyribonucleoside kinases where specific interactions occur with side chains. The TK1 structure differs fundamentally from the structures of the other deoxyribonucleoside kinases, indicating a different evolutionary origin.
Aquaporins are membrane channels that facilitate the flow of water across biological membranes. Two conserved regions are central for selective function: the dual asparagine, proline, alanine (NPA) ...aquaporin signature motif and the aromatic/arginine selectivity filter (SF). Here we present the crystal structure of a yeast aquaporin at 0.88 Å resolution. We visualize the H-bond donor interactions of the dual NPA motif asparagine residues to passing water molecules; observe a polarized water-water H-bond configuration within the channel; assign the tautomeric states of the SF histidine and arginine residues; and observe four SF water positions too closely spaced to be simultaneously occupied. Strongly correlated movements break the connectivity of SF waters to other water molecules within the channel and prevent proton transport
via
a Grotthuss mechanism.
This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from ...expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
Thymidine kinases have been found in most organisms, from viruses and bacteria to mammals. Ureaplasma urealyticum (parvum), which belongs to the class of cell‐wall‐lacking Mollicutes, has no de novo ...synthesis of DNA precursors and therefore has to rely on the salvage pathway. Thus, thymidine kinase (Uu‐TK) is the key enzyme in dTTP synthesis. Recently the 3D structure of Uu‐TK was determined in a feedback inhibitor complex, demonstrating that a lasso‐like loop binds the thymidine moiety of the feedback inhibitor by hydrogen bonding to main‐chain atoms. Here the structure with the substrate deoxythymidine is presented. The substrate binds similarly to the deoxythymidine part of the feedback inhibitor, and the lasso‐like loop binds the base and deoxyribose moieties as in the complex determined previously. The catalytic base, Glu97, has a different position in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5′‐OH of the substrate to form a hydrogen bond. The phosphorylation of and inhibition by several nucleoside analogues were investigated and are discussed in the light of the substrate binding pocket, in comparison with human TK1. Kinetic differences between Uu‐TK and human TK1 were observed that may be explained by structural differences. The tight interaction with the substrate allows minor substitutions at the 3 and 5 positions of the base, only fluorine substitutions at the 2′‐Ara position, but larger substitutions at the 3′ position of the deoxyribose.
Abstract Accumulating evidence suggests that dysregulated glycerol metabolism contributes to the pathophysiology of obesity and type 2 diabetes. Glycerol efflux from adipocytes is regulated by the ...aquaglyceroporin AQP7, which is translocated upon hormone stimulation. Here, we propose a molecular mechanism where the AQP7 mobility in adipocytes is dependent on perilipin 1 and protein kinase A. Biochemical analyses combined with ex vivo studies in human primary adipocytes, demonstrate that perilipin 1 binds to AQP7, and that catecholamine activated protein kinase A phosphorylates the N-terminus of AQP7, thereby reducing complex formation. Together, these findings are indicative of how glycerol release is controlled in adipocytes, and may pave the way for the future design of drugs against human metabolic pathologies.
Thymidine kinase (TK) is the key enzyme in salvaging thymidine to produce thymidine monophosphate. Owing to its ability to phosphorylate nucleoside analogue prodrugs, TK has gained attention as a ...rate‐limiting drug activator. We describe the structures of two bacterial TKs, one from the pathogen Bacillus anthracis in complex with the substrate dT, and the second from the food‐poison‐associated Bacillus cereus in complex with the feedback inhibitor dTTP. Interestingly, in contrast with previous structures of TK in complex with dTTP, in this study dTTP occupies the phosphate donor site and not the phosphate acceptor site. This results in several conformational changes compared with TK structures described previously. One of the differences is the way tetramers are formed. Unlike B. anthracis TK, B. cereus TK shows a loose tetramer. Moreover, the lasso‐domain is in open conformation in B. cereus TK without any substrate in the active site, whereas in B. anthracis TK the loop conformation is closed and thymidine occupies the active site. Another conformational difference lies within a region of 20 residues that we refer to as phosphate‐binding β‐hairpin. The phosphate‐binding β‐hairpin seems to be a flexible region of the enzyme which becomes ordered upon formation of hydrogen bonds to the α‐phosphate of the phosphate donor, dTTP. In addition to descriptions of the different conformations that TK may adopt during the course of reaction, the oligomeric state of the enzyme is investigated.
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