Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ-phosphate of ATP to 2′-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C-terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design

Water channel pore size determines exclusion properties but not solute selectivity

Aquaporins (AQPs) are a ubiquitous family of transmembrane water channel proteins. A subgroup of AQP water channels also facilitates transmembrane diffusion of small, polar solutes. A constriction within the pore, the aromatic/arginine (ar/R) selectivity filter, is thought to control solute permeability: previous studies on single representative water channel proteins suggest narrow channels conduct water, whilst wider channels permit passage of solutes. To assess this model of selectivity, we used mutagenesis, permeability measurements and in silico comparisons of water-specific as well as glycerol-permeable human AQPs. Our studies show that single amino acid substitutions in the selectivity filters of AQP1, AQP4 and AQP3 differentially affect glycerol and urea permeability in an AQP-specific manner. Comparison between in silico-calculated channel cross-sectional areas and in vitro permeability measurements suggests that selectivity filter cross-sectional area predicts urea but not glycerol permeability. Our data show that substrate discrimination in water channels depends on a complex interplay between the solute, pore size, and polarity, and that using single water channel proteins as representative models has led to an underestimation of this complexity

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis

Crystal Structure of a Yeast Aquaporin at 1.15 Å Reveals a Novel Gating Mechanism

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

Human aquaporins: regulators of transcellular water flow

Background: Emerging evidence supports the view that (AQP) aquaporin water channels are regulators of transcellular water flow. Consistentwith their expression in most tissues, AQPs are associatedwith diverse physiological and pathophysiological processes. Scope of review: AQP knockout studies suggest that the regulatory role of AQPs, rather than their action as passive channels, is their critical function. Transport through all AQPs occurs by a common passive mechanism, but their regulation and cellular distribution varies significantly depending on cell and tissue type; the role of AQPs in cell volumeregulation (CVR) is particularly notable. This reviewexamines the regulatory role of AQPs in transcellular water flow, especially in CVR.We focus on key systems of the human body, encompassing processes as diverse as urine concentration in the kidney to clearance of brain oedema. Major conclusions: AQPs are crucial for the regulation of water homeostasis, providing selective pores for the rapidmovement ofwater across diverse cellmembranes and playing regulatory roles in CVR. Gatingmechanisms have been proposed for human AQPs, but have only been reported for plant andmicrobial AQPs. Consequently, it is likely that the distribution and abundance of AQPs in a particular membrane is the determinant of membrane water permeability and a regulator of transcellular water flow. General significance: Elucidating the mechanisms that regulate transcellular water flow will improve our understanding of the human body in health and disease. The central role of specific AQPs in regulating water homeostasis will provide routes to a range of novel therapies. This article is part of a Special Issue entitled Aquaporins

Beyond water homeostasis:diverse functional roles of mammalian aquaporins

Background - Aquaporin (AQP) water channels are best known as passive transporters of water that are vital for water homeostasis. Scope of review - AQP knockout studies in whole animals and cultured cells, along with naturally occurring human mutations suggest that the transport of neutral solutes through AQPs has important physiological roles. Emerging biophysical evidence suggests that AQPs may also facilitate gas (CO2) and cation transport. AQPs may be involved in cell signalling for volume regulation and controlling the subcellular localization of other proteins by forming macromolecular complexes. This review examines the evidence for these diverse functions of AQPs as well their physiological relevance. Major conclusions - As well as being crucial for water homeostasis, AQPs are involved in physiologically important transport of molecules other than water, regulation of surface expression of other membrane proteins, cell adhesion, and signalling in cell volume regulation. General significance - Elucidating the full range of functional roles of AQPs beyond the passive conduction of water will improve our understanding of mammalian physiology in health and disease. The functional variety of AQPs makes them an exciting drug target and could provide routes to a range of novel therapies

Structural studies of thymidine kinases from Bacillus anthracis and Bacillus cereus provide insights into quaternary structure and conformational changes upon substrate binding

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 beta-hairpin. The phosphate-binding beta-hairpin seems to be a flexible region of the enzyme which becomes ordered upon formation of hydrogen bonds to the alpha-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

Affinity tags can reduce merohedral twinning of membrane protein crystals

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