Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen Trypanosoma brucei brucei

Abstract

Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis, or sleeping sickness. The causative parasite, Trypanosoma, encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. The latter are acidic membrane-enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg 2؉ and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from Tb b VSP1 crystal structure is further confirmed by SAXS and TEM data. SAXS data suggest structural flexibility in EF hand domains indicative of conformational plasticity within Tb b VSP1.African trypanosomiasis, commonly known as sleeping sickness, affects ϳ50,000 inhabitants of sub-Saharan Africa yearly (1) with 60 million people at risk of infection (2). Sleeping sickness is caused by two subspecies of T. brucei: T. brucei gambiense and T. brucei rhodesiense. The former alone accounts for ϳ98% of the cases in humans and livestock (1). T. brucei brucei is another subspecies of T. brucei that is used as an experimental model in laboratory to study sleeping sickness, and this parasite infects animals causing a disease called nagana (3). Left untreated, sleeping sickness is fatal, and there are currently two drugs used to treat the initial phase of the disease: suramin and pentamidine. These are employed in treatment of trypanosomiasis caused by T. brucei rhodesiense and T. brucei gambiense infections (4). For the treatment of second or neurological phase, an arsenic-based drug called melarsopol is used. However, this drug causes severe side effects and can sometimes be lethal (5). A newer and much more expensive drug, eflornithine, is effective only against T. brucei gambiense. (6). A combination of another drug, nifurtimox, with eflornithine has been used for treatment, but unfortunately it is not effective against T. brucei gambiense (6). Therefore, there is a pressing case to find new, safe, inexpensive, and broad spectrum drugs for treating sleeping sickness in humans and livestock.Soluble inorganic pyrophosphatase (PPase, EC 3.6.1.1) 3 is a ubiquitous and essential enzyme that hydrolyzes the PP i generated during cellular processes such as DNA replication and protein translation (7-9). Soluble PPases comprise two families that differ in both sequence and structure (10, 11). Family/class I PPases occur in all types of cells from bacteria to humans, whereas class II occurs exclusively in bacteria (11-12). Both classes of enzymes require divalent metal cations for catalysis (13-15). PPases from wide sources have been characterized, but those from Saccharomyces cerevisi...

Pepsi-SAXS: an adaptive method for rapid and accurate computation of small-angle X-ray scattering profiles

“…This will allow a thorough evaluation of existing computational methods, which falls outside the scope of the current work. Nonetheless, Pepsi-SAXS has already been successfully applied to select putative near-native conformations of the Tb b VSP1 complex (Jamwal et al, 2015) from 10 000 models generated with the Ensemble Optimization Method (Tria et al, 2015). The best selected conformation overlaid well with the ab initio model calculated using DAMAVER (Volkov & Svergun, 2003).…”

Crystallographic and Modeling Study of the Human PPA1 (Inorganic Pyrophosphatase 1): a Potential Anti-cancer Drug Target

“…Other eukaryotic PPases either do not have a C-terminal extension (Tg-PPase and Tb b VSP1) or has a C-terminal extension in different configurations (Pf-PPase). The dimerization modes in Tg-PPase, Tb b VSP1, and Pf-PPase are different from each other, and different from the conserved mode among human PPA1, Sj-PPase, and Y-PPase [50]. The available crystal structures of eukaryotic PPases show that diverse modes of dimerization exist in eukaryotic soluble Family I PPases.…”

“…All but one know eukaryotic PPases structures form dimers [40][41][42][43][44][45]48]. The exception is Tb b VSP1, which forms a tetramer (dimer of dimer) [50]. The crystal structure of human PPA1 reveals a dimerization mode that is conserved in the Sj-PPase and Y-PPase.…”

“…Extensive structural and biochemical studies had been reported on Family I PPases from Escherichia coli (E-PPase) [37][38][39] and Saccharomyces cerevisiae (Y-PPase) [40][41][42][43][44][45]. X-ray crystallographic studies had also been carried out on soluble PPases from other species, including Thermus thermophilus (T-PPase) [46], Pyrococcus horikoshii (Pho-PPase) [47], human parasites Plasmodium falciparum (Pf-PPase) and Toxoplasma gondii (Tg-PPase) [48], Mycobacterium tuberculosis (Mt-PPase) [36,49], Trypanosoma brucei brucei (also called vacuolar soluble protein TbbVSP1) [50], and S. japonicum (Sj-PPase, not published).…”

The Tetrameric Structure of Nucleotide-regulated Pyrophosphatase and Its Modulation by Deletion Mutagenesis and Ligand Binding

“…Formation of tetramers of 21-25-kDa subunits has been claimed in reports of a preliminary nature on Family I PPases from thermophilic and acidophilic bacteria [24][25][26]. Conclusive evidence for homotetramer formation has been only reported for a unique Family I vacuolar PPase from Trypanosoma brucei, which has an additional EF hand domain in its 47-kDa subunit [27]. CBS-PPase is thus unique among different PPases by the type and size of its oligomer structure.…”