Cloning, expression, solubilization, and purification of a functionally active recombinant cAMP-dependent protein kinase catalytic subunit-like protein PKAC1 from Trypanosoma equiperdum.

The gene encoding the cAMP-dependent protein kinase (PKA) catalytic subunit-like protein PKAC1 from the Venezuelan TeAp-N/D1 strain of Trypanosoma equiperdum was cloned, and the recombinant TeqPKAC1 protein was overexpressed in bacteria. A major polypeptide with an apparent molecular mass of ∼38 kDa was detected by SDS-polyacrylamide gel electrophoresis, and immunoblotting using antibodies against the human PKA catalytic subunit α. Unfortunately, most of the expressed TeqPKAC1 was highly insoluble. Polypeptides of 36-38 kDa and 45-50 kDa were predominantly seen by immunoblotting in the bacterial particulate and cytosolic fractions, respectively. Since the incorporation of either 4% Triton X-100 or 3% sarkosyl or a mixture of 10 mM MgCl2 and 1 mM ATP (MgATP) improved the solubilization of TeqPKAC1, we used a combination of Triton X-100, sarkosyl and MgATP to solubilize the recombinant protein. TeqPKAC1 was purified by first reconstituting a hybrid holoenzyme between the recombinant protein and a mammalian poly-His-tagged PKA regulatory subunit that was immobilized on a Ni2+-chelating affinity resin, and then by eluting TeqPKAC1 using cAMP. TeqPKAC1 was functional given that it was capable of phosphorylating PKA catalytic subunit substrates, such as kemptide (LRRASLG), histone type II-AS, and the peptide SP20 (TTYADFIASGRTGRRNSIHD), and was inhibited by the peptide IP20 (TTYADFIASGRTGRRNAIHD), which contains the inhibitory motif of the PKA-specific heat-stable inhibitor PKI-α. Optimal enzymatic activity was obtained at 37 °C and pH 8.0-9.0; and the order of effectiveness of nucleotide triphosphates and divalent cations was ATP ¼ GTP â‰… ITP and Mg2+ â‰… Mn2+ â‰… Fe2+ ¼ Ca2+ â‰… Zn2, respectively.

Weak O2 binding and strong H2O2 binding at the non-heme diiron center of trypanosome alternative oxidase.

Alternative oxidase (AOX) catalyzes the four-electron reduction of dioxygen to water as an additional terminal oxidase, and the catalytic reaction is critical for the parasite to survive in its bloodstream form. Recently, the X-ray crystal structure of trypanosome alternative oxidase (TAO) complexed with ferulenol was reported and the molecular structure of the non-heme diiron center was determined. The binding of O2 was a unique side-on type compared to other iron proteins. In order to characterize the O2 binding state of TAO, the O2 binding states were searched at a quantum mechanics/molecular mechanics (QM/MM) theoretical level in the present study. We found that the most stable O2 binding state is the end-on type, and the binding states of the side-on type are higher in energy. Based on the binding energies and electronic structure analyses, O2 binds very weakly to the TAO iron center (ΔE =6.7 kcal mol-1) in the electronic state of Fe(II)…OO, not in the suggested charge transferred state such as the superoxide state (Fe(III)OO· -) as seen in hemerythrin. Coordination of other ligands such as water, Cl-, CN-, CO, N3- and H2O2 was also examined, and H2O2 was found to bind most strongly to the Fe(II) site by ΔE = 14.0 kcal mol-1. This was confirmed experimentally through the measurement of ubiquinol oxidase activity of TAO and Cryptosporidium parvum AOX which was found to be inhibited by H2O2 in a dose-dependent and reversible manner.

Nucleotide Binding, Evolutionary Insights, and Interaction Partners of the Pseudokinase Unc-51-like Kinase 4.

Unc-51-like kinase 4 (ULK4) is a pseudokinase that has been linked to the development of several diseases. Even though sequence motifs required for ATP binding in kinases are lacking, ULK4 still tightly binds ATP and the presence of the co-factor is required for structural stability of ULK4. Here, we present a high-resolution structure of a ULK4-ATPγS complex revealing a highly unusual ATP binding mode in which the lack of the canonical VAIK motif lysine is compensated by K39, located N-terminal to αC. Evolutionary analysis suggests that degradation of active site motifs in metazoan ULK4 has co-occurred with an ULK4-specific activation loop, which stabilizes the C helix. In addition, cellular interaction studies using BioID and biochemical validation data revealed high confidence interactors of the pseudokinase and armadillo repeat domains. Many of the identified ULK4 interaction partners were centrosomal and tubulin-associated proteins and several active kinases suggesting interesting regulatory roles for ULK4.

Dominant IgM synthesis against the soluble form of the prevailing variant surface glycoprotein from TeAp-N/D1 Trypanosoma equiperdum throughout the experimental acute infections of horses with non-tsetse transmitted Trypanozoon parasites.

Two horses were infected with distinct non-tsetse transmitted Trypanozoon Venezuelan stocks, namely TeAp-N/D1 Trypanosoma equiperdum and TeAp-El Frio01 Trypanosoma evansi. Preceding reports have revealed that a 64-kDa antigenic glycopolypeptide (p64), which is the soluble form of the predominant variant surface glycoprotein from TeAp-N/D1 T. equiperdum, can be used as a good antigen for immunodiagnosis of animal trypanosomosis. Here, the course of the experimental acute infection in both horses was monitored by evaluating total anti-p64 IgG and particular anti-p64 γ-specific IgG and µ-specific IgM isotypes in sera using indirect enzyme-linked immunosorbent assays. Both equines showed a maximum of whole anti-p64 antibody generation, which dropped to readings below the maximum but always above the positive cutoff point. Levels of specific IgG and IgM isotypes oscillated throughout the course of the experiments. Essentially, the γ-specific IgG response remained very close to the cutoff point, whereas the µ-specific IgM response displayed values that were mostly above the positive cutoff point, showing a major peak that coincided with the maximum of complete anti-p64 IgG production. These results showed that horses infected with non-tsetse transmitted Trypanozoon parasites developed an immune reaction characterized by a dominant IgM generation against the p64 antigen.

Targeting Trypanosoma evansi with disulphide-rich peptides derived from a phage display library.

A phage-display library was generated using a Bus thalamus scorpion toxin (BTK-2) as a peptide scaffold. BTK-2 belongs to the disulfide-rich family of proteins with pronounced structural stability due to the presence of three disulfide bridges that connects antiparallel beta-sheets and one alpha helix. Using BTK-2 as a phage display scaffold, we introduced mutations in five residues located in the alpha-helix and two residues located in the smaller loop, keeping intact the disulfide bridges to create a peptide phage-displayed library with disulfide-rich family properties. The library was subjected to in vivo and in vitro phage display selections against Trypanosoma evansi, the etiological agent of "Surra", a disease that affects a wide range of mammals. The development of T. evansi specific biomarkers is essential to improve diagnostic methods and epidemiological studies leading to a more accurate clinical decision for the treatment of this disease of economic impact for commercial livestock production. In this study, we identified two disulfide-rich peptides targeting T. evansi parasites. Further specificity studies are necessary to investigate the potential of selected peptides as new biomarkers to aid diagnostic and treatment procedures of T. evansi infections.

New Cysteine Protease Inhibitors: Electrophilic (Het)arenes and Unexpected Prodrug Identification for the Trypanosoma Protease Rhodesain.

Electrophilic (het)arenes can undergo reactions with nucleophiles yielding π- or Meisenheimer (σ-) complexes or the products of the SNAr addition/elimination reactions. Such building blocks have only rarely been employed for the design of enzyme inhibitors. Herein, we demonstrate the combination of a peptidic recognition sequence with such electrophilic (het)arenes to generate highly active inhibitors of disease-relevant proteases. We further elucidate an unexpected mode of action for the trypanosomal protease rhodesain using NMR spectroscopy and mass spectrometry, enzyme kinetics and various types of simulations. After hydrolysis of an ester function in the recognition sequence of a weakly active prodrug inhibitor, the liberated carboxylic acid represents a highly potent inhibitor of rhodesain (Ki = 4.0 nM). The simulations indicate that, after the cleavage of the ester, the carboxylic acid leaves the active site and re-binds to the enzyme in an orientation that allows the formation of a very stable π-complex between the catalytic dyad (Cys-25/His-162) of rhodesain and the electrophilic aromatic moiety. The reversible inhibition mode results because the SNAr reaction, which is found in an alkaline solvent containing a low molecular weight thiol, is hindered within the enzyme due to the presence of the positively charged imidazolium ring of His-162. Comparisons between measured and calculated NMR shifts support this interpretation.

Evolution of mitochondrial protein import - lessons from trypanosomes.

The evolution of mitochondrial protein import and the systems that mediate it marks the boundary between the endosymbiotic ancestor of mitochondria and a true organelle that is under the control of the nucleus. Protein import has been studied in great detail in Saccharomyces cerevisiae. More recently, it has also been extensively investigated in the parasitic protozoan Trypanosoma brucei, making it arguably the second best studied system. A comparative analysis of the protein import complexes of yeast and trypanosomes is provided. Together with data from other systems, this allows to reconstruct the ancestral features of import complexes that were present in the last eukaryotic common ancestor (LECA) and to identify which subunits were added later in evolution. How these data can be translated into plausible scenarios is discussed, providing insights into the evolution of (i) outer membrane protein import receptors, (ii) proteins involved in biogenesis of α-helically anchored outer membrane proteins, and (iii) of the intermembrane space import and assembly system. Finally, it is shown that the unusual presequence-associated import motor of trypanosomes suggests a scenario of how the two ancestral inner membrane protein translocases present in LECA evolved into the single bifunctional one found in extant trypanosomes.

Practical Synthesis of Cap-4 RNA.

Eukaryotic mRNAs possess 5' caps that are determinants for their function. A structural characteristic of 5' caps is methylation, with this feature already present in early eukaryotes such as Trypanosoma. While the common cap-0 (m7 GpppN) shows a rather simple methylation pattern, the Trypanosoma cap-4 displays seven distinguished additional methylations within the first four nucleotides. The study of essential biological functions mediated by these unique structural features of the cap-4 and thereby of the metabolism of an important class of human pathogenic parasites is hindered by the lack of reliable preparation methods. Herein we describe the synthesis of custom-made nucleoside phosphoramidite building blocks for m62 Am and m3 Um, their incorporation into short RNAs, the efficient construction of the 5'-to-5' triphosphate bridge to guanosine by using a solid-phase approach, the selective enzymatic methylation at position N7 of the inverted guanosine, and enzymatic ligation to generate trypanosomatid mRNAs of up to 40 nucleotides in length. This study introduces a reliable synthetic strategy to the much-needed cap-4 RNA probes for integrated structural biology studies, using a combination of chemical and enzymatic steps.

The domain architecture of the protozoan protein J-DNA-binding protein 1 suggests synergy between base J DNA binding and thymidine hydroxylase activity.

J-DNA-binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J (ß-d-glucosyl-hydroxymethyluracil), an epigenetic modification of thymidine (T) confined to pathogenic protozoa such as Trypanosoma and Leishmania JBP1 has two known functional domains: an N-terminal T hydroxylase (TH) homologous to the 5-methylcytosine hydroxylase domain in TET proteins and a J-DNA-binding domain (JDBD) that resides in the middle of JBP1. Here, we show that removing JDBD from JBP1 results in a soluble protein (Δ-JDBD) with the N- and C-terminal regions tightly associated together in a well-ordered structure. We found that this Δ-JDBD domain retains TH activity in vitro but displays a 15-fold lower apparent rate of hydroxylation compared with JBP1. Small-angle X-ray scattering (SAXS) experiments on JBP1 and JDBD in the presence or absence of J-DNA and on Δ-JDBD enabled us to generate low-resolution three-dimensional models. We conclude that Δ-JDBD, and not the N-terminal region of JBP1 alone, is a distinct folding unit. Our SAXS-based model supports the notion that binding of JDBD specifically to J-DNA can facilitate T hydroxylation 12-14 bp downstream on the complementary strand of the J-recognition site. We postulate that insertion of the JDBD module into the Δ-JDBD scaffold during evolution provided a mechanism that synergized J recognition and T hydroxylation, ensuring inheritance of base J in specific sequence patterns following DNA replication in kinetoplastid parasites.

High immunological response against a Trypanosoma equiperdum protein that exhibits homology with the regulatory subunits of mammalian cAMP-dependent protein kinases.

Previously, we have identified a protein in Trypanosoma equiperdum that possesses homology with the regulatory (R) subunits of the mammalian cAMP-dependent protein kinase (PKA). The recombinant T. equiperdum PKA R-like protein was expressed in bacteria and purified to homogeneity. Mice polyclonal antibodies were raised against the recombinant R-like protein to serologically evaluate its humoral immune response. High titers of specific sera antibodies were obtained against the parasite R-like protein by indirect enzyme-linked immunosorbent assay (ELISA), and immunoblots revealed that this protein was specifically recognized by the hyperimmune mice sera. Cellular proliferation assays using splenic B cells from the immunized mice showed higher values when the recombinant T. equiperdum R-like protein was employed than when concanavalin A was utilized as an unspecific mitogen. Two healthy horses that were experimentally infected using either T. equiperdum or Trypanosoma evansi showed a curve response characterized by the appearance of anti-T. equiperdum PKA R-like protein antibody production in sera using indirect ELISA. The recombinant parasite PKA R-like protein was also recognized by sera from naturally trypanosome-infected horses using western blotting. These findings demonstrated that the T. equiperdum PKA R-like protein is an antigen that exhibits cross-reaction with T. equiperdum and T. evansi.

Identification and characterization of a calmodulin binding domain in the plasma membrane Ca2+-ATPase from Trypanosoma equiperdum.

The plasma membrane Ca2+-ATPase (PMCA) from trypanosomatids lacks a classical calmodulin (CaM) binding domain, although CaM stimulated activities have been detected by biochemical assays. Recently we proposed that the Trypanosoma equiperdum CaM-sensitive PMCA (TePMCA) contains a potential 1-18 CaM-binding motif at the C-terminal region of the pump. In the present study, we evaluated the potential CaM-binding motifs using CaM from Trypanosoma cruzi and either the recombinant full length TePMCA C-terminal sequence (P14) or synthetic peptides comprising different regions of the C-terminal domain. We demonstrated that P14 and a synthetic peptide corresponding to residues 1037-1062 (which contains the predicted 1-18 binding motif) competed efficiently for binding to TcCaM, exhibiting similar IC50s of 200 nM. A stable complex of this peptide and TcCaM was formed in the presence of Ca2+, as determined by native-polyacrylamide gel electrophoresis. A predicted structure obtained by molecular docking showed an interaction of the 1-18 binding motif with the Ca2+/CaM complex. Moreover, when the peptide was incubated with CaM and Ca2+, a blue shift in the tryptophan fluorescence spectrum (from 350 to 329 nm) was observed. Substitutions at W1039 and F1056, strongly decreased both CaM-peptide interaction and the complex assembly. Our results demonstrated the presence of a functional 1-18 motif at the TePMCA C-terminal domain. Furthermore, on the basis of spectrofluorometric assays and the resulting structure modeled by docking we propose that the L1042 and W1060 residues might also participate as anchors to form a 1-4-18-22 motif.

The inositol-1,2-cyclic phosphate moiety of the cross-reacting determinant, carbohydrate chains, and proteinaceous components are all responsible for the cross-reactivity of trypanosome variant surface glycoproteins.

Salivarian trypanosomes evade the host immune system by continually swapping their protective variant surface glycoprotein (VSG) coat. Given that VSGs from various trypanosome stocks exhibited cross-reactivity (Camargo et al., Vet. Parasitol. 207, 17-33, 2015), we analyzed here which components are the antigenic determinants for this cross-reaction. Soluble forms of VSGs were purified from four Venezuelan animal trypanosome isolates: TeAp-N/D1, TeAp-ElFrio01, TeAp-Mantecal01, and TeGu-Terecay323. By using the VSG soluble form from TeAp-N/D1, we found that neither the inositol-1,2-cyclic phosphate moiety of the cross-reacting determinant nor the carbohydrate chains were exclusively responsible for its cross-reactivity. Then, all four purified glycoproteins were digested with papain and the resulting peptides were separated by high-performance liquid chromatography. Dot blot evaluation of the fractions using sera from trypanosome-infected animals yielded peptides that possessed cross-reaction activity, demonstrating for the first time that proteinaceous epitopes are also responsible for the cross-reactivity of trypanosome VSGs.

Comparative Proteomic Analysis of Lysine Acetylation in Trypanosomes.

Protein acetylation is a post-translational modification regulating diverse cellular processes. By using proteomic approaches, we identified N-terminal and ε-lysine acetylated proteins in Trypanosoma cruzi and Trypanosoma brucei, which are protozoan parasites that cause significant human and animal diseases. We detected 288 lysine acetylation sites in 210 proteins of procyclic form, an insect stage of T. brucei, and 380 acetylation sites in 285 proteins in the form of the parasite that replicates in mammalian bloodstream. In T. cruzi insect proliferative form we found 389 ε-lysine-acetylated sites in 235 proteins. Notably, we found distinct acetylation profiles according to the developmental stage and species, with only 44 common proteins between T. brucei stages and 18 in common between the two species. While K-ac proteins from T. cruzi are enriched in enzymes involved in oxidation/reduction balance, required for the parasite survival in the host, in T. brucei, most K-ac proteins are enriched in metabolic processes, essential for its adaptation in its hosts. We also identified in both parasites a quite variable N-terminal acetylation sites. Our results suggest that protein acetylation is involved in differential regulation of multiple cellular processes in Trypanosomes, contributing to our understanding of the essential mechanisms for parasite infection and survival.

Comparative interactomics provides evidence for functional specialization of the nuclear pore complex.

The core architecture of the eukaryotic cell was established well over one billion years ago, and is largely retained in all extant lineages. However, eukaryotic cells also possess lineage-specific features, frequently keyed to specific functional requirements. One quintessential core eukaryotic structure is the nuclear pore complex (NPC), responsible for regulating exchange of macromolecules between the nucleus and cytoplasm as well as acting as a nuclear organizational hub. NPC architecture has been best documented in one eukaryotic supergroup, the Opisthokonts (e.g. Saccharomyces cerevisiae and Homo sapiens), which although compositionally similar, have significant variations in certain NPC subcomplex structures. The variation of NPC structure across other taxa in the eukaryotic kingdom however, remains poorly understood. We explored trypanosomes, highly divergent organisms, and mapped and assigned their NPC proteins to specific substructures to reveal their NPC architecture. We showed that the NPC central structural scaffold is conserved, likely across all eukaryotes, but more peripheral elements can exhibit very significant lineage-specific losses, duplications or other alterations in their components. Amazingly, trypanosomes lack the major components of the mRNA export platform that are asymmetrically localized within yeast and vertebrate NPCs. Concomitant with this, the trypanosome NPC is ALMOST completely symmetric with the nuclear basket being the only major source of asymmetry. We suggest these features point toward a stepwise evolution of the NPC in which a coating scaffold first stabilized the pore after which selective gating emerged and expanded, leading to the addition of peripheral remodeling machineries on the nucleoplasmic and cytoplasmic sides of the pore.

Interplay between Protein Thermal Flexibility and Kinetic Stability.

Kinetic stability is a key parameter to comprehend protein behavior and it plays a central role to understand how evolution has reached the balance between function and stability in cell-relevant timescales. Using an approach that includes simulations, protein engineering, and calorimetry, we show that there is a clear correlation between kinetic stability determined by differential scanning calorimetry and protein thermal flexibility obtained from a novel method based on temperature-induced unfolding molecular dynamics simulations. Thermal flexibility quantitatively measures the increment of the conformational space available to the protein when energy in provided. The (ß/α)8 barrel fold of two closely related by evolution triosephosphate isomerases from two trypanosomes are used as model systems. The kinetic stability-thermal flexibility correlation has predictive power for the studied proteins, suggesting that the strategy and methodology discussed here might be applied to other proteins in biotechnological developments, evolutionary studies, and the design of protein based therapeutics.

Direct identification of trypanosomatids by matrix-assisted laser desorption ionization-time of flight mass spectrometry (DIT MALDI-TOF MS).

Accurate and rapid determination of trypanosomatids is essential in epidemiological surveillance and therapeutic studies. Matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a useful and powerful technique to identify bacteria, fungi, metazoa and human intact cells with applications in clinical settings. Here, we developed and optimized a MALDI-TOF MS method to profile trypanosomatids. trypanosomatid cells were deposited on a MALDI target plate followed by addition of matrix solution. The plate was then subjected to MALDI-TOF MS measurement to create reference mass spectra library and unknown samples were identified by pattern matching using the BioTyper software tool. Several m/z peaks reproducibly and uniquely identified trypanosomatids species showing the potentials of direct identification of trypanosomatids by MALDI-TOF MS. Moreover, this method discriminated different life stages of Trypanosoma cruzi, epimastigote and bloodstream trypomastigote and Trypanosoma brucei, procyclic and bloodstream. T. cruzi Discrete Typing Units (DTUs) were also discriminated in three clades. However, it was not possible to achieve enough resolution and software-assisted identification at the strain level. Overall, this study shows the importance of MALDI-TOF MS for the direct identification of trypanosomatids and opens new avenues for mass spectrometry-based detection of parasites in biofluids. Copyright © 2016 John Wiley & Sons, Ltd.

Direct-methods structure determination of a trypanosome RNA-editing substrate fragment with translational pseudosymmetry.

Using direct methods starting from random phases, the crystal structure of a 32-base-pair RNA (675 non-H RNA atoms in the asymmetric unit) was determined using only the native diffraction data (resolution limit 1.05 Å) and the computer program SIR2014. The almost three helical turns of the RNA in the asymmetric unit introduced partial or imperfect translational pseudosymmetry (TPS) that modulated the intensities when averaged by the l Miller indices but still escaped automated detection. Almost six times as many random phase sets had to be tested on average to reach a correct structure compared with a similar-sized RNA hairpin (27 nucleotides, 580 non-H RNA atoms) without TPS. More sensitive methods are needed for the automated detection of partial TPS.

Three-dimensional structures in the design of therapeutics targeting parasitic protozoa: reflections on the past, present and future.

Parasitic protozoa cause a range of diseases which threaten billions of human beings. They are responsible for tremendous mortality and morbidity in the least-developed areas of the world. Presented here is an overview of the evolution over the last three to four decades of structure-guided design of inhibitors, leads and drug candidates aiming at targets from parasitic protozoa. Target selection is a crucial and multi-faceted aspect of structure-guided drug design. The major impact of advances in molecular biology, genome sequencing and high-throughput screening is touched upon. The most advanced crystallographic techniques, including XFEL, have already been applied to structure determinations of drug targets from parasitic protozoa. Even cryo-electron microscopy is contributing to our understanding of the mode of binding of inhibitors to parasite ribosomes. A number of projects have been selected to illustrate how structural information has assisted in arriving at promising compounds that are currently being evaluated by pharmacological, pharmacodynamic and safety tests to assess their suitability as pharmaceutical agents. Structure-guided approaches are also applied to incorporate properties into compounds such that they are less likely to become the victim of resistance mechanisms. A great increase in the number of novel antiparasitic compounds will be needed in the future. These should then be combined into various multi-compound therapeutics to circumvent the diverse resistance mechanisms that render single-compound, or even multi-compound, drugs ineffective. The future should also see (i) an increase in the number of projects with a tight integration of structural biology, medicinal chemistry, parasitology and pharmaceutical sciences; (ii) the education of more `medicinal structural biologists' who are familiar with the properties that compounds need to have for a high probability of success in the later steps of the drug-development process; and (iii) the expansion of drug-development capabilities in middle- and low-income countries.

Kinetic and biochemical characterization of Trypanosoma evansi nucleoside triphosphate diphosphohydrolase.

Nucleoside triphosphate diphospho-hydrolases (NTPDases) catalyze the hydrolysis of several nucleosides tri and diphosphate playing major roles in eukaryotes including purinergic signaling, inflammation, hemostasis, purine salvage and host-pathogen interactions. These enzymes have been recently described in parasites where several evidences indicated their involvement in virulence and infection. Here, we have investigated the presence of NTPDase in the genome of Trypanosoma evansi. Based on the genomic sequence from Trypanosoma brucei, we have amplified an 1812 gene fragment corresponding to the T. evansi NTPDase gene. The protein was expressed in the soluble form and purified to homogeneity and enzymatic assays were performed confirming the enzyme identity. Kinetic parameters and substrate specificity were determined. The dependence of cations on enzymatic activity was investigated indicating the enzyme is stimulated by divalent cations and carbohydrates but inhibited by sodium. Bioinformatic analysis indicates the enzyme is a membrane bound protein facing the extracellular side of the cell with 98% identity to the T. brucei homologous NTPDase gene.

Sequence variation and structural conservation allows development of novel function and immune evasion in parasite surface protein families.

Trypanosoma and Plasmodium species are unicellular, eukaryotic pathogens that have evolved the capacity to survive and proliferate within a human host, causing sleeping sickness and malaria, respectively. They have very different survival strategies. African trypanosomes divide in blood and extracellular spaces, whereas Plasmodium species invade and proliferate within host cells. Interaction with host macromolecules is central to establishment and maintenance of an infection by both parasites. Proteins that mediate these interactions are under selection pressure to bind host ligands without compromising immune avoidance strategies. In both parasites, the expansion of genes encoding a small number of protein folds has established large protein families. This has permitted both diversification to form novel ligand binding sites and variation in sequence that contributes to avoidance of immune recognition. In this review we consider two such parasite surface protein families, one from each species. In each case, known structures demonstrate how extensive sequence variation around a conserved molecular architecture provides an adaptable protein scaffold that the parasites can mobilise to mediate interactions with their hosts.