Highlights d Crystal structure of the malaria parasite lipocalin d Comparative analysis of lipocalin superfamily members in alveolate genomes d Localization of PfLipocalin to the parasitophorous vacuole and food vacuole d Reverse genetics reveal PfLipocalin function in oxidative damage control
Single-molecule force spectroscopy by atomic force microscopy exploits the use of multimeric protein constructs, namely, polyproteins, to decrease the impact of nonspecific interactions, to improve data accumulation, and to allow the accommodation of benchmarking reference domains within the construct. However, methods to generate such constructs are either time- and labor-intensive or lack control over the length or the domain sequence of the obtained construct. Here, we describe an approach that addresses both of these shortcomings that uses Gibson assembly (GA) to generate a defined recombinant polyprotein rapidly using linker sequences. To demonstrate the feasibility of this approach, we used GA to make a polyprotein composed of alternating domains of I27 and TmCsp, (I27-TmCsp)3-I27)GA, and showed the mechanical fingerprint, mechanical strength, and pulling speed dependence are the same as an analogous polyprotein constructed using the classical approach. After this benchmarking, we exploited this approach to facilitiate the mechanical characterization of POTRA domain 2 of BamA from E. coli (EcPOTRA2) by assembling the polyprotein (I27-EcPOTRA2)3-I27GA. We show that, as predicted from the α + β topology, EcPOTRA2 domains are mechanically robust over a wide range of pulling speeds. Furthermore, we identify a clear correlation between mechanical robustness and brittleness for a range of other α + β proteins that contain the structural feature of proximal terminal β-strands in parallel geometry. We thus demonstrate that the GA approach is a powerful tool, as it circumvents the usual time- and labor-intensive polyprotein production process and allows for rapid production of new constructs for single-molecule studies. As shown for EcPOTRA2, this approach allows the exploration of the mechanical properties of a greater number of proteins and their variants. This improves our understanding of the relationship between structure and mechanical strength, increasing our ability to design proteins with tailored mechanical properties.
The human pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis resulting in over 1 million fatalities every year, despite decades of research into the development of new anti-TB compounds. Unlike most other organisms M. tuberculosis has six putative genes for epoxide hydrolases (EH) of the α/β-hydrolase family with little known about their individual substrates, suggesting functional significance for these genes to the organism. Due to their role in detoxification, M. tuberculosis EH’s have been identified as potential drug targets. Here, we demonstrate epoxide hydrolase activity of M. thermoresistibile epoxide hydrolase A (Mth-EphA) and report its crystal structure in complex with the inhibitor 1,3-diphenylurea at 2.0 Å resolution. Mth-EphA displays high sequence similarity to its orthologue from M. tuberculosis and generally high structural similarity to α/β-hydrolase EHs. The structure of the inhibitor bound complex reveals the geometry of the catalytic residues and the conformation of the inhibitor. Comparison to other EHs from mycobacteria allows insight into the active site plasticity with respect to substrate specificity. We speculate that mycobacterial EHs may have a narrow substrate specificity providing a potential explanation for the genetic repertoire of epoxide hydrolase genes in M. tuberculosis.
Proteins of the lipocalin family are known to bind small hydrophobic ligands and are involved in various physiological processes ranging from lipid transport to oxidative stress responses. The genome of the malaria parasite Plasmodium falciparum contains a single protein PF3D7_0925900 with a lipocalin signature. Using crystallography and small-angle X-ray scattering, we show that the protein has a tetrameric structure of typical lipocalin monomers, hence we name it P. falciparum lipocalin (PfLCN), the first lipocalin structurally and functionally characterized in a single-celled eukaryote. We show that PfLCN is expressed in the intraerythrocytic stages of the parasite and localizes to the parasitophorous and food vacuoles. Conditional knockdown of PfLCN impairs parasite development, which can be rescued by treatment with the radical scavenger Trolox or by temporal inhibition of hemoglobin digestion. This suggests a key function of PfLCN in counteracting oxidative stress induced cell damage during multiplication of parasites within red blood cells.Keywords: blood stage / food vacuole / lipocalin / malaria / oxidative stress compound 2. Images and time-lapse movies were acquired on microscopes of the CSSB imaging facility.
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