The distribution of different classes of nuclear localization signals (NLSs) in diverse organisms and the utilization of the minor NLS-binding site in plant nuclear import factor importin-α

The specific recognition between the import receptor importin-α and the nuclear localization signals (NLSs) is crucial to ensure the selective transport of cargoes into the nucleus. NLSs contain one or two clusters of positively-charged amino-acids, which usually bind to the major (monopartite NLSs) or both minor and major NLS-binding sites (bipartite NLSs). In our recent study, we determined the structure of importin-α1a from rice (Oryza sativa), and made two observations that suggest an increased utilization of the minor NLS-binding site in this protein. First, unlike the mammalian protein, both the major and minor NLS-binding sites are auto-inhibited in the unliganded rice protein. Second, we showed that NLSs of the 'plant-specific' class preferentially bind to the minor NLS-binding site of rice importin-α. Here, we show that a distinct group of 'minor site-specific' NLSs also bind to the minor site of the rice protein. We further show a greater enrichment of proteins containing these plant-specific' and 'minor site-specific' NLSs in the rice proteome. However, the analysis of the distribution of different classes of NLSs in diverse eukaryotes shows that in all organisms, the minor site-specific NLSs are much less prevalent than the classical monopartite and bipartite NLSs

Crystallization and preliminary X-ray analysis of the chemokine-binding protein from orf virus (Poxviridae)

The chemokine-binding protein from orf virus was purified and crystallized. The morphology and diffraction behaviour of these crystals was significantly improved through the use of additives known as Silver Bullets

Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major.

Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections