Structural Analysis of the Partially Disordered Protein EspK from Mycobacterium Tuberculosis

For centuries, tuberculosis has been a worldwide burden for human health, and gaps in our understanding of its pathogenesis have hampered the development of new treatments. ESX-1 is a complex machinery responsible for the secretion of virulence factors that manipulate the host response. Despite the importance of these secreted proteins for pathogenicity, only a few of them have been structurally and functionally characterised. Here, we describe a structural study of the ESX-secretion associated protein K (EspK), a 74 kDa protein known to be essential for the secretion of other substrates and the cytolytic effects of ESX-1. Small-Angle X-ray Scattering (SAXS) data show that EspK is a long molecule with a maximal dimension of 228 Å. It consists of two independent folded regions at each end of the protein connected by a flexible unstructured region driving the protein to coexist as an ensemble of conformations. Limited proteolysis identified a 26 kDa globular domain at the C-terminus of the protein consisting of a mixture of α-helices and β-strands, as shown by circular dichroism (CD) and SAXS. In contrast, the N-terminal portion is mainly helical with an elongated shape. Sequence conservation suggests that this architecture is preserved amongst the different mycobacteria species, proposing specific roles for the N- and C-terminal domains assisted by the middle flexible linker.