New insights into the domain of unknown function DUF of EccC5, the pivotal ATPase providing the secretion driving force to the ESX5 secretion system

Abstract

Type VII secretion (T7S) systems, also referred to as ESAT6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implication in cell homeostasis and host pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis and a pandemic accounting for more than 1 million deaths every year. The ESX5 system is exclusively found in slow-growing pathogenic mycobacteria, where it mediates the secretion of a large family of virulence factors, the PE and PPE proteins. The secretion driving force is provided by EccC5, a multidomain ATPase operating through four globular cytosolic domains, an N-terminal domain of unknown function (EccCDUF) and three FtsK/SpoIIIE ATPase domains. Recent structural and functional studies of ESX3 and ESX5 systems have revealed EccCDUF as an ATPase-like fold domain with potential ATPase activity, and whose functionality is essential for secretion. Here we report the crystal structure of MtbEccC5DUF domain at 2.05 [A] resolution, which unveils a nucleotide-free structure with degenerated cis-acting and trans-acting elements involved in ATP-binding and hydrolysis. Our crystallographic study, together with a biophysical assessment of MtbEccC5DUF interaction with ATP/Mg2+, supports the absence of ATPase activity proposed for this domain. We show that this degeneration is also present in DUF domains of other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, and based on an in-silico model of MtbEccC5 N-terminal region, we propose that MtbEccC5DUF is a degenerated ATPase domain that may have retained the ability to hexamerise. Observations that call the attention on DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process.