Characterization of MsmX: A multitask ATPase from Bacillus subtilis

Transport across biological membranes is fundamental for cell survival and is mainly accomplished by specialized membrane proteins known as transporters. The ABC-type transporters constitute one of the largest and most diverse transporter superfamilies and are found in all three domains of life (Archaea, Bacteria and Eukarya). These permeases utilize the binding and hydrolysis of ATP to power the directional transport of a wide variety of substrates across membranes, ranging from ions to macromolecules. The ABC transporters have a modular architecture comprising two transmembrane domains (TMDs) that form the translocation pore and two nucleotide-binding domains (NBDs) that hydrolyse the necessary ATP for the translocation process. Until the last few years, NBDs were thought to be exclusive of each transporter complex, however it was discovered that several bacterial species ABC transporters share the same energy-generating component. In the Gram-positive model organism Bacillus subtilis ten ABC transporters are predicted to be involved in sugar uptake and eight of these systems do not display a gene encoding a putative NBD protein in their vicinity. Recent studies, demonstrated that MsmX interacts with several of these eight distinct ABC sugar importers. Thus, unlike other NBDs, MsmX was shown to be multitask serving as energy-generating component to several sugar importers. Other examples of multitask ATPases are also found in Streptococcus and Streptomyces species and are involved in the uptake of carbohydrates. So a better understanding of these multitask ATPases may have an important impact in therapy development for pathogenic bacteria since carbohydrate utilization is essential for microorganisms survival. In this work we demonstrate that B. subtilis can be used as a model to study multitask ATPases from other Gram-positive pathogenic species. A genetic system was developed to test in vivo the functionality of MsmX homologs from pathogenic species: Bacillus thuringiensis, Staphylococcus aureus and Streptococcus pneumoniae. The results show that the proteins are capable to play the role of MsmX in the cell, although with a different degree of efficiency. Functional analysis studies of MsmX indicate that modifications in the N- and C-terminal amino acid sequence do not affect its function. Moreover, by mutagenesis we show both in vitro an in vivo that the Lys 43 of MsmX, a conserved amino acid of NBDs, is essential for ATP hydrolysis. Additionally, the MsmX production and purification protocol was improved for further crystallography studies and biochemical characterization