MmpL3, the trehalose monomycolate transporter, is stable in solution in several detergents and can be reconstituted into peptidiscs

Kien Lam Ung, Husam Alsarraf, Laurent Kremer, Mickaël Blaise*

*Corresponding author af dette arbejde

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Abstract

Mycobacteria possess a complex and waxy cell wall comprising a large panel of glycolipids. Among these, trehalose monomycolate (TMM) represents abundant and crucial components for the elaboration of the mycomembrane. TMM is synthesized in the cytoplasmic compartment and translocated across the inner membrane by the MmpL3 transporter. Inhibitors impeding TMM transport by targeting MmpL3 show great promises as new antimycobacterials. The recent X-ray or Cryo-EM structures of MmpL3 complexed to TMM or its inhibitors have shed light on the mechanisms of TMM transport and inhibition. So far, purification procedures mainly involved the use of n-Dodecyl-ß-D-Maltopyranoside to solubilize and stabilize MmpL3 from Mycobacterium smegmatis (MmpL3Msm) or Lauryl Maltose Neopentyl Glycol for MmpL3 from Mycobacterium tuberculosis. Herein, we explored the possibility to solubilize and stabilize MmpL3 with other detergents. We demonstrate that several surfactants from the ionic, non-ionic and zwitterionic classes are prone to solubilize MmpL3Msm expressed in Escherichia coli. The capacity of these detergents to stabilize MmpL3Msm was evaluated by size-exclusion chromatography and thermal stability. This study unraveled three new detergents DM, LDAO and sodium deoxycholate that favor solubilization and stabilization of MmpL3Msm in solution. In addition, we report a protocol that allows reconstitution of MmpL3Msm into peptidiscs.

OriginalsprogEngelsk
Artikelnummer106014
TidsskriftProtein Expression and Purification
Vol/bind191
ISSN1046-5928
DOI
StatusUdgivet - mar. 2022

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