Dissecting the membrane lipid binding properties and lipase activity of the 1 Mycobacterium tuberculosis LipY domains

21 The Mycobacterium tuberculosis LipY protein, a prototype of the proline-glutamic acid (PE) family, 22 exhibits a triacylglycerol (TAG) hydrolase activity that contributes to host cell-lipid degradation and 23 persistence of the bacilli. LipY is found either as a full-length intracytosolic form or as a mature 24 extracellular form lacking the N-terminal PE domain. Even though the contribution of the 25 extracellular form in TAG consumption has been partly elucidated, very little information is available 26 regarding the potential interactions of each form of LipY with either the cytoplasmic membrane for 27 the full-length protein or with the outer membrane for the matured form. Herein, several LipY 28 variants truncated in their N-terminal domain were produced and biochemically characterized in 29 lipid-protein interaction assays using the monomolecular film technique and Fourier transform 30 infrared spectroscopy. Comparison of the catalytic activities of these recombinant proteins showed 31 that LipY∆149, corresponding to the extracellular form of LipY lacking the PE domain, is more 32 active than the full-length protein. This confirms previous studies reporting that the PE domain 33 negatively modulates the TAG hydrolase activity of LipY. Lipid-protein interaction studies indicate 34 that the PE domain anchors LipY onto membrane lipids. Consistent with these findings, LipY∆149 35 was loosely-associated with the mycobacterial cell wall and that this interaction is mediated by the 36 sole lipase domain. Altogether, our results bring new information regarding the molecular 37 mechanisms by which LipY either binds and hydrolyzes host cell lipids or degrades TAG, the major 38 source of lipids within mycobacterial intracytosolic lipid inclusions.