posted on 2021-04-29, 10:49authored byCamilla
A. K. Lundgren, Michael Lerche, Charlotta Norling, Martin Högbom
The very-long-chain
fatty acyl-CoA synthetase FadD13 from Mycobacterium tuberculosis activates fatty acids for further
use in mycobacterial lipid metabolism. FadD13 is a peripheral membrane
protein, with both soluble and membrane-bound populations in vivo. The protein displays a distinct positively charged
surface patch, suggested to be involved in membrane association. In
this paper, we combine structural analysis with liposome co-flotation
assays and membrane association modeling to gain a more comprehensive
understanding of the mechanisms behind membrane association. We show
that FadD13 has affinity for negatively charged lipids, such as cardiolipin.
Addition of a fatty acid substrate to the liposomes increases the
apparent affinity of FadD13, consistent with our previous hypothesis
that FadD13 can utilize the membrane to harbor its very-long-chain
fatty acyl substrates. In addition, we unambiguously show that FadD13
adopts a dimeric arrangement in solution. The dimer interface partly
buries the positive surface patch, seemingly inconsistent with membrane
binding. Notably, when cross-linking the dimer, it lost its ability
to bind and co-migrate with liposomes. To better understand the dynamics
of association, we utilized two mutant variants of FadD13, one in
which the positively charged patch was altered to become more negative
and one more hydrophobic. Both variants were predominantly monomeric
in solution. The hydrophobic variant maintained the ability to bind
to the membrane, whereas the negative variant did not. Taken together,
our data indicate that FadD13 exists in a dynamic equilibrium between
the dimer and monomer, where the monomeric state can adhere to the
membrane via the positively charged surface patch.