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Solution and membrane interaction dynamics of Mycobacterium tuberculosis fatty Acyl-CoA synthetase FadD13

DOI: 10.1021/acs.biochem.0c00987 DOI Help

Authors: Camilla A. K. Lundgren (Stockholm University; University of Oxford) , Michael Lerche (Stockholm University) , Charlotta Norling (Stockholm University) , Martin Högbom (Stockholm University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemistry

State: Published (Approved)
Published: April 2021
Diamond Proposal Number(s): 11265

Open Access Open Access

Abstract: 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.

Journal Keywords: Vesicles; Lipids; Bacteria; Oligomers; Membranes

Diamond Keywords: Tuberculosis (TB); Bacteria; Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: B21-High Throughput SAXS

Added On: 02/05/2021 22:08


Discipline Tags:

Pathogens Infectious Diseases Health & Wellbeing Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Scattering Small Angle X-ray Scattering (SAXS)