Structure of MlaFB uncovers novel mechanisms of ABC transporter regulation

Abstract

ABC transporters facilitate the movement of diverse molecules across cellular membranes, but how their activity is regulated post-translationally is not well understood. Here we report the crystal structure of MlaFB from E. coli, the cytoplasmic portion of the larger MlaFEDB ABC transporter complex, which drives phospholipid trafficking across the bacterial envelope to maintain outer membrane integrity. MlaB, a STAS domain protein, binds the ABC nucleotide binding domain, MlaF, and is required for its stability. Our structure also implicates a unique C-terminal tail of MlaF in self-dimerization. Both the C-terminal tail of MlaF and the interaction with MlaB are required for the proper assembly of the MlaFEDB complex and its function in cells. This work leads to a new model for how an important bacterial lipid transporter may be regulated by small proteins, and raises the possibility that similar regulatory mechanisms may exist more broadly across the ABC transporter family.

Structure and lipid dynamics in the A. baumannii maintenance of lipid asymmetry (MLA) inner membrane complex

“…1), again similar to the G5G8 sterol exporter complex. MlaF is bound to MlaB away from the nucleotide binding site, similar to the recently-reported E.coli MlaBF structure (14), with C-termini of MlaF binding the opposing MlaB subunit by a "handshake" mode ( Fig. 1E).…”

Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

“…This rotation is similar to motions described in other ABC transporters (Karpowich et al, 2001;Smith et al, 2002;Orelle et al, 2010). An unusual C-terminal extension of each MlaF protomer wraps around the neighboring MlaF subunit and docks near the MlaFB interface, almost identical to the domain-swapped "handshake" motif observed in the crystal structure of the MlaF 2 B 2 subcomplex (Figure 1figure supplement 4B) (Kolich et al, 2020). While the MlaFEB subcomplex exhibits near-perfect 2-fold rotational symmetry at this resolution, the MlaD ring is clearly tilted relative to MlaE, resulting in a misalignment of the 2-fold symmetry axis of MlaFEB and the pseudo-6-fold axis of MlaD by approximately 6 degrees ( Figure 1F).…”

“…Serial dilutions of the strains in 96 well plates were manually spotted (2 uL each) on plates containing LB agar or LB agar supplemented with 0.2% SDS and 0.30-0.35 mM EDTA, and incubated for 16 hours at 37 °C. We find that this growth assay is very sensitive to the reagents used, particularly the LB agar (see Kolich et al, 2020). For the experiments reported here, we used Difco LB agar pre-mix (BD Difco #244510), a 10% stock solution of SDS (Sigma L5750), and a 500 mM stock solution of EDTA, pH 8.0 (Sigma ED2SS).…”

Evolutionary history of ATP‐binding cassette proteins

“…The maintenance of asymmetric ATPase sites in ABC proteins over evolution might suggest that ATP is not simply a source of Gibbs free energy from hydrolysis but rather ATP‐binding and hydrolysis play specific biophysical roles in conformational cycling [40–46]. Sequence variation in NBDs outside the core ‘ABC_tran’ domain are also functionally relevant for interactions with regulatory domains that provide post‐translational control over the transporter [47]. Though these extensions are fused to the NBD proteins outside the ‘ABC_tran’ region, they can change the relative orientation of the NBDs, directly controlling the formation of the sandwich dimer required for ATP binding and hydrolysis [47].…”

“…Sequence variation in NBDs outside the core ‘ABC_tran’ domain are also functionally relevant for interactions with regulatory domains that provide post‐translational control over the transporter [47]. Though these extensions are fused to the NBD proteins outside the ‘ABC_tran’ region, they can change the relative orientation of the NBDs, directly controlling the formation of the sandwich dimer required for ATP binding and hydrolysis [47].…”