Structure and mechanism of an active LLO-flippase

The transportation of phospholipids from one side of the phospholipid bilayer to the other, called flipping, is energetically unfavourable, and thus requires the assistance of ATP-dependent flippases.  However, not much is known about their mechanisms.

To illuminate a possible mechanism for this type of flippase, PglK, which flips a chemically similar LLO (GlcGalNAc5Bac-PP-undecaprenyl) in C. jejuni; the Pglk gene, cloned into a vector along with a His-10 tag (fused to the N-terminus), was over expressed in E. Coli and purified by affinity chromotagraphy. The protein is identified as a homodimeric ATP-Binding Cassette (ABC) transporter in X-ray crystallography, necessary for bacterial protein N-glycosylation. 

ATPase Activity of PglK

The Pglk ATP-hydrolysing domians are consisted of two short motifs in their primary structure:

• Walker A: GXXGXGKS/T (X can be varied)
• Walker B: hhhhDQ
• linker peptide: LSGGQ

Mutagenesis from a glutamate to a glutamine residue at 510 in the Nucleotide Binding Domain (NBD) the walker-B motif, exhibited decreased flipping rate by almost 100-fold.

ATP hydrolysis is necessary to the flipping of LLO by PglK, as evinced by the fact that the PglK ATPase activity in the presence of LLO follows Michaelis-Menten kinetics, with a similar Km value compare to ATP-dependent trisaccharide LLO (tLLO) flipping. The binding of the native substrate in ABC exporters stimulates ATPase activity: PglK-catalysed ATP hydrolysis is stimulated by 2.5 fold by full-length LLO and tLLO in detergent and liposome, showing the terminal GalNAc and the branching glucose are not relevant for ATPase stimulation or flipping. The ATPase activity of mutant (E510Q) is also stimulated by LLO, suggesting the interaction with LLO is not affected by the E510Q mutation. ATPase activity of PglK seems to be specific to LLO/tLLO; however, the ATPase activity may be limited by shorter polyprenyl tails.

Figure 1. The ADP-bounded state of  walker-A motif  in a homologous ABC transporter, similar to walker A motifs found in PglK. The motif is rich in glycine residues; however, the lysine residue, as well as the NH groups in the main chain, is essential for phosphate binding

Structures of PglK in distinct states

PglK can take on several conformations, including two inward-facing (apo1 and apo2) and one outward-facing conformation, which is pertinent for flipping. The oligosaccharide moiety passes through the transporter; the hydrophobic tail interacts primarily with the lipid bilayer. The three crystal structures of PglK mutant (E510Q) have been determined at resolution ranging from 2.9Å to 5.9Å. The outward occluding conformation was an ADP-bound PglK. Other ABC transporters have transmembrane-helices that move in pairs, which helped to establish the model below.

Figure 2. The three main conformations of PglK: showing the interactions of the two subunits. The main conformational change involves the two subunits moving together, in a 'scissoring motion' which is powered by basal ATPase activity. 

Figure 3. The loop connecting  TM1 and TM2 (blue) of PglK contains External helices (EH) (red), at the periplasmic boundary of the membrane. The orientation of the helix relative to the transmembrane domain is defined by several hydrophobic, hydrophilic and cation-pi interactions. Two grooves between the external membrane leaflet are formed between the helices and TM1 and TM2. The main conformational change involves TM4 and TM5 (yellow) moving together towards TM6 (green) and tilting by 40° (apo1 to outward).

Role of EH in PglK – LLO interaction

The interaction between aromatic residues (Y50/Y56) and positively charged residues (R53/K55) results a hydrophobic grooves on each sub-unit, along with other hydrophobic residues. These grooves are thought to be contributing to the binding of LLO to PglK which anchor the lipid tail of LLO and favour the positioning of the PP-moiety for entering a translocation cavity, initiating the flipping reaction.
The EH links LLO binding to ATPase activity: alanine scanning mutagenesis revealed that the EH mutant had unaltered basal ATPase activity, however, no stimulated ATPase activity is found compared to wild type.

Figure 4. The hydrophobic grooves (green) are present within the membrane and key amino acid residues are shown.

The inward-facing structure contains a large cavity facing the cytoplasmic side and the inner leaflet of the bilayer. During LLO flipping, the polyprenyl tail enters the inward-facing cavity from the lipid bilayer to fold into the hydrophobic bottom of the cavity (indicated green in Figure 4).

Role of Arginine residues: 

Multiple arginines residues (R86, R260, R302 and R309) line the interior of the outward-facing cavity, accounting for a total of 8 positive charges. These arginines are buried/form salt-bridges in the inward-facing conformations but become accessible when conformation changes to outward-facing state. When flipping starts, the arginine will form salt bridges with the pyrophosphate-moiety of LLO to serve as binding partners.

Figure 5. The four arginine residues shown in one subunit of PglK (86, 260, 302 and 309) line the interior of the cavity. When any of the residues are mutated to alanine, the cavity no longer binds the oligosaccharide moiety and flipping does not occur

Mechanism

The two conformations actually involved in the mechanism of PglK-catalysed LLO flipping are the outward-occluded (ADP-bound) and outward-facing (ATP-bound) ones. The model of outward-open state is based on the structure of Sav1866 since the state is transient (crystal cannot be formed). In both states, the outward-facing cavity spans almost the entire membrane, whereas the outward-occluded conformation would not provide sufficient space to contain the pp and oligosaccharide moieties of LLO, the fully outward-facing conformation could.

Figure 6. The subunits of PglK are coloured blue and orange. Orange sphere denotes di-N-acetylbacillosamine; yellow square denotes N-acetylgalactosamine, and grey circle denotes glucose. The proposed molecular events indicated by arrows are: A, polyprenyl tail (green) interacting with hydrophobic region including and around EH (red); B, The pp moiety (circles containing 'P') enters the outward-facing cavity and form salt bridges with 8 arginine residues (brown); C, ATP hydrolysis, LLO head-group release on external side of the membrane; D, release of polyprenyl tail 

The driving force for the flipping reaction has two components:

• the NBDs are pushed apart due to ATP hydrolysis, releasing the polyprenyl tail: this motion is transmitted to the TMDs and can exert pressure on the substrate in the translocation pathway;
• As the oligosaccharide and the pyrophosphate moieties have diffused out of the translocation pathway, PglK will adopt an outward-occluded conformation, preventing the substrate moves backwards.

The energy from ATP hydrolysis is primarily used to break the interaction of the pyrophosphate with the arginine residues.

Conclusion 

The flipping of LLOs by PglK is ATP-dependent and requires the conformational change of PglK from outward-occluding to outward-open (as well as apo1 and apo2, which are not relevant to flipping). The hydrophobic tail of the LLO continues to interact with the membrane bilayer during flipping, while the oligosaccharide moitey interacts with the internal cavity. CryoTEM might be the final solution to confirm the mechanism above, it allows the observation of the transient state of Pglk which is not in crystal, but showing it in its native environment.