Oligosaccharride Flippase in C.jejuni

Alex Schaal and Toby SantaMaria


I. Introduction

This is the ABC transporter molecule PgIK--or, oligosaccharide flippase--in the apo-inward configuration for Camplylobacter jejuni. Flippases are essential for cellular functions from cell-cell signaling to cellular transport. In general, flippases catalyze the flipping of the polar head groups of oligosaccharides LLO embedded in the cell membrane to the opposite orientation in the membrane mosaic. Oligosaccharride flippase is an ATP bound molecule which flips the orientation of the membrane embedded lipids. This flippase is composed of 4 identical subunits--a homotetramer.

In order to flip the membrane,the pyrophosphate oligosaccharride head group enters the translocation cavity and interacts with positively charged side chains while the lipidic polyphrenyl tail binds and activates the transporter, while interacting with the lipid bilayer. 

II. General Structure

PgIK is a homotetramer . It contains 4 alpha helices (TM1, TM2, TM4, and TM5 ) along with one short alpha helix called EH. Although there are some beta sheets within the molecule, the external sides of the molecule are mainly composed of alpha helices and these helices are what catalyze the flipping reaction. Usually, molecules of PgIK are found in liposomes or near the cellular membrane. The molecule itself is a sort of dimer of dimers; one subunit adopts a "closed" conformation in which the amino- and carboxy-termini are closer together than in the "open" subunit. Each subunit is composed of two distinct domains connected by a hinge region. The N-terminal domain is responsible for dimerization and the carboxy-terminal domain contains a helix-turn helix binding motif, and is also responsible for bending. Another important aspect of this flippase is the motif. The LLO-- or, lipid linked oligosaccharide-- consists of decaprenyl chain connected to a GlcGalNAc5Bac-phosphate-phosphate chain. This structure is found in most lipid bilayers, and has its own binding domain in the molecule PgIK-- the outward facing cavity. The domain also serves as an ATP biding site, where ATP is hydrolzyed to catalyze the flipping motion of the protein.  

III. ATPase Stimulation and Binding

An important site for LLO mediated ATPase stimulation is the External Helix. The EH is a short alpha helix at the periplasmic boundary which contains both hydrophobic and hydrophyllic residues . The EH links the LLO structure to the external facing cavity of PgIK to initiate ATPase stimulation of PgIK. Specifically, the hydrophobic grooves within the EH act as an anchor to hold the lipid tail of the LLO and push it into position within the outward cavity, positioning the pyrophosphate moiety into the proper position to initiate ATP hydrolysis and initiate the flipping reaction.These hydrophobic and hydrophyllic sections within the EH are crucial to the LLO mediated ATPase activity on PgIK-- without the hydrophobic entities within the EH, LLO would not be attracted to the outward facing cavity of PgIK. Then there would be no stimulation by ATPase by the LLO site. Without the EH, the overall flipping mechanism of the protein is greatly reduced, although not completely diminished. Without the EH to bring the LLO into contact with the external cavity and position correctly within PgIK, PgIK would have to wait for the LLO to diffuse naturally into the outward facing cavity. If EH cannot induce the LLO into ATPase activity, this is called binding failure.

IV. ATP hydrolysis by PgIK and Conformational Changes

PgIK has both inward and outward facing cavities. The inward facing cavity houses the 55-carbon long polyprenyl tail of the oligosaccharide being flipping in the lipid bilayer--essentially, the tail curls up within the very bottom and hydrophobic section of the internal cavity. The outward-facing portion og PgIK is key to ATP hydrolysis and conformational change neccessary for the flipping reaction. This wide opening that faces towards the periplasmic side of the lipid membrane has sufficient space inside to include the pyrophosphate and oligosaccharide moeities of the LLO-- these moities serve as both an anchor point for the pyrophosphate linked oligosaccharide being flipped, and as a domain where ATP can be hydrolyzed. There are also multiple arginine residues ( Arg86, Arg260, Arg302, and Arg309) which line the interior of the outward cavity, giving the outer cavity a net positive charge. The arginines act as anchor points for the pyrophosphate chains within the LLO structure so that the hydrolysis of ATP can occur once the LLO itself is within the outward facing cavity.These arginines later form salt-bridges within the inner cavity of the PgIK, helping to make the scissoring motion required for flipping of oligosaccharides in the cell membrane.

V. Flipping Reaction and Crystal Packing.

The flipping reaction involves the apo-inward or apo-occluded, ADP bound state changing conformation to the apo-outward state (ATP bound) state. The polyprenyl tails of the LLO interacts with the polar surfaces of the EH; transfering a pyrophosphate into the outward cavity of the apo-outward state PgIK. Here, the pyrophosphate has many electrostatic interactions with the postively charged arginine chains, which in turn pull in the oligosaccharide to the cavity. The hydrolysis of ATP then changes the confirmation of PgIK, causing the molecule to "scissor"--flipping the oligosaccharide in the process. However, PgIK tetramers are usually found in "crystal packed" formations Crystal Packing : meaning that multiple tetramers or multiple molecules can interact and be bound to one another. Depending on the nature of the flipping reaction, PgIK can be packed together at EH-EH units, from hinge-hinge structures, or from EH-hinge structures. There are also polar and non-polar groups on the PgIK homotetramer that allow for asymmetrical packing-- loosely attracted to one another due to the weak electrostatic interactions between polar sections or van derWaal's between hydrophobic sections. The beta sheets of the non-binding domain (NBD) do not participate in the binding of LLOs but are thought to participate in these non-polar interactions. These NBD-NBD interactions are important in crystal packing .

VI. References

Caimlio Perez, Sabrina Gerber, Jeremy Boilevin, Monika Bucher, Tamis Barbre, Markus Aebi, Jean-Louis Reymond and Kaspar P. Locher. Structure and mechanism of an active lipid-linked oligosaccharide flippase Nature 524:433-438.

Thomas Pomorski, Joost C.m. Holthuis,Andrea Herrmann, and Gerrit van Meer.Tracking down lipid flippases and their functions. Journal of Cell Science 117: 805-813.

Cristina Alaimo, Ina Catrein, Laura Morf, Cristina L. Marolda, Nico Callewaert, Miguel A. Valvano, Mario F. Feldman, and Markus Aebi. Two distinct but interchangeable mechanisms for the flipping of lipid-linked oligosaccharides. The European Molecular Biology Organization Journal 25:967-976.

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