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Sandbox Reserved 812

This Sandbox is Reserved from 06/12/2018, through 30/06/2019 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1480 through Sandbox Reserved 1543.
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MsbA is a lipid flippase composed of 8 polypeptidic chains, essential for the cell viability and characterized in the E.Coli eubacteria. Its role is both to facilitate the transport of lipopolysaccharides (LPS) and phospholipids from the cytoplasmic to the periplasmic side of the cell membrane (flipping mechanism), and to transport them from the inner to the outer membrane of gram negative bacterias[1]. These functions are achieved by the protein’s transmembrane structure, and an adenosine triphosphate (ATP) binding cassette (ABC) which provides the necessary energy for the transport. It is essential for the transport of newly synthesized lipids to the outer membrane, and to maintain the asymmetrical composition of the plasmic membrane. MsbA belongs to the ABC super-family of transporters and it is more specifically a member of the MDR (Multidrug resistance)-ABC transporter group by sequence homology. MsbA is more closely related to the mammalian P-glycoprotein than any other bacterial ABC transporter.

Structure of MsbA of E.Coli

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Structural organization of MsbA flippaseStructural organization of MsbA flippase

All known ABC transporters are composed of four modules, including two membranes panning regions and two NBDs. The msbA gene encodes a half transporter that contains a single membrane spanning region fused with a NBD (Nucleotide binding domain). MsbA is assembled as a homodimer with a total molecular mass of 129.2 kD. Hydropathy analysis indicates six transmembrane α-helices (TM1 to TM6) and the NBD located on the cytoplasmic side of the cell membrane. Researchers have identified a third domain bridging the transmembrane and nucleotide-binding domains, which is called ICD (intracellular domain).[2]

Transmembrane domainTransmembrane domain

All the are tilted between 30ᵒand 40ᵒ from the normal of the membrane, forming a cone shaped structure with two substantial openings on either side facing the lipid bilayer. These openings lead into a large cone-shaped chamber in the interior of the molecule's transmembrane domain. The primary role of the transmembrane domain is to recognize and transport substrates across the lipid bilayer. The residues lining the proposed chamber opening (TM2, TM5, and TM6) play an important role in substrate recognition.

NBD structureNBD structure

The ABC is located in the and couples the energy of ATP hydrolysis to substrate translocation. The NBD is the most conserved feature of the MDR-ABC transporter family and contains the Walker A/B motif along with the ABC signature motif. In the absence of ATP or nucleotide analog in this structure, residues 341 to 418, which includes the Walker A motif, are disordered in electron density maps.

ICD structureICD structure

A distinctive feature of the MDR-ABC transporter family is two extensive intracellular regions called (residues 97 to 139) (residues 193 to 252) and (residues 302 to 327). ICD1, which is sandwiched between ICD2 and the NBD, is composed of three α-helices connected by short loops to form a "U"-like structure. ICD3 links TM6 and the NBD and forms two α-helices connected by short loops.

Chamber structureChamber structure

The chamber is a symmetric structure that is formed from two MsbA transmembrane domains. The chamber has an opening (25 Å) on either side facing the bilayer providing free access of substrate from the cytoplasmic leaflet of the lipid bilayer while excluding molecules from the outer leaflet. The openings to the chamber are defined by intermolecular interactions between TM2 of one monomer and TM5 of another. Residues 268 to 273 of TM2 lining the opening of the chamber are partially shielded from the bilayer by TM5. The residues lining the chamber are contributed by all 12 transmembrane α-helices and could be highly solvated. The inner membrane leaflet side of the chamber contains a cluster of positively-charged residues (Arg14, Arg 83, Lys187, Arg190, Lys194, and Arg296) (Fig. 6B), which contrasts with the significantly less charged and more hydrophobic environment within the outer membrane leaflet side.


Function of flippase MsbAFunction of flippase MsbA

Lipid translocation across the inner membraneLipid translocation across the inner membrane

LPS and phospholipids are synthesized at the cytoplasmic side of the inner bacterial membrane. In order to maintain the asymmetrical composition of the membrane, they need to be translocated to the periplasmic side of the inner membrane. MsbA is responsible for this translocation.[3] As MsbA is an ABC transporter, the lipid translocation is ATP-dependent. It was found that an ATP concentration of 5 mM provided substantial flippase activity, whereas an ATP concentration of 10 mM provided a maximal flippase activity [4]. The maximal flippase specific activity for MsbA was reported to be 0.39 nmol.min-1.mg-1.

Lipid translocation to the outer membraneLipid translocation to the outer membrane

In order to maintain the outer bacterial membrane of gram negative bacteria, lipids need to be translocated from the inner to the outer membrane. This is achieved through MsbA ATP-dependent transport. An important substrate for MsbA is lipid A. Lipid A are anchors for LPS, and are used for antigen-O recognition. Its transport to the outer membrane is therefore essential.

Upon binding of a lipid A molecule, conformational changes relayed from the transmembrane domain to the intracellular domain initiate nucleotide hydrolysis by the NBD. Movement of TM2, TM5, TM6, and the two nucleotide-binding domains serves to recruit the substrate and close the chamber. Hydrolysis of ATP by the NBD may result in a conformational shift that promotes the interaction of the adjacent NBDs. The cluster of charges lining the chamber on the inner membrane leaflet side creates an energetically unfavorable microenvironment for a hydrophobic substrate. The asymmetric charge distribution in the interior of the chamber is consistent with the vectored transport of substrate from the inner to the outer membrane leaflet. Faced with both the charge and the highly polar contribution of potentially bound solvent, the lipid A molecule "flips" into an energetically more favorable position within the outer membrane leaflet side of the chamber where it can form hydrophobic interactions. The substrate is now properly orientated to enter the outer bilayer leaflet. The flipping of substrate signals the chamber to undergo structural rearrangements that include the separation of the NBDs and repositioning of TM2 and TM5, enabling the expulsion of the substrate to the outer membrane leaflet.

ATPase activityATPase activity

The ATPase activity of MsbA was characterized by Doerrler et al. [5] . ATP hydrolysis occurs through an ATP-binding cassette, and the obtained energy is used to catalyze the translocation of lipids. After purification, MsbA displayed a Km of 878 μM and a Vmax of 37 nmol.min-1.mg-1 for ATP. Mg2+ was found to be needed as cofactor for this hydrolysis process to occur. Lipid A is an activator of MsbA ATPase activity, lowering the Km to 379 μM, and raising the Vmax to 154 nmol.min-1.mg-1. Phospholipids were also found to stimulate MsbA activity.

Amphipathic drugs transportAmphipathic drugs transport

It was found that MsbA is able to bind amphipathic drugs (like daunorubicin) with an affinity comparable to that of the binding of lipid A [6]. The ABC transporter MsbA is able to bind lipid A and daunorubicin simultaneously, which reflects the presence of two different binding sites, one for lipid A, and the other for amphipathic drugs. Those two sites can be independently bound by their substrate, but prior binding of lipid A leads to a higher affinity of MsbA for daunorubicin. Binding of both substrates has an additive effect on MsbA activity. It was finally found that each binding site communicate with each other and with the nucleotide binding site, as binding of one substrate affects the affinity of the other sites for their substrates. This strongly suggests that MsbA, apart from being a lipid flippase, could also be a multidrug transporter. The structure study can help elucidate the mechanism underlying the multidrug resistance phenotype, which could have a profound impact on the development of novel therapeutics used to treat cancer and infectious disease.

ContributorsContributors

THEDIE Daniel/ VIGNES Hélène ESBS 1A

ReferencesReferences

  1. ↑ Zhou Z, White KA, Polissi A, Georgopoulos C, Raetz CR. Function of Escherichia coli MsbA, an essential ABC family transporter, in lipid A and phospholipid biosynthesis. J Biol Chem. 1998 May 15;273(20):12466-75. PMID:9575204
  2. ↑ Eckford PD, Sharom FJ. The reconstituted Escherichia coli MsbA protein displays lipid flippase activity. Biochem J. 2010 Jul 1;429(1):195-203. doi: 10.1042/BJ20100144. PMID:20412049 doi:http://dx.doi.org/10.1042/BJ20100144
  3. ↑ Doerrler WT, Gibbons HS, Raetz CR. MsbA-dependent translocation of lipids across the inner membrane of Escherichia coli. J Biol Chem. 2004 Oct 22;279(43):45102-9. Epub 2004 Aug 10. PMID:15304478 doi:http://dx.doi.org/10.1074/jbc.M408106200
  4. ↑ Eckford PD, Sharom FJ. The reconstituted Escherichia coli MsbA protein displays lipid flippase activity. Biochem J. 2010 Jul 1;429(1):195-203. doi: 10.1042/BJ20100144. PMID:20412049 doi:http://dx.doi.org/10.1042/BJ20100144
  5. ↑ Doerrler WT, Raetz CR. ATPase activity of the MsbA lipid flippase of Escherichia coli. J Biol Chem. 2002 Sep 27;277(39):36697-705. Epub 2002 Jul 15. PMID:12119303 doi:http://dx.doi.org/10.1074/jbc.M205857200
  6. ↑ Siarheyeva A, Sharom FJ. The ABC transporter MsbA interacts with lipid A and amphipathic drugs at different sites. Biochem J. 2009 Apr 15;419(2):317-28. doi: 10.1042/BJ20081364. PMID:19132955 doi:http://dx.doi.org/10.1042/BJ20081364
  • Geoffrey CHANG and Christopher B.ROTH. Structure of MsbA from E. coli: A Homolog of the Multidrug Resistance ATP Binding

Cassette (ABC) Transporters. Science, New Series, Vol. 293, No. 5536 (Sep. 7, 2001)

  • Andrew WARD, Christopher L. REYEST, Jodie YU, Christopher B. ROTH, and Geoffrey CHANG. Flexibility in the ABC transporter MsbA: Alternating

access with a twist. PNAS(2007)

  • Jinhui DONG and al. Structural Basis of Energy Transduction in the Transport Cycle of MsbA.Science 308, 1023 (2005)

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA, Hélène Vignes, Daniel Thédié
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