User:Centralyoga/Dual Topology of animal Membrane Proteins

Definition:

The term, Dual Topology, has recently been coined to characterize the mirror-image orientation of two identical subunits across the membrane that has been found to play vital role in membrane function. Several membrane proteins of animal origin have recently been demonstrated to be homodimers in the functionally active state having mirror-image (dual topology) symmetry across the membrane. A number of approaches have thus far been used to study the topological layouts of intrinsic membrane proteins relative to their activity and functional stability in eukaryotic cells. Among them the use of tightly sealed gastric microsomal vesicles of uniform (inside-out) orientation provided first direct evidence (1) for the topology of the related active sites within the functional H, K-ATPase (proton-pump) system.

Dual Topology of the plasmalemmal P-2 ATPase Pumps:

The use of Valinomycin-K sensitive microsomal vesicles having H, K-ATPase dependent H/K-transport activity offered unique opportunity to study sidedness of the associated ATP and pNPP hydrolytic sites, enlightening the topological layout of the molecule (1). The studies revealed a high affinity (hydrolysable) and a low affinity (non-hydrolysable) ATP site on the functional H, K-ATPase complex with opposite orientation across the bilayer (1). Based on the evidence that the non-hydrolysable ATP binding (regulatory) site and the high affinity K binding site are located across the bilayer separated from the known ATP catalytic site, and the same sidedness of the individual low affinity K site crucial for pNPP hydrolysis on either side of the bilayer (1) it was clear that two α-subunits are juxtaposed in unity in mirror image having a dual topology (1). Concurrently, decisive evidence for an identical dual topology model for another member of the P-2 ATPase family, the Na-pump (Na, K-ATPase system), was gathered from studies with isolated gastric epithelial cells showing ouabain-inhabitable Rb uptake mediated by the ubiquitous Na-pump (2). Thus, similar to the gastric H, K-ATPase system mentioned above, discrete orientation of the intracellular ATPase (catalytic) site and the extracellular pNPPase site, and separate location of the K-effector sites for the enhanced activities of the respective ATPase and pNPPase were observed (2) providing strong support for a dual topology Na-pump in the isolated viable cell system.

A unified dual topology model for the P-2 ATPase has since been constructed and is shown in figure 1. The practicality of the basic dual topology model in H (or Na), K-ATPase function has been apparent over the years given that in contrast to the traditional Post-Albers single topology scheme the dual topology model explains numerous published data in the field since its inception. The model also defines the function of the associated K-pNPPase activity which is clearly unrelated to the actual turn-over of the H (or Na), K- ATPase, and has been found to correlate well with the ion channel function (figure 1). The ion channels located in each of the alpha subunit are embedded adjacent to each other in mirror images within the bilayer (figure 1) and carry out the simultaneous transport of H (or Na) and K across (3, 4). In addition to providing a mechanism for the simultaneous transport of ions the dual topology model (figure 1) has a non-hydrolysable ATP binding-site (5-7) accessible from the lumen that has been suggested (1, 2) to be involved in ion-channel modulation.

Figure 1: Dual topology model for the univalent cation transporting ATPase system showing bilayer orientation of the α and β subunits Two identical catalytic subunits, α1 and α2 (about 100 k. Da each), are shown in intimate communion as mirror images across the membrane bilayer with relevant embedded-ion channel in close contact, and are held together along with two closely associated β-subunits facing the lumen. The cytosolic ATP hydrolytic site (separate from the cis-pNPPase site at or near the ion channel) on the α1, and trans-cytosolic (luminal) low affinity (nonhydrolysable) ATP-binding site and corresponding trans-pNPPase site on the α2 are shown. The high affinity K site responsible for the ATPase stimulation is located across the bilayer on α2 (designated by half square) and the corresponding high affinity H or Na site (designated also by half square) is on the cytosolic side of the α1; both being present in a relatively hydrophilic environment. The low affinity ion binding sites responsible for the release of the transported ions from respective channels are present at or near the related channel of the α-domain on each side of the bilayer. These low affinity sites appear to be responsible for the observed K-pNPPase reactions related to the ion channel activity. Appropriate cooperative interactions between the two α subunits are essential for the ATPase mediated active cation transport although either the α1 or the α2 subunit can independently manifest the K-pNPPase reaction and the ATP binding process. It is apparent that each α-subunit holds its own built-in lipid-embedded ion channel made up of the corresponding transmembrane helixes arranged along the channel periphery that carry H (or Na) and K respectively in opposite direction across the bilayer. The process of active ion transport is mediated by a series of intra- and inter-subunit events within the ATPase complex that involve binding of ATP, binding of H (or Na) and K, resultant phosphorylation, K induced dephosphorylation, and simultaneous release of cations (H or Na) due to the regeneration of E. The activity of K-pNPPase appears unrelated to the ATPase reaction, yet somehow related to the ion channel function (Ray and Nandi, 1986). Precise nature and significance of the ATPase associated pNPPase activity remains to be elucidated. Binding of high affinity K to the designated α2-K-site across the membrane bilayer releases the proton produced from ATP hydrolysis mediated by the enzyme’s phosphorylated intermediates (E-P) and, in turn mediates the conformation-driven ion translocation. The conformational tension that is created by E-P within the lipid-embedded helix-domain of α1 would in turn induce changes in the adjoining (α2) helixes. Concurrently, the binding of high affinity K to its specific site on α2 would provide the free energy needed to release the existing molecular (E-P) tension with simultaneous dephosphorylation of the intermediate, thus making the energy available for the H and K ion-transport. The non-hydrolysable ATP binding site on α2 within the lumen is analogous to the high affinity ATP hydrolytic (α1) site in the cytosol that has been changed in nature due to the steric influence of the overlaying glycoproteins (β-subunits) and now appears to act as a channel facilitator for H (or Na).

Dual Topology of the MRAP:

Another dual topology protein of animal cell origin is the melanocortin-2 receptor accessory protein (MRAP) that is essential for trafficking the G-protein coupled melanocortin-2 (MC2) receptor to the adrenal cortex plasma membrane for ACTH binding. The MRAP is a single span transmembrane protein that is only active in a dual topology dimmeric form for correct trafficking. The following evidences establishing the dual topology of the MRAP have been reported (8, 9). First, epitopes on both the N- and C-terminal ends of MRAP are localized on the external face of CHO cells using antibodies raised against N- and C-terminal MRAP peptides. Second, about half of MRAP are glycosylated at the single endogenous N-terminal glycosylation site. Third, coimmunoprecipitation of differentially tagged MRAPs established that MRAP is a dimer. Finally, by selectively immunoprecipitating cell surface MRAP in one or the other orientation, it has been demonstrated that MRAP homodimers are antiparallel and form a stable complex with MC2 receptor.

Gene duplication as the basis of Dual Topology configuration: It may be noted in this connection that based on a comprehensive review of E. coli membrane proteins the dual topology model has been suggested to be a common occurrence in membrane function and has been suggested to evolve via a small number of steps through gene duplication (Rapp et al, 2007, Lolkema et al, 2008).

References

1.	Ray, TK and Nandi J (1986) K-stimulated pNPPase is not a partial reaction of the gastric (H, K)-transporting ATPase: Evidence supporting a new model for the univalent cation transporting ATPase systems, Biochem. J. 233, 231-238 2.	Nandi, J., Das, P. K., Levine, R. A. and Ray, T. K. (1988) Half of the Na, K-transporting ATPase-associated K-stimulated p-nitrophenyl phosphatase activity of gastric epithelial cells is exposed to the surface exterior. Biochem J. 252, 29-34 3.  Antolovic, R., Hamer, E.,  Serpersu, E. H., Kost, H., Linnertz, H., Kovarik,      Z. and Schoner, W (1999) Affinity labelling with MgATP analogues reveals coexisting Na+ and K+ forms of the {alpha}-subunits of Na+/K+-ATPase FEBS J. 261(1), 181 – 189 4.	Pauls, H.., Serpersu, E.H., Kirch, U. & Schoner, W. (1986) Chromium (III) ATP inactivating (Na++K+)-ATPase supports Na+-Na+ and Rb+-Rb+ exchanges in everted red blood cells but not Na+,K+-transport. Eur. J. Biochem. 157, 585–595 5. Thoenges, G. and Schoner, W. (1997) 2-O- Dansyl analogues of ATP bind with high affinity to the low affinity site of the Na, K-ATPase and reveal the interaction of two ATP sites during catalysis. J Biol Chem, 272, 16315-13321 65. 6. Ward, D. G. and Cavieres, J. D. (1998) Affinity labeling of two nucleotide sites on Na, K-ATPase using TNP-8N3-(alpha 32P) as a photo activatable probe  J Biol Chem. 273, 33759-33765 7. Ward, D.G. & Cavieres, J.D. (1996) Binding of 2’(3’)-O-(2,4,6-trinitrophenyl) ADP to soluble   protomers of Na,K-ATPase modified with isothiocyanate. Evidence for two distinct nucleotide binding sites. J. Biol. Chem. 271, 12317–12321 8. Sebag, J. A. and Hinkle, P. M. (2009) Region of melanocortin 2 (MC2) receptor accessory protein for dual topology and MC2 receptor trafficking and signaling. J. Biol. Chem. 284, 610-618 9. Sebag, J. A. and Hinkle, P. M. (2007) Melanocortin-2 receptor accessory  protein MRAP forms antiparallel homodimer PNAS 104, 20244-20249, 2007 10. Rapp, M., Seppälä, S., Granseth, E., von Heijne, G. (2007) Emulating membrane protein evolution by rational design. Science 315,1282-1284 11. Lolkema, J. S., Dobrowolski, A. and Dirk-Jan, S. (2008) Evolution of anti-parallel two-domain membrane proteins: Tracking Multiple Gene Duplication Events in the DUF606 Family. J. Mol. Biol. 378, 596-606