Spectral Study On The Structure And Orientation Of Slcllal Transmembrane Domains In Lipid Membranes

Slcll family (also named as Nramp), which is a highly conserved transmembrane protein family, has been proved to play an important role in maintaining homeostasis of divalent metal ions from bacteria to human. They can transport a broad range of divalent metal ions including Zn2+,Ni2+,Pb2+,Mn2+,Cu2+ Cd2+and Co2+. This protein family has two members in mammals, Slcllal and Slclla2, which share 64% amino acid sequence identity and 78% similarity. Disabled Slcllal causes macrophages sensitive to several intracellular pathogens (such as pulmonary tuberculosis and leprosy) and defective Slclla2 results in anemia.We investigated the structures and transmembrane topologies of the transmembrane segments 1-5 of Slcllal in phospholipid membranes using CD, fluorescence and ATR-FTIR spectroscopic methods. We studied effects of the headgroup charge, the state of lipid chains (gel or liquid cryctalline phase) and cholesterol on the structure, orientation and location of these peptides in lipid membranes. On the basis of the orientation information of helices 1-5. one may suppose the orientations of corresponding segments in helices 6-10 according to the symmetric model of two domains. This study will shed some light on the three dimensional structure and transmembrane topology of the highly conserved core of Slcllal in biological membranes.All TMDs form a helix structure when they were bound to POPG. The helical contents of TMD1-5 in POPG were 27%,51%,37%,54% and 50%. respectively. The a-helices of these TMDs are shortened with increasing amount of POPC component in lipid membrane. Whereas the helix is still a predominant structure in TMD2, TMD4 and TMD5; it is no longer dominant in TMD3 and even lesser in TMD1. When a value of 27°was used as the angle between the C=O dipole and the helix axis, the order parameters of helices, SH, are 0.69,0.21, 0.57 and 0.75 for POPG-bound TMD2-TMD5, respectively, which corresponds to the a-helices tilting relative to the normal of the germanium plate at the angles of 27°,47°.32°and 24°. respectively. Of these helices of TMDs in POPG membrane, TMD2. TMD4 and TMD5 are less tilted relative to membrane normal and TMD3 is much more tilted. The order of tilted angles of these peptides is TMD3>>MD4>TMD2>TMD5. Larger tilted angle of TMD3 helix in POPG membrane may be attributed to the deprotonation of the side chain of Glul2 in TMD3. With increasing POPC component, the tilted angles of TMD2 and TMD4 increased and the tilted angle of TMD5 changed, too. while their tilted angles was still in the order of TMD4>TMD2>TMD5.We depicted Slcllal structural model in POPG based on their structures and orientations obtained by the CD and ATR results. This model is different from Courville et.al in two points:the first one is the relative helix lengths of these peptides. An order of TMD3≈TMD2>TMD5>TMD4>TMD1 was predicted by Courville et.al model, while the length order obtained by our CD results was TMD4≥TMD2≥TMD5>>TMD3>TMD1, the helix length of TMD3 is far shorter than that of Courville et.al model; the second one is the relative orientation angle. The ATR results showed that the orientation angles of four helix are in an order of TMD3>>TMD4>TMD2>TMD5. while the order was TMD3>>TMD2≈TMD5>TMD4 in Courville et.al model.Different from POPG and POPC membranes in liquid crystal phase, lipids DPPG and DPPC form membranes of gel state. In DPPG and DPPC membranes, helix length of TMD1-5 were shortened sharply compared with the cases in POPG and POPC membranes (except for TMD4 in PG lipids). The a-helical structures of TMD1 and TMD3 almost disappeared. Even though, there are still more a-helical contents in negatively charged DPPG for TMD2,TMD4 and TMDS. In DPPG, the tilted angle of TMD2 and TMD4 are 21°and 27°, respectively and TMD5 are even less tilted. They are less tilted in the gel state than in liquid crystal lipids. Their relative order of the orientation angles, which may be determined by their intrinsic characteristics, were not changed, still in an order of TMD4>TMD2>TMD5 in gel phase lipid.The state of the lipid chains can affect the secondary structure and orientation of these peptides. Lipids in liquid crystal phase do much favor for these transmembrane peptides for forming a-helical structure, but can decrease their ordering in membranes. The relative order of the tilted angles was maintained because they were changed nearly in the same way. This suggests that compared with the state of the lipids, the intrinsic characteristics of primary structure of TMDs were more important in determining their relative orientation. The state of the lipid chains has little effect on the location of TMD1-4 in lipid membranes. Besides, the properties of lipid head groups can affect the secondary structure and orientation of TMDs. Compared with lipids with zwitterionic head groups (POPC,DPPC), negative charged head groups (POPG,DPPG) are more favorable for the formation of helix for all the TMDs, and in most cases, the tilted angle of helix increased in the presence of negatively charged lipids.For TMD2 and TMD4 in DPPG and DPPC lipid membranes, the presence of cholesterol reduced peptide a-helix contents, while for TMD5 cholesterol has little effect on helicity of the peptide. Among these TMD peptides, TMD4 demonstrated a dramatic decrease in the a-helix content in DPPC membrane along with an increase in helix ordering in the presence of cholesterol, implying that TMD4 may interact with cholesterol. This may be associated to the specific sequence of the transmembrane domain that includes a cholesterol recognition amino acid consensus in the C-terminus (L/V-(X)(1-5)-Y-(X)(1-5)-R/K). The interaction between cholesterol and the consensus sequence of TMD4 may result in the reduction of TMD4 in helicity and tilted angle.