Studies On The Structure And Aggregation Of Transmembrane Domains Of Human Copper Transport1and On The Interactions Of Model Peptide With Lipid Bilayers

The SLC31family contains two members: copper transporter proteins andcopper transporting phosphorylated ATPases, and the latter includ ATP7A and ATP7Bproteins. At the protein level, two families of membrane proteins control cellularcopper uptake and secretion: copper efflux is governed by the ATP-dependent pumpsATP7A and ATP7B, whereas copper influx is mediated by the copper transporter (Ctr)proteins. The SLC31family and intracellular chaperone co-regulate intracellularmetabolic balance of copper ions. The copper transport protein of mammalian isessential for the embryonic development and the congenital absence will causediseases such as Menkes syndrome and Wilson etc.. Resent studies have confirmedthat the SLC31family not only mediates cellular uptake of copper, but also are linkedto the cellular uptake of Pt-based chemotherapeutic anticancer drugs like cisplatin.The genes encoding high affinity copper ion transport proteins were firstindentified in the plasma membrane by studies in yeast cells. The human high-affinitycopper transporter (hCtr1) was subsequently isolated by the functionalcomplementation of Ctr1-deficient yeast (Zhou and Gitschier et al.,1997). Thesecond human Ctr family member, hCtr2(SLA31A2), was isolated by sequencehomology to hCtr1, and the function of it may be the release of copper from internalcompartments in human cells. The hCtr1is comprised of190amino acids, includingthree putative transmembrane domains, an extracellular N-terminal region and anintracellular C-terminal region. Because of lacking high-resolution structuralinformation on the atomic level, until now the mechanisms of transporting copper andPt-containing drugs remain obscure.In our thesis, we firstly used the CD and NMR spectral methods to study thestructure of the three transmembrane domains (TMD) from hCtr1protein, respectively, and try to characterize the aggregation of the three transmembrane domains bySDS-PAGE and FRET methods. The CD results showed that the secondary structuresof the three transmembrane domains in both40%HFIP/60%H₂O and POPC lipidvesicles are similar. We determined the three dimensional structure andoligomerization of the transmembrane domains in40%HFIP/60%H₂O usingsolution-state NMR spectroscopy. And firstly revealed that TMD1forms an-helicalstructure from Gly67to Glu84and is dimerized by close packing of its C-terminalhelix; TMD2forms an amphiphilic-helical structure from Leu134to Thr155and thepolar residues including the residues (His, Ser, Met) that may be important forfunction lie in the same face of the helix, the other face occupied by non-polarresidues (Leu,Val,Phe). The amphiphilic structure may be related to the spatialstructure formation of the hCtr1protein, ion selection and transferring in the processof transporting or regulation of ion channel switch. The chemical shifts of HN protons(HN) in peptides and DOSY experiments demonstrated that the trimerization ofTMD2is induced by the interactions of the apolar residues in the region from thecenter (Ile11) to the C-terminal end (Met24), therefore, the pore size at the C-terminalend of trimer (extracellular end of TMD2in hCtr1) is smaller than that of theN-terminal end. The aggregate result is complied with the previous cryo-EM results.The TMD3adopts a discontinuous helix structure, known as―-helix-coiledsegment--helix‖, and is dimerized by the interaction between the N-terminal parthelices. The motif GxxxG in TMD3is not fully involved in the helix, but partiallyunstructured between helices as a linker.The SDS-PAGE results suggest that the second transmembrane domain haveintense propensity to aggregate than the first and the third transmembrane domains.The experiments also display that the aggregation of TMD2depends on theconcentration in solution, which is different from the TMD1and TMD3. In the FRETexperiments with lower concentration of peptides (5.5μМ), all the threetransmembrane domains exist as monomer. The intense propensity of aggregation, thedenpendence of aggragation on the concentration and the amphiphilic structure maymake TMD2important in aggregation of hCtr1protein. Different spatial structure and aggregation ability may have three transmembranedomains different both in trimeric assembly and the role playing in process of iontransporting. The hCTR1forms cone-shaped pore by the packing of TMD2helices inthe inner and by close contact between TMD1and TMD3from different subunit ofhCtr1. The pore size may be regulated by the relative orientation of TMD2heliceslining the inner of the pore, which could induce a cooperative change in theorientation of TMD3helices because the loop between TMD2and TMD3is veryshort (only3residues). The flexible linker between the helices of TMD3accommodates a partial change of the N-terminal half helix in the orientation.However, the detailed mechanism by which the TMDs play roles in the switch of thehCtr1channel is still unknown.Studies have confirmed that the hydrophobic mismatch between the hydrophobiclength of transmembrane peptide and hydrophobic thickness of lipid membrane mayinduce the changes in membrane protein orientation or tilt angle. In another part ofthis thesis, we combined fluorescence, CD and ATR-IR spectroscopic methods toinvestigate the behaviors of the peptide and lipids under hydrophobic mismatch usinga model peptide from the fourth transmembrane domain of Nramp1, thephosphatidylcholines (PCs) and phosphatidylglycerols (PGs) with different lengths ofacyl chains (14:0,16:0and18:0). The experimental results show that in all PG lipidmembranes, the peptide forms stable-helix structure and the helix axis is parallel tolipid chains. The helical span and orientation are nearly unchanged in varyingthickness of PG membranes, while the lipid chains can deform to accommodate thehydrophobic surface of embedded peptide. In contrast, the helical structures of themodel peptide in PC lipid membranes are less stable. Upon incorporation with PClipid membranes, the peptide can deform itself to accommodate the hydrophobicthickness of lipid membranes in response to hydrophobic mismatch. In addition,hydrophobic mismatch can increase aggregation propensity of the peptide in both PCand PG lipid membranes and the peptide in PC membranes has more aggregationtendency than in PG membranes.The structural studies of hCtr1-TMDs in our thesis may be meaningful to understand the transporting mechanism of the entire hCtr1protein, and the study onthe hydrophobic mismatching will be helpful to better understanding of the interactionof membrane with transmembrane peptides.