Abstract
The first transmembrane (TM1) helix in the red cell anion exchanger (AE1, Band 3, or SLC4A1) acts as an internal signal anchor that binds the signal recognition particle and directs the nascent polypeptide chain to the endoplasmic reticulum (ER) membrane where it moves from the translocon laterally into the lipid bilayer. The sequence N-terminal to TM1 forms an amphipathic helix that lies at the membrane interface and is connected to TM1 by a bend at Pro403. Southeast Asian ovalocytosis (SAO) is a red cell abnormality caused by a nine-amino acid deletion (Ala400–Ala408) at the N-terminus of TM1. Here we demonstrate, by extensive (∼4.5 μs) molecular dynamics simulations of TM1 in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane, that the isolated TM1 peptide is highly dynamic and samples the structure of TM1 seen in the crystal structure of the membrane domain of AE1. The SAO deletion not only removes the proline-induced bend but also causes a “pulling in” of the part of the amphipathic helix into the hydrophobic phase of the bilayer, as well as the C-terminal of the peptide. The dynamics of the SAO peptide very infrequently resembles the structure of TM1 in AE1, demonstrating the disruptive effect the SAO deletion has on AE1 folding. These results provide a precise molecular view of the disposition and dynamics of wild-type and SAO TM1 in a lipid bilayer, an important early biosynthetic intermediate in the insertion of AE1 into the ER membrane, and extend earlier results of cell-free translation experiments.
Highlights
The anion exchanger AE1 is an abundant glycoprotein in the plasma membrane of the red cell where it mediates the electro-neutral exchange of chloride and bicarbonate ions.[1−3] Human protein contains 911 amino acids with a single N-glycosylation site at Asn[642]
transmembrane segment 1 (TM1) moves laterally into the lipid bilayer after synthesis of TM2 or after synthesis of TM2 and -3, which are transiently in the endoplasmic reticulum lumen followed by TM4 that acts as a stop-transfer sequence. (D) Structure of this sequence in the AE1 crystal structure, consisting of a cytosolic helix H1 connected by a sharp bend to the TM1 helix that begins at P403 and terminates at T431 with a slight kink at Pro419. (E) The ensemble of 21 wild-type structures of residues 389−430 resolved by nuclear magnetic resonance has a high degree of structural variability, especially in their N- and C-terminal regions
A single transmembrane helix, such as TM1 of AE1, is likely to be more dynamic on its own in a lipid bilayer, as will be the case during biosynthesis, than when confined within the full transmembrane protein. This variation in conformation sampled by the sequence Arg384−Lys[430], which includes TM1, makes it well-suited to being studied by molecular dynamics simulation
Summary
Hydrophobicity plot analyses suggest that TM1 begins at Val[405] and extends to Phe[423], creating a 19-amino acid hydrophobic segment long enough to span the hydrophobic core of a lipid bilayer as an α-helix.[8]. The GROMOS53a6 atomistic force field was used.[46] A short 0.1 ns molecular dynamics simulation with the position of the protein restrained was run before a 10 ns unrestrained molecular dynamics simulation Both simulations used an integration time step of 2 fs with the lengths of all bonds involving a hydrogen restrained using the LINCS algorithm. Simulations were not included in the final analysis either because they failed to complete the pipeline, usually because the conversion back to atomistic coordinates was not successful, or because the sequence did not adopt a transmembrane orientation This was defined as the sequence having Cα atoms 1.4 nm above and below the midplane of the bilayer at the end of the selfassembly process. Graphs were plotted using gnuplot, and all images were rendered using VMD
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