Abstract
Salt bridges in lipid bilayers play a decisive role in the dynamic assembly and downstream signaling of the natural killer and T-cell receptors. Here, we describe the identification of an inter-subunit salt bridge in the membrane within yet another key component of the immune system, the peptide-loading complex (PLC). The PLC regulates cell surface presentation of self-antigens and antigenic peptides via molecules of the major histocompatibility complex class I. We demonstrate that a single salt bridge in the membrane between the transporter associated with antigen processing TAP and the MHC I-specific chaperone tapasin is essential for the assembly of the PLC and for efficient MHC I antigen presentation. Molecular modeling and all-atom molecular dynamics simulations suggest an ionic lock-switch mechanism for the binding of TAP to tapasin, in which an unfavorable uncompensated charge in the ER-membrane is prevented through complex formation. Our findings not only deepen the understanding of the interaction network within the PLC, but also provide evidence for a general interaction principle of dynamic multiprotein membrane complexes in immunity.
Highlights
Understanding of the function of the endoplasmic reticulum (ER)-lumenal domains, we have to date only a limited knowledge of the molecular recruitment mechanism of tapasin into the peptide-loading complex (PLC)
We generated a set of 16 single-cysteine mutants of TMD0TAP1 with four consecutive mutations in the middle region of each of the four predicted transmembrane helices (TMs) of Cys-less TAP, which is fully functional in antigen translocation and major histocompatibility complex class I (MHC I) loading[22]
In the absence of tapasin, a highly stable intra-molecular salt bridge is formed between D32 of TM1 and R64 of TM2, stabilizing the negative charge buried in the hydrophobic membrane environment. This interaction is broken and replaced by a new, inter-molecular salt bridge between D32 and tapasin K428 (Supporting Movie S1). We propose that this ionic lock-switch mechanism fulfills two functions: the D32/R64 salt bridge stabilizes individual TMD0 interaction hubs in the absence of tapasin (Fig. S5), whereas the D32 TMD0TAP1 / K428 tapasin interaction is crucial for the specific recruitment of tapasin
Summary
Understanding of the function of the ER-lumenal domains, we have to date only a limited knowledge of the molecular recruitment mechanism of tapasin into the PLC. Despite sharing only 17% sequence identity[14,15], both TMD0s adopt a four-helix topology[16,17]. Both domains fulfill the same function and are targeted to the ER membrane independently of the coreTAP transporter, consisting of the remaining 2 × 6 transmembrane helices (TMs), each followed by a nucleotide-binding domain[18]. Despite the pivotal role of the TMD0TAP1/2 in PLC assembly, their atomic structures and interaction sites for tapasin binding are not yet determined. A few residues in its TMD were described to be involved in TAP binding[19,20]. Molecular modeling and all-atom molecular dynamics (MD) simulations provide information on the structural organization of this essential protein-protein interface in the membrane
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