Specific chemical modification explores dynamic structure of the NqrB subunit in Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae

摘要

The Na+-pumping NADH-ubiquinone oxidoreductase (Na+-NQR) is a main ion transporter in many pathogenic bacteria. We previously proposed that N-terminal stretch of the NqrB subunit plays an important role in regulating the ubiquinone reaction at the adjacent NqrA subunit in Vibrio cholerae Na+-NQR. However, since approximately three quarters of the stretch (NqrB-Met(1)-Pro(37)) was not modeled in an earlier crystallographic study, its structure and function remain unknown. If we can develop a method that enables pinpoint modification of this stretch by functional chemicals (such as spin probes), it could lead to new ways to investigate the unsettled issues. As the first step to this end, we undertook to specifically attach an alkyne group to a lysine located in the stretch via protein-ligand affinity-driven substitution using synthetic ligands NAS-K1 and NAS-K2. The alkyne, once attached, can serve as an "anchor" for connecting functional chemicals via convenient click chemistry. After a short incubation of isolated Na+-NQR with these ligands, alkyne was predominantly incorporated into NqrB. Proteomic analyses in combination with mutagenesis of predicted target lysines revealed that alkyne attaches to NqrB-Lys(22) located at the nonmodeled region of the stretch. This study not only achieved the specific modification initially aimed for but also provided valuable information about positioning of the nonmodeled region. For example, the fact that hydrophobic NAS-Ks come into contact with NqrB-Lys(22) suggests that the nonmodeled region may orient toward the membrane phase rather than protruding into cytoplasmic medium. This conformation may be essential for regulating the ubiquinone reaction in the adjacent NqrA.