Structural Requirements of a Glycolipid MPIase for Membrane Protein Integration.

MPIase is a glycolipid involved in membrane protein integration in the inner membrane of Escherichia coli. To overcome the trace amounts and heterogeneity of natural MPIase, we systematically synthesized MPIase analogs. Structure-activity relationship studies revealed the contribution of distinctive functional groups and the effect of the MPIase glycan length on membrane protein integration activity. In addition, both the synergistic effects of these analogs with the membrane chaperone/insertase YidC, and the chaperone-like activity of the phosphorylated glycan were observed. These results verified the translocon-independent membrane integration mechanism in the inner membrane of E. coli, in which MPIase captures the highly hydrophobic nascent proteins via its characteristic functional groups, prevents protein aggregation, attracts the proteins to the membrane surface, and delivers them to YidC in order to regenerate its own integration activity.

Intermolecular Interactions between a Membrane Protein and a Glycolipid Essential for Membrane Protein Integration.

Inducing newly synthesized proteins to appropriate locations is an indispensable biological function in every organism. Integration of proteins into biomembranes in Escherichia coli is mediated by proteinaceous factors, such as Sec translocons and an insertase YidC. Additionally, a glycolipid named MPIase (membrane protein integrase), composed of a long sugar chain and pyrophospholipid, was proven essential for membrane protein integration. We reported that a synthesized minimal unit of MPIase possessing only one trisaccharide, mini-MPIase-3, involves an essential structure for the integration activity. Here, to elucidate integration mechanisms using MPIase, we analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate, the Pf3 coat protein, using physicochemical methods. Surface plasmon resonance (SPR) analyses revealed the importance of a pyrophosphate for affinity to the Pf3 coat protein. Compared with mini-MPIase-3, natural MPIase showed faster association and dissociation due to its long sugar chain despite the slight difference in affinity. To focus on more detailed MPIase substructures, we performed docking simulations and saturation transfer difference-nuclear magnetic resonance. These experiments yielded that the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles leading to interactions with the Pf3 coat protein. The high affinity of MPIase to the hydrophobic region and the basic amino acid residues of the protein was suggested by docking simulations and proven experimentally by SPR using protein mutants devoid of target regions. These results demonstrated the direct interactions of MPIase with a substrate protein and revealed detailed mechanisms of membrane protein integration.