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

Abstract

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.

Role of a bacterial glycolipid in Sec-independent membrane protein insertion

“…S11 ). In a previous study, we observed that the interaction rate between mini-MPIase and Pf3 coat protein were accelerated when mini-MPIase-3 was reconstructed in DAG-containing EPL LUVs, suggesting that it assembles on the membrane cooperatively 18 . We also observed that mini-MPIase-3 electrostatically interacts with DAG on the membranes and inhibited the flip-flop motion of DAG 21 .…”

“…Recently, we demonstrated the importance of a pyrophosphate linker in MPIase for affinity with Pf3 coat protein in an aqueous environment by surface plasmon resonance (SPR) analyses and docking simulations 18 . These results suggest that the pyrophosphate linker of MPIase is one of the key structures in membrane insertion activity.…”

“…6 ). The flexible long sugar chains interact with the protein and prevent protein aggregation after a substrate protein is released from the ribosomal tunnel 8 , 10 , 18 . The double negative charge of the pyrophosphate linker part of MPIase strongly attracts the basic amino acid residues at the membrane surface and immediately delivers the protein into the membrane.…”

“…Local thinning of lipid bilayers due to short transmembrane domains of YidC and/or membrane potential enable the hydrophilic regions of the protein to cross the bilayer 11 , 15 – 17 . Our recent study revealed that the protein released from the ribosome is captured by sugar chains of MPIase and attracted to the membrane surface through the pyrophosphate group 18 . In the presence of MPIase, some hydrophobic proteins such as the 3L-Pf3 coat, a mutant version of bacteriophage Pf3 coat protein, are inserted into the membrane without YidC, while YidC accelerates the insertion in combination with MPIase 19 , 20 .…”

Structural Requirements of a Glycolipid MPIase for Membrane Protein Integration

“…[15] DS studies deduced that the GlcNAc 6-OAc and NHAc groups interact with the hydrophobic amino acid residues of the substrate protein, and that pyrophosphate and uronate interact electrostatically with basic amino acids. [15] In this study, the importance of the GlcNAc 6-OAc and carboxy groups was experimentally proven using trisaccharide analogs. Since the effect of individual functional groups was weak, alteration of one functional group did not completely abolish activity.…”