Abstract
Interactions that facilitate transmembrane domain (TMD) dimerization have been identified mainly using synthetic TMDs. Here, we investigated how inherent properties within natural TMDs modulate their interaction strength by exploiting the sequence variation in the nine neuraminidase subtypes (N1-N9) and the prior knowledge that a N1 TMD oligomerizes. Initially, consensus TMDs were created from the influenza A virus database, and their interaction strengths were measured in a biological membrane system. The TMD interactions increased with respect to decreasing hydrophobicity across the subtypes (N1-N9) and within the human N1 subtype where the N1 TMDs from the pandemic H1N1 strain of swine origin were found to be significantly less hydrophobic. The hydrophobicity correlation was attributed to the conserved amphipathicity within the TMDs as the interactions were abolished by mutating residues on the polar faces that are unfavorably positioned in the membrane. Similarly, local changes enhanced the interactions only when a larger polar residue existed on the appropriate face in an unfavorable membrane position. Together, the analysis of this unique natural TMD data set demonstrates how polar-mediated TMD interactions from bitopic proteins depend on which polar residues are involved and their positioning with respect to the helix and the membrane bilayer.
Highlights
Transmembrane domain (TMD) interactions in bitopic proteins are less understood than in multispanning proteins
The hydrophobicity correlation was attributed to the conserved amphipathicity within the transmembrane domain (TMD) as the interactions were abolished by mutating residues on the polar faces that are unfavorably positioned in the membrane
The three potential outlier TMDs that had relative interaction strengths above the linear threshold (ϳ80%) were analyzed separately at lower IPTG levels (0.02 mM) to show that the correlation was still valid (Fig. 2B, inset). These results indicate that the TMD interaction strength across the different neuraminidase subtypes in influenza A viruses (IAVs) is driven by their polar characteristics, making them inversely proportional to their hydrophobicity
Summary
Transmembrane domain (TMD) interactions in bitopic proteins are less understood than in multispanning proteins. It has been quantitatively demonstrated how each of the 20 amino acids vary in their contribution to membrane insertion based on their positioning in the membrane bilayer [31, 32] We combined these two principles to investigate whether a correlation exists between the strength of TMD oligomerization and their membrane integration properties using the large number of natural neuraminidase TMDs from the IAV database. It was determined that the neuraminidase TMD interactions are dictated by their polar properties and this correlated with a conserved amphipathic pattern of two adjacent polar faces within the TMDs (except for those from N2) Using this information, the polar residues responsible for the interactions were identified based on their location within the TMD helix and how favorable their positioning is in the membrane with respect to integration. These results provide the first example of a predictable TMD interaction strength for a protein and put polar mediated TMD interactions in the context of their polar residue positioning within the helix and the membrane bilayer
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