Specificity in Transmembrane Helix-Helix Interactions Mediated by Aromatic Residues

Neta Sal-Man,Doron Gerber,Itai Bloch,Yechiel Shai

doi:10.1074/jbc.m610368200

Neta Sal-Man, Doron Gerber + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m610368200

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Abstract

Aromatic residues have been previously shown to mediate the self-assembly of different soluble proteins through pi-pi interactions (McGaughey, G. B., Gagne, M., and Rappe, A. K. (1998) J. Biol. Chem. 273, 15458-15463). However, their role in transmembrane (TM) assembly is not yet clear. In this study, we performed statistical analysis of the frequency of occurrence of aromatic pairs in a bacterial TM data base that provided an initial indication that the appearance of a specific aromatic pattern, Aromatic-XX-Aromatic, is not coincidental, similar to the well characterized QXXS motif. The QXXS motif was previously shown to be both critical and sufficient for stabilizing TM self-assembly. Using the ToxR system, we monitored the dimerization propensities of TM domains that contain mutations of interacting residues to aromatic amino acids and demonstrated that aromatic residues can adequately stabilize self-association. Importantly, we have provided an example of a natural TM domain, the cholera toxin secretion protein EpsM, whose TM self-assembly is mediated by an aromatic motif (WXXW). This is, in fact, the first evidence that aromatic residues are involved in the dimerization of a wild type TM domain. The association mediated by aromatic residues was found to be sensitive to the TM sequence, suggesting that aromatic residue motifs can provide a general means for specificity in TM assembly. Molecular dynamics provided a structural explanation for this backbone sequence sensitivity.

Highlights

To date, the non-covalent association of native TM domains was reported to be mediated by (i) a heptad motif of leucines through their side chain residues packing interaction [6]; (ii) a GXXXG motif, which was first found in the glycophorin A TM domain [4, 8, 9]; or (iii) polar residues through the formation of hydrogen bonds (10 –14)
We performed a statistical analysis that revealed that an Aromatic-XX-Aromatic pattern is over-represented in a bacterial TM data base
We have examined whether replacement of one or both polar residues by aromatic residues will support dimerization

Summary

Aromatic Residues Mediate TM Interactions

WXXW, YXXY, and FXXF patterns were performed on a bacterial TM domain data base. The source of the TM sequences was the annotated non-redundant data base Swiss-Prot updated to February 2005. The data base contains 41,916 bacterial TM domains with lengths ranging between 15 and 30 amino acids. Construction of the ToxR Chimeras—A NheI-BamHI TMDNA cassette encoding 16 residues of the Tar-1 wild type (WT) TM domain (13MVLGVFALLQLISGSL28) or the EpsM WT TM domain (26MGALTVLAIAYWGIWQ41) were inserted between the ToxR transcription activator and the E. coli maltose binding protein MalE within the ToxR-MalE plasmid. A Tar-1 TM domain encoding the DNA cassette was grafted between the ToxR transcription activator and the maltose binding protein in the ToxR-MalE plasmid. For each TM dimer, the 100 lowest energetic structures were subjected to a simulated annealing protocol from 500,000 to 300,000 in 50 steps (each step lasts 100 fs) This was followed by 1000 steps of energy minimization. The structure with the minimal calculated energy from the lower energetic cluster was selected as the final structure of each TM sequence for further analysis

RESULTS

Designation of TM domain

Name of inserted TM domain

DISCUSSION

Ser from the dimerization motif

These results demonstrate the ability of aromatic residues to