Predicting the Role of a Single Amino Acids in the Dimerization of Transmembrane Helices

Abstract

Single-span membrane proteins take part in biological processes. In many cases their function has been shown to require dimerization via their transmembrane domains (TMDs). Research into the dimerization of TMDs has revealed sequence dependence, driven by a combination of amino acids. The helix interactions of multi-pass membrane proteins are well understood, as analysis of crystal structures shows that helix interfaces tend to contain small, polar and highly conserved residues. Unfortunately, for single-pass proteins structural data is scarce.The aim of this study is to characterize interfaces of TMDs that show strong self-interaction. In one study of this kind, we examine amino acid composition and conservation in order to identify patterns characteristic of interface residues. Our aim is to develop a method to predict the TMD interfaces from sequences alone.We used the ToxR assay in Escherichia coli to measure TMD homodimerization. In this assay, dimerization driven by TMDs results in the transcriptional activation of ctx promoter, inducing the expression of a reporter enzyme. The amount of reporter enzyme activity is a measure of the strength of the dimerization.Here, we present the scanning mutagenesis followed by ToxR analysis of 10 TMDs with strong self-interaction. Mutations were made to all amino acids within the TM helix. Some TMD interfaces were found to contain sequence patterns previously associated with parallel helix interactions, such as the GxxxG motif. However, in other cases motif searching would not have identified the interface, and the key residues in the interactions were diverse. The conservation of each amino acid in each TMD was calculated based on amino acid alignments against homologs. Preliminary analysis shows that amino acid conservation is a good indicator for the interface of some, but not all interacting TMDs.