Self-Interacting Transmembrane Helices from the Human Single-Pass Membrane Proteome: The Impact of Primary Structure and Lipids on Affinity and Stoichiometry

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Single-pass membrane proteins comprise a functionally diverse set of proteins that corresponds to >10% (~2200 proteins) of the human proteome. Many of these proteins are already known to homomerize via transmembrane domains (TMDs). The underlying helix-helix interfaces are frequently built from complex motifs formed from different types of amino acids. It is currently not known how many different types of interfacial amino acid motifs exist and how they relate to the respective protein quaternary structure and function. Our recently published comprehensive bioinformatic analyses of the entire human single-pass membrane proteome that were connected to experimental assessments of TMD-TMD interactions can briefly be summarized as follows. First, based on TMD sequence homology, a significant fraction (13.5 %) of the TMDs can be grouped into clusters mostly consisting of paralogs. Some of these clusters are characterized by high-affinity TMD self-interaction not reported before. Second, hundreds of TMD helices exhibit non-random one-sided residue conservation that is useful in predicting correlated homotypic interactions.In this follow-up application, we will exploit these previous results. In Goal 1, we plan to apply scanning mutagenesis and the in vivo ToxR assay to comprehensively investigate the sequence specificity and the role of amino acid motifs in TMD-TMD interaction. Besides expanding the list of high-affinity TMDs, bioinformatic analyses of the results will reveal the connection between the evolutionary conservation of amino acids and their role in the helix-helix contacts. In Goal 2, we will develop a novel in vitro homoFRET assay to determine the stoichiometry of interaction for these TMDs, and investigate the influence of positively charged flanking residues and negatively charged lipids in homodimerization. By combining both in vivo and in vitro assays in this comparative study, we hope to make broad conclusions regarding some of the predominant questions in the field.