Ala‐insertion scanning mutagenesis of the glycophorin a transmembrane helix: A rapid way to map helix‐helix interactions in integral membrane proteins

Abstract

Alanine insertions into the glycophorin A transmembrane helix are found to disrupt helix-helix dimerization in a way that is fully consistent with earlier saturation mutagenesis data, suggesting that Ala-insertion scanning can be used to rapidly map the approximate location of structurally and/or functionally important segments in transmembrane helices.Keywords: a-helix; helix packing; membrane protein; mutagenesis According to the "two-stage" model for membrane protein assembly (Popot & Engelman, 1990), individually stable transmembrane a-helices develop during the initial insertion of a nascent chain into the membrane, and these preformed helices then pack together during the second stage to form the fully folded structure. Formation of the secondary structure (i.e., the transmembrane helices) and the tertiary structure (i.e., the tightly packed helix bundle) are thus effectively decoupled processes, with little feedback from the tertiary to the secondary structure level.Although the sequence characteristics that control the membrane insertion step are quite well understood (von Heijne, 1994), much less is known about the molecular interactions that drive helix-helix packing in a lipid environment. This is due both to the lack of good assays for helix-helix interactions, and to the difficulty of obtaining high-resolution structural information for membrane proteins.As an alternative to direct structure determination methods, molecular genetic approaches to the problem of helix-helix packing have been developed over the past few years (Popot & Saraste, 1995). In particular, saturation mutagenesis has been used to map the helix-helix interfaces in the glycophorin A (GpA) homodimer (Lemmon et al., 1992b) and in the phospholamban homopentamer (Arkin et al., 1994). In the case of GpA, the analysis is especially simple because the homodimer is stable at high temperatures even in detergents such as SDS (Lemmon ~.

Structure-based prediction of the stability of transmembrane helix–helix interactions: The sequence dependence of glycophorin A dimerization

“…Given that insertions within the motif must rotate and translate the residues that participate in the wild-type interface relative to one another, this class of mutants would not be expected to dimerize (22,23). In agreement with these expectations, our model suggests that insertion of one or two alanines after position 81 disrupts dimerization because of steric clashes (Fig.…”

“…Fig. 7 shows the excellent correlation between calc and the apparent free energy of dissociation, ⌬G app , for wild type and all 33 mutants reported by von Heijne and coworkers that bear up to four inserted residues (22,23). Like the examples in Fig.…”

“…If the agreement between calc and the experimental stabilities described above reflects the presence of chemical information in the empirical parameters, then the effects of multiple sequence changes might also be predicted by this model. A series of single and multiple alanine insertion mutations of GpA generated by von Heijne and coworkers (22,23) provides the opportunity to test this idea. We analyze the single alanine insertion variants (22) as well as all sequence variants in ref.…”

“…A series of single and multiple alanine insertion mutations of GpA generated by von Heijne and coworkers (22,23) provides the opportunity to test this idea. We analyze the single alanine insertion variants (22) as well as all sequence variants in ref. 23 having up to four residues inserted.…”

Transmembrane domains of the syndecan family of growth factor coreceptors display a hierarchy of homotypic and heterotypic interactions

“…1E), the SNSyn2 fusion migrates as Ͼ90% dimer with trace amounts of monomer, SNSyn3 migrates as Ϸ50% dimer, and SNSyn4 migrates as Ϸ60% dimer. [The SNGpA fusion forms Ϸ85% dimer under these conditions (30).] In contrast with the other SNase syndecan TMD fusions, SNSyn1 migrates as Ͼ90% monomer.…”