Biologically Active Ultra-Simple Proteins Reveal Principles of Transmembrane Domain Interactions

Abstract

Specific interactions between the helical membrane-spanning domains of transmembrane proteins play central roles in the proper folding and oligomerization of these proteins. However, the relationship between the hydrophobic amino acid sequences of transmembrane domains and their functional interactions is in most cases unknown. Here, we use ultra-simple artificial proteins to systematically study the sequence basis for transmembrane domain interactions. We show that most short homopolymeric polyleucine transmembrane proteins containing single amino acid substitutions can activate the platelet-derived growth factor β receptor or the erythropoietin receptor in cultured mouse cells, resulting in cell transformation or proliferation. These proteins displayed complex patterns of activity that were markedly affected by seemingly minor sequence differences in the ultra-simple protein itself or in the transmembrane domain of the target receptor, and the effects of these sequence differences are not additive. In addition, specific leucine residues along the length of these proteins are required for activity, and the positions of these required leucines differ based on the identity and position of the central substituted amino acid. Our results suggest that these ultra-simple proteins use a variety of molecular mechanisms to activate the same target and that diversification of transmembrane domain sequences over the course of evolution minimized off-target interactions.

Keywords: BPV E5 protein; BaF3 cells; erythropoietin receptor; helix; traptamer.

Fig. 1.

PolyLeucine traptamers with amino acid substitutions at position 13 activate the PDGFβR. (a) Ultra-simple traptamer sequence in single-letter amino acid code where red “X” denotes one of the 20 standard amino acids, blue residues comprise the FLAG epitope tag, and green residues comprise a flexible linker. (b) C127 cells were infected with empty vector (MSCV) or retroviruses expressing each of the polyLeucine traptamers with different residues substituted at position 13. Cellular transformation was quantitated by counting foci after three weeks in culture. Results were normalized for viral titer and are shown as a percentage of positive control polyLeu-I13, plus and minus standard error of the mean. Significance from ordinary one-way ANOVA Dunnett’s multiple comparison test: ****, p<.0001, *, p<.05, ns=not significant for each sample compared to cells infected with MSCV. (c) Extracts were prepared from C127 cells expressing MSCV vector or an ultra-simple traptamer with the indicated amino acid at the 13^th position. Extracts were immunoprecipitated with anti-PDGFβR antibody and blotted for phosphotyrosine (Upper) or PDGFβR (Lower). M and P indicate mature and precursor forms of the receptor, respectively. (d) Extracts were prepared from HFFs expressing MSCV or an ultra-simple traptamer with the indicated amino acid at the 13^th position. Phospho-RTK array membranes were incubated with cell extracts, probed with anti-phosphotyrosine, and visualized by chemiluminescence. The paired spots representing PDGFβR are circled in red on all arrays. EGF receptor is circled in blue on the empty vector membrane. The dark pairs of spots in the corners are standards.

Fig. 2.

PolyLeucine traptamers with amino acid substitutions at position 13 activate the human and mouse EPORs. (a) BaF3 cells expressing the hEPOR, the hEPOR bearing the TMD of the mEPOR (hmh), or the hEPOR L238S TMD mutant were retrovirally infected to express ultrasimple traptamers with each of the 20 standard amino acids at position 13. The heat maps summarize the relative activity (left panels) and expression (right panels) of these traptamers. All values are shown as a percentage of expression of polyLeu-P13 (the highest value) in the case of expression levels, or as percentage of positive control (MSCV plus 0.06 units/ml EPO for EPORs; polyLeu-I13 for PDGFβR (PRβ)) in activity, where darker blues indicate values closer to the positive control, and white boxes indicate a value of zero or 20% or less in activity measurements. Primary data for hEPOR/hmhEPOR activity shown in Fig. S3. Activity in PDGFβR cells is the focus forming activity in C127 cells from Fig. 1B. (b) TMD sequences of the mouse PDGFβR, hEPOR, and mEPOR. Residues in red in the mEPOR sequence differ between mouse and human forms of the receptor.

Fig. 3.

Biochemical analysis of hEPOR activation by polyLeucine traptamers. (a) Extracts were prepared from BaF3 cells expressing the hEPOR together with MSCV, polyLeucine, or polyLeu with the indicated substitution at position 13. Lysates were subjected to SDS-PAGE and blotted for HA-tag on the hEPOR (input). Traptamers were immunoprecipitated using anti-FLAG magnetic beads and then electrophoresed and blotted as above (IP). Lysates were prepared in 1% Triton X-100 (left panel) or in 1% Triton X-100 (T) or in 1% NP40 (N) (right panel). (b) BaF3 cells expressing the hEPOR together with MSCV or different ultra-simple traptamers or positive control traptamer TC2–3 were starved of IL-3 for 3 hours. MSCV plus EPO sample was then acutely treated with 0.6 units/ml EPO for 5 min. Extracts were prepared and proteins were separated by SDS-PAGE and blotted for phosphorylated STAT5 (top panel), stripped, and reprobed for total STAT5 (bottom panel).

Fig. 4.

polyLeucine traptamers with an amino acid substitution at several individual positions activate the EPORs. (a) Heat maps show ability of ultra-simple traptamers to confer growth factor independence in cells expressing the hEPOR (left) or hmhEPOR (right). Numbers on the bottom indicate the position of the substitution within the polyLeucine stretch, and letters along the left indicate the amino acid present at the positions within the polyLeu TMD. All values are given as a percent of cells incubated in soluble EPO (0.06 units/ml). Darker values represent greater activity. Primary data provided in Fig. S3. An arbitrary cutoff was set at 20%. Activity was assessed by counting live cells four days after IL-3 removal. (b) IL-3 independence assay in BaF3 cells expressing hEPOR, mEPOR, or mEPOR S238L TM mutant together with MSCV, polyLeucine or polyLeu-G13. MSCV plus 0.06 units/ml was used as positive control. Activity was assessed by counting live cells four days after IL-3 removal. Significance from ordinary one-way ANOVA Dunnett’s multiple comparison test: ****, p<.0001 for each sample compared to MSCV in the absence of EPO. (c) Representative IL-3 independence assay in BaF3 cells expressing hEPOR, mEPOR, or mEPOR TM mutant L236V, S238L, or L239V together with MSCV, polyLeucine, polyLeu-G11, -Q11, -G13, -F10, or -D14. MSCV plus 0.06 units/ml EPO was used for positive control. Pictures of flasks show the relative growth after eight days without IL-3. Color change from pink to yellow reflects the pH in dense cell cultures, illustrating traptamer activity.

Fig. 5.

Identification of flanking leucines required for activity with hEPOR. (a) Relative activity of polyLeu-G13 and -Q13 and mutants with individual leucine-to-isoleucine mutations at every position in the polyLeucine stretch. Red leucines indicate positions where ability to induce IL-3 independence in BaF3/hEPOR cells was lost (<5% wild-type) for mutants containing a leucine-to-isoleucine mutation at that position. (b) The activity of wild-type traptamers is set at 100% in dark blue, and the activity of each mutant relative to wild-type is shown in shades of blue. White boxes represent a complete lack of activity. Numbers on bottom show absolute positions of leucine residues; numbers on top show positions relative to position 13. Activity was assessed by counting live cells four days after IL-3 removal in multiple independent experiments. (c) Helical wheel diagrams summarizing the results in panel (a) for a canonical alpha helix. Position 13 is in green, and pink dots represent required leucines that are located upstream of the substituted amino acid, while red dots represent those that are downstream. Red arcs denote the regions of the helix where required leucines are located.

Fig. 6.

Leucine-isoleucine mutagenesis of polyLeucine traptamers with glutamine or glycine at neighboring positions. (a) Red leucines indicate positions where the ability to induce IL-3 independence in BaF3/hEPOR cells was lost, as in Fig. 5a. Bottom panel shows sequences aligned according to glutamine. (b) Helical wheel diagrams as in Fig. 5c. (c) Activity of the indicated polyLeu traptamers with individual leucine-to-isoleucine mutations, relative to the cognate wild-type traptamer, as in Fig. 5b. Sequences are aligned to the glutamine or glycine, with numbers denoting position relative to glutamine or glycine. (d) Table showing the similarity in activity profiles for each pair of traptamers aligned to the glutamine or glycine. The difference at each position between a mutant with wild-type activity and one with no activity is set as 100. Numbers show difference in the activity averaged over all positions for the indicated pair. Results are also color-coded in shades of green where the most different pairs are darker, with completely identical patterns set at white.

Fig. 7.

Biochemical consequences of leucine-to-isoleucine mutations. (a) Cell extracts were prepared from BaF3 cells expressing the hEPOR together with MSCV, polyLeucine, polyLeu-Q10, or polyLeu-Q10 with leucine-to-isoleucine mutations at positions +1 to +7 relative to position 10. Co-IP was carried out as described in Fig. 3a. (b) Extracts were prepared from BaF3 cells expressing the hEPOR together with MSCV, polyLeucine, polyLeu-Q10, or polyLeu-Q10 with leucine-to-isoleucine mutations at positions +1 to +7. STAT5 phosphorylation was determined as described as in Fig. 3B. Activity indicates whether or not conditions confer IL-3-independent growth.