Interaction patterns of methoprene-tolerant and germ cell-expressed Drosophila JH receptors suggest significant differences in their functioning

Methoprene-tolerant (Met) and germ cell-expressed (Gce) proteins were shown to be juvenile hormone (JH) receptors of Drosophila melanogaster with partially redundant functions. We raised the question of where the functional differentiation of paralogs comes from. Therefore, we tested Met and Gce interaction patterns with selected partners. In this study, we showed the ability of Gce and its C-terminus (GceC) to interact with 14-3-3 in the absence of JH. In contrast, Met or Met C-terminus (MetC) interactions with 14-3-3 were not observed. We also performed a detailed structural analysis of Met/Gce interactions with the nuclear receptor fushi tarazu factor-1 (Ftz-F1) ligand-binding domain. We showed that GceC comprising an Ftz-F1-binding site and full-length protein interacts with Ftz-F1. In contrast to Gce, only MetC (not full-length Met) can interact with Ftz-F1 in the absence of JH. We propose that the described differences result from the distinct tertiary structure and accessibility of binding sites in the full-length Met/Gce. Moreover, we hypothesize that each interacting partner can force disordered MetC and GceC to change the structure in a partner-specific manner. The observed interactions seem to determine the subcellular localization of Met/Gce by forcing their translocation between the nucleus and the cytoplasm, which may affect the activity of the proteins. The presented differences between Met and Gce can be crucial for their functional differentiation during D. melanogaster development and indicate Gce as a more universal and more active paralog. It is consistent with the theory indicating gce as an ancestor gene.

Tables' abbreviations: nucl -nucleus, indir dim -indirect dimension, sw -spectral width, sgs -sampling grid size, ni -number of non-uniform sampling complex points (randomly chosen from the sampling grid).The MetC chemical shifts were deposited in the BMRB [65], under the accession number 51720.Tables' abbreviations: nucl -nucleus, indir dim -indirect dimension, sw -spectral width, sgs -sampling grid size, ni -number of non-uniform sampling complex points (randomly chosen from the sampling grid) -for non-uniform sampling experiment (HNCO only), conv exp -conventional experiment: all grid points were collected.

Table S4. Sequences of primers used in PCR
The primers used for protein cDNA amplification introduces restriction site sequences for the selected endonucleases (underlined in the primer sequences).The upper-case letters in the primer sequence represent the sequence present in the protein sequence.The reverse primers for Met, Gce, MetC, and GceC introduced the C-terminal FLAG protein sequence (DYKDDDDK, marked in blue).

Figure S2. P-GceC dephosphorylation.
SDS-PAGE analysis of the P-GceC digested with phosphatase for 2h.Lane 1, not digested P-GceC; lane 2, protein after digestion with phosphatase for 1h; lane 3, protein after digestion with phosphatase for 2h; lane 4, molecular mass standards.The upper band, with reduced mobility, refers to a phosphorylated protein (P-GceC), while the lower band refers to non-phosphorylated GceC.Arrows indicate P-GceC, GceC and phosphatase.

Figure S1 .
Figure S1.Met and Gce sequences alignment.In contrast to bHLH and PAS domains (marked with pink and blue) presenting high sequence homology (78% for bHLH, 68% for PAS-1, and 86% for PAS-2)(Moore et al., 2000), C-termini of Met and Gce (MetC and GceC, marked with red) are highly differentiated.Specific sequences localized in MetC and GceC are marked: NRbox with purple; Met QR sequence with grey, and Gce NLS with yellow.The alignment is based on the SIM server(Huang and  Miller, 1991).

Figure S3 .
Figure S3.MetC assignment.The 2D 15 N-HSQC spectrum of MetC presents features typical for IDPs with a very narrow chemical shift range (8-8.6 ppm in proton dimension).Most of observed signals strongly overlap.Single more dispersed signals (like 50GLU, 68GLU, 149VAL, 210ARG) may correspond to aa residues involved in the formation of local and transient motifs of the secondary structure (like MoREs).Assigned aa residues are indicated and signed with residue type and position in the protein sequence.The buffer used for MetC NMR analysis was 20 mM sodium phosphate pH 6.8, 50 mM NaCl, 1 mM TCEP, 0.5 mM EDTA.

Figure S4 .
Figure S4.GceC chemical shift perturbation NMR spectrum.A) GceC NMR reference spectrum (blue) and GceC spectrum obtained in the presence of equimolar concentration of Ftz-F1 LBD (red)(Kolonko et al., 2020).Specific aa shifts are observed.B) P-GceC NMR reference spectrum (blue) and P-GceC spectrum obtained in the presence of equimolar concentration of14- 3-3 (red).Specific aa shifts are observed.The differences between two partners are indicated with green circles.The buffer used for GceC NMR analysis was PBS pH = 7.4, 2 mM DTT.

Figure S5 .
Figure S5.ITC profiles for Met PEP binding to Ftz-F1 LBD.The top panel shows the baseline-subtracted thermograms of two independent measurements (wine and blue lines).The bottom panel represents the binding isotherms together with errorresiduals.Data were fitted to independent model A + B ⇌ AB using global fitting mode.All measurements were obtained in 20 mM HEPES buffer, pH 6.8, 150 mM NaCl, and 1 mM TCEP.

Figure S6 .
Figure S6.Pull-down experiment controls.COS-7 cells were transfected with a vector encoding one protein pulled down with ANTI-FLAG M2 Affinity gel.Finally, the samples were analysed for the presence of protein by Western blotting and detected by anti-GFP antibodies.A and B -negative controls, no bands are detected in the elution fraction.CFP-FTZ-F1 LBD and CFP-14-3-3 did not bind to the ANTI-FLAG M2 Affinity gel (marked with orange arrows).C and D -positive controls, a band corresponding to YFP-Met-FLAG, YFP-Gce-FLAG, YFP-MetC-FLAG, or YFP-GceC-FLAG it is present in the elution fraction for each protein (marked with blue arrows).Proteins tagged with FLAG have bound to the ANTI-FLAG M2 Affinity gel.SUP -supernatant; FT -proteins not bound to the resin; W1-3 -wash fraction; elution -elution fraction.Observed fusion proteins with MM are marked with arrows (blue for proteins with FLAG tag, binding to the ANTI-FLAG M2 Affinity gel and orange for untagged proteins, not binding to the ANTI-FLAG M2 Affinity gel).All experiments were repeated twice or more.

Table S2 .
Experimental parameters of resonance assignment experiments for MetC protein.

Table S3 .
Experimental parameters of resonance assignment experiments for