Orchestration of signaling by structural disorder in class 1 cytokine receptors

Abstract

Background: Class 1 cytokine receptors (C1CRs) are single-pass transmembrane proteins responsible for transmitting signals between the outside and the inside of cells. Remarkably, they orchestrate key biological processes such as proliferation, differentiation, immunity and growth through long disordered intracellular domains (ICDs), but without having intrinsic kinase activity. Despite these key roles, their characteristics remain rudimentarily understood.

Methods: The current paper asks the question of why disorder has evolved to govern signaling of C1CRs by reviewing the literature in combination with new sequence and biophysical analyses of chain properties across the family.

Results: We uncover that the C1CR-ICDs are fully disordered and brimming with SLiMs. Many of these short linear motifs (SLiMs) are overlapping, jointly signifying a complex regulation of interactions, including network rewiring by isoforms. The C1CR-ICDs have unique properties that distinguish them from most IDPs and we forward the perception that the C1CR-ICDs are far from simple strings with constitutively bound kinases. Rather, they carry both organizational and operational features left uncovered within their disorder, including mechanisms and complexities of regulatory functions.

Conclusions: Critically, the understanding of the fascinating ability of these long, completely disordered chains to orchestrate complex cellular signaling pathways is still in its infancy, and we urge a perceptional shift away from the current simplistic view towards uncovering their full functionalities and potential. Video abstract.

Keywords: CIDER; Cytokine receptors; Disorder; IDDomainSpotter; IDPs; IDRs; NMR; SAXS; SLiM; Signaling; Structural biology; Transmembrane receptors.

Fig. 1

The C1CR family - structures and ICD isoforms. a Structural model of the full-length human PRLR [35] in the membrane, with the ECD in light grey, the TMD in pink and a total of six ICD chains shown in blue shades to represent its disordered conformational ensemble. b Left: A representative sketch of a prototypical C1CR (blue) in a membrane (green) having a long ICD with its length given in %. Box1 and Box2 are highlighted in orange. The red shades highlight the approximate extent and position of small ICD fragments of different C1CR-ICDs having their structure solved in complex with signaling proteins. Right: Three-dimensional structures of signaling proteins (grey surfaces) in complex with C1CR-ICD fragments (red cartoon and sticks), being the FERM-SH2 domain of JAK2 in complex with EPOR-ICD_279–334 (pdb code 6E2Q) (top), SOCS2 in complex with GHR-ICD_591–603 (pdb code 6I5J, 6I5N) (middle top), the PTB domain of IRS-1 in complex with IL-4Rα_490–500 (pdb code 1IRS) (middle bottom) and the PDZ domain from syntenin in complex with IL-5Rα_417–420 (pdb code 1OBZ) (bottom). The grey arrows point to the approximate positions of the ICD fragments (red shades) on the representative ICD sketch. c The 29 receptors of the C1CR family with an ICD and their isoforms. The length of the ICDs are to scale and red indicates the length of unique sequences differing from the long-form. The receptors are divided into 5 groups according to [33].

Fig. 2

Intrinsic disorder and compositional bias of C1CR-ICDs. a Disorder prediction for C1CR common receptors and group 1. The disorder propensity, ranging from 0 to 1 was predicted using IUPred2A (blue), ANCHOR (black) and Pondr-fit VSL2 (red) and is plotted as a function of residue number. The boundaries between the ECD (white background), TMD (yellow background) and ICD (orange background) were predicted using TMHMM v. 2.0. The sequence numbering includes the signal peptide. b Fractional differences in composition between the C1CR-ICDs (dark grey) or a set of IDPs (light grey), and a set of folded proteins calculated for each amino acid type (see methods for details). Negative values denote that the amino acid is less frequent than in folded proteins, and positive values denote that the amino acid is more frequent than in folded proteins. The arrows indicate the directions of “more” abundant than in folded proteins, and “less” abundant than in folded proteins. The error bars indicate the 90% confidence interval of the estimated frequencies, calculated using a per-protein bootstrapping procedure with 1000 iterations [105]. c IDDomainSpotter profiles of group 1 C1CR-ICDs. Profiles display scores for +Arg,Lys-Asp,Glu (green), +Cys (brown), +Pro (purple), + Ile,Leu,Val (red) and + Phe,Tyr,Gly (blue) calculated over 15 residue windows for each of the ICDs

Fig. 3

Short linear motifs form the basis for signaling choreography. a Sequence logo of the amino acid distribution in C1CR group 1 Box1 and Box2 motifs. Letter-height indicates the relative abundance of the given amino acid at the specific position. Red indicates an acidic residue, blue - a basic residue, green – a polar residue, black – a hydrophobic residue and pink – a neutral residue [102]. b SLiM and phosphorylation site mapping in C1CR group 1 ICDs. Regions with SLiMs are highlighted and colored according to the amount of overlap. Black indicates regions with no overlap. Yellow indicates regions with two overlapping SLiMs, whereas red indicates regions with more than two overlapping SLiMs. Predicted phosphorylation sites are indicated by grey diamonds [107] and confirmed and well-characterized sites by black diamonds. c Zoom on the membrane proximal 67-residue region of GHR-LF-ICD containing overlapping binding sites. Box1 and Box2 are highlighted in blue. Proteins were illustrated in IBS 1.0.3 [114]

Fig. 4

Network rewiring by disorder. Top; Comparison of a long and short isoform of PRLR. a Structural propensities of the PRLR-LF-ICD compared to PRLR-SF1b-ICD illustrated by differences in NMR secondary chemical shifts (SCSs) of C^α. Red coloring indicates lower C^α SCSs in PRLR-SF1b-ICD compared to PRLR-LF-ICD, and hence less helicity. b NMR chemical shift perturbations upon addition of POPC/POPS SUVs to PRLR-SF1b-ICD and c intensity ratios of PRLR-SF1b-ICD in the absence and the presence of POPC/POPS SUVs. Red circles highlight prolines and/or unassigned residues and shaded areas loss of NMR signal upon addition. Bottom; Predicted and selected binding SLiMs in the isoforms of d PRLR, e IL-31Rα (GLMR), and f LEPR (OBR). Introduction of potential new binding SLiMs in the unique sequences are indicated by the sequence of the SLiM, and the presence of SLiMs in the LF of the isoforms is indicated with a “+”. When a SLiM is not present, this is indicated by a “-”. The length of the ICD is indicated by the scale bars on top, and red lines illustrate the length of the unique sequence. “*” indicates a different SLiM compared to the binding site identified by mutations in [148]. Alternative names for the isoforms are given in the second row. For other C1CR isoforms, see Table S2

Fig. 5

Conformational properties of C1CR-ICDs. a Plot of conformational parameters provided by CIDER analyses of the longest isoforms of the C1CR-ICDs. For GM-SCFRα, both isoform 1 and 2 were analyzed as they differed > 50%. Each receptor group is highlighted with different colors; 1 – blue, 2 – green, 3 -yellow, 4 – red, and 5 - purple. When sequences have a Pro warning, i.e. a Pro fraction > 0.15, the ICD is highlighted with a P on top. The numbers at the top are the compositional region assigned by CIDER. # residues are the numbers of residues in the ICD, FCR is the fraction of charged residues (FCR = f⁺ + f⁻), NCPR is the net charge per residue (NCPR = f⁺ − f⁻), κ is a measure of the charge mixing across the sequence (normalized between 0 and 1) and Ω additionally takes Pro mixing into account (normalized between 0 and 1). The dotted lines at 0.4, 0.3, and 0.1 in the FCR plot represent the borders for very highly charged (above), highly charged (above) and highly depleted (lower) in charges, respectively [160]. The area between the dotted lines at 0.1 and − 0.1 in the NCPR plot represents the range occupied by 70% of human IDPs [160]. b Experimental SAXS data (spheres) and Gaussian random coil model fit (lines) for PRLR-LF-ICD at different concentrations of NaCl (see color key), with the residuals from the fit shown below in units of standard deviations. c Pair distance distribution functions for the PRLR-LF-ICD SAXS data. The color coding is the same as in b)