Order and disorder-An integrative structure of the full-length human growth hormone receptor

Abstract

Because of its small size (70 kilodalton) and large content of structural disorder (>50%), the human growth hormone receptor (hGHR) falls between the cracks of conventional high-resolution structural biology methods. Here, we study the structure of the full-length hGHR in nanodiscs with small-angle x-ray scattering (SAXS) as the foundation. We develop an approach that combines SAXS, x-ray diffraction, and NMR spectroscopy data obtained on individual domains and integrate these through molecular dynamics simulations to interpret SAXS data on the full-length hGHR in nanodiscs. The hGHR domains reorient freely, resulting in a broad structural ensemble, emphasizing the need to take an ensemble view on signaling of relevance to disease states. The structure provides the first experimental model of any full-length cytokine receptor in a lipid membrane and exemplifies how integrating experimental data from several techniques computationally may access structures of membrane proteins with long, disordered regions, a widespread phenomenon in biology.

Fig. 1. The hGHR has a dynamic ECD with a broad structural ensemble.

(A) A schematic representation of homodimeric hGHR (blue) in the membrane in complex with hGH (green). ECD, Extracellular domain; TMD, transmembrane domain; and ICD, intracellular domain. (B) SEC profiles of hGHR-ECD and hGH in 20 mM Na₂HPO₄/NaH₂PO₄ (pH 7.4) and 150 mM NaCl at ratios 1:0 (hGH:hGHRECD 1:0), 0:1 (hGH:hGHR-ECD 0:1), 1:2 (hGH:hGHR-ECD 1:2), and 4:1 (hGH:hGHR-ECD 4:1). Absorption was measured at 280 nm. (C) Concentration-normalized SAXS data from hGHR-ECD (concentrations in legend) with the p(r) from the sample (3.5 mg/ml) shown as inset. a.u., absorbance units. (D) SAXS data from hGHR-ECD at 3.5 mg/ml (black dots) together with fits of the theoretical scattering curves from a crystal structure of hGRH-ECD (blue; PDB 3HHR), the same crystal structure with missing loops completed (purple), and the average (green) and reweighted average [red; reweighted against the experimental data using the Bayesian maximum entropy approach (see Materials and Methods)] of scattering curves of the 500 hGHR-ECD models with added N- and C-terminal tails. Residuals are plotted below. (E) An ensemble model of the hGHR-ECD with a representative reweighted subensemble of 100 models highlighting the N-terminal (cyan) and C-terminal (green) dynamic tails.

Fig. 2. The single-pass α-helical hGHR-TMD.

(A) Extent of the hGHR-TMD α helix by NMR (black), TMHMM (blue), Phobius (red), METSAT-SVM (purple), and UniProt (green) (fig. S2). Cylinder represents length of the hGHR-TMD α helix by NMR with the first and last helical residue numbered. (Left) Predicted numbers. (B) MICS α helix probability (top) and R₂ relaxation rates (bottom) of hGHR-TMD in DHPC micelles. Red diamonds, insufficient data quality or prolines. (C) Models of hGHR-TMD α helix by CYANA (blue) or CS-Rosetta (magenta). (D) Energy versus C^α-RMSD from CS-Rosetta modeling of hGHR-TMD. (E) Electron density profiles (EDPs) of lipid bilayers [POPC:POPS 3:1 mole percent (mol %)] with hGHR-TMD at 0.2 and 2 mol %, respectively. (F) Difference EDPs and best-fit profiles for the hGHR-TMD CS-Rosetta and the CYANA models, respectively. (G) Hermans orientation (left) and Lamellar spacing (right) of membranes at varying concentrations of hGHR-TMD. *, onefold change; ***, threefold change. (H) Illustration of membrane curvature. (I) Top: Oriented CD (OCD) spectra of 6 μg of hGHR-TMD in POPC, with L:P ratios varied from 1:40 to 1:200. Bottom: OCD spectra of 6 μg of hGHR-TMD in POPC, POPC:POPS (3:1), or DOPC at an L:P ratio of 50:1. The dashed lines represent nonreliable data due to too high high-tension voltage values.

Fig. 3. Properties of the hGHR-ICD ensemble.

(A) ¹H-¹⁵N-HSQC spectra at 5°C of hGHR-ICD (black) and hGHR-ICD-GFP-H₁₀ (red) at 150 and 100 μM, respectively. (B) Concentration normalized SAXS data from hGHR-ICD (black dots; 1.1 mg/ml) and hGHR-ICD-GFP-H₁₀ (red dots; 2.2 mg/ml). Fits to the data are shown for a Gaussian random coil model (orange) and from averaged scattering profiles from 5200 conformations taken from the hGHR-ICD_{ub_pws10} simulation (one every nanosecond) (blue). Residuals are plotted below. (C) R_H of hGHR-ICD and hGHR-ICD-GFP-H₁₀ determined from pulse-field gradient NMR. Signal decays of hGHR-ICD (black) and hGHR-ICD-GFP-H₁₀ (red) are shown as a function of gradient strength together with the corresponding fits. (D) Concentration normalized p(r)’s derived from the above SAXS data from hGHR-ICD (black) and hGHR-ICD-GFP-H₁₀ (red). A subensemble of 200 conformations representative of the hGHR-ICD_{metaD_pws10} simulation is shown in the right side of the plot.

Fig. 4. Incorporation of hGHR-GFP into MSP1D1, functional, and structural analysis.

(A) SEC profile of hGHR-GFP-loaded MSP1D1. The areas highlighted in gray indicate fractions (F1 to F3) used for the SDS-PAGE analysis in (B). (B) SDS-PAGE analysis of hGHR-GFP and MSP1D1 standards along with hGHR-GFP-loaded MSP1D1. Fractions F1 to F3 were taken from the indicated positions of the SEC-purified hGHR-GFP-loaded MSP1D1 shown in (A). The illustration above the gel shows the stoichiometry of the hGHR-GFP-loaded MSP1D1. (C) Intact mass spectra of hGHR-GFP show a single protein population with an average mass of 100,951 ± 64 Da. Asterisks denote detergent peaks. (D) MST determination of equilibrium binding constants for hGH to hGHR-GFP-loaded MSP1D1. The mean values and SD were obtained by fitting a 1:1 binding model (full line) as described in Materials and Methods. Concentration-normalized (E) SAXS data and (F) SANS data of the nanodisc-embedded hGHR-GFP corresponding to the highlighted SEC frames in fig. S4 (C and D).

Fig. 5. Model of the full-length hGHR-GFP in nanodiscs.

(A) Schematic representation of the semianalytical Gaussian random coil (SA-GRC) model. (B) Fits of the SA-GRC to the SAXS data of nanodisc-embedded hGHR-GFP (with GFP) (blue), the ensemble of 6000 conformations taken from the hGHR-GFP +POPC_pws10 simulations embedded in the nanodisc model (gray), their ensemble average (green), and reweighted ensemble average (red). (C) Representative snapshot from one of the hGHR-GFP + POPC_pws10 simulations (see Materials and Methods). POPC lipids are shown as gray sticks; protein is depicted in surface representation. Some lipids and all water and ions are omitted for clarity. (D) Distribution of R_g from 6000 all-atom conformations obtained from the hGHR-GFP + POPC_pws10 simulation after reweighting against the SAXS data. The values are shown both for the full-length protein (blue) and for the individual structural components: ECD (orange), ICD (green), and ICD-GFP (red).

Fig. 6. The ensemble structure of membrane-embedded full-length hGHR-GFP.

(A) Representative ensemble of conformations obtained from the last 1.5 μs of each of the 20 runs of 2-μs hGHR-GFP + POPC_pws10 simulations. Color scheme and representations as in Fig. 5C. (B) Examples of the multitude of domain orientations of hGHR-GFP in the membrane. In the first panel, the structures of hGH (PDB 3HHR_A; orange) and of JAK2-FERM-SH2 (PDB 4Z32; red) are shown. Color scheme and representation of hGHR and POPC as in Fig. 5C.