Presentation is loading. Please wait.

Presentation is loading. Please wait.

Volume 24, Issue 8, Pages (August 2016)

Similar presentations


Presentation on theme: "Volume 24, Issue 8, Pages (August 2016)"— Presentation transcript:

1 Volume 24, Issue 8, Pages 1271-1281 (August 2016)
Conformational Changes in the GM-CSF Receptor Suggest a Molecular Mechanism for Affinity Conversion and Receptor Signaling  Sophie E. Broughton, Timothy R. Hercus, Tracy L. Nero, Mara Dottore, Barbara J. McClure, Urmi Dhagat, Houng Taing, Michael A. Gorman, Jack King-Scott, Angel F. Lopez, Michael W. Parker  Structure  Volume 24, Issue 8, Pages (August 2016) DOI: /j.str Copyright © 2016 Elsevier Ltd Terms and Conditions

2 Structure 2016 24, 1271-1281DOI: (10.1016/j.str.2016.05.017)
Copyright © 2016 Elsevier Ltd Terms and Conditions

3 Figure 1 Structure of the GM-CSF Binary Complex
(A) The wrench-like architecture adopted by GMRα (colored gray in all panels) when binding the four α-helical bundle of GM-CSF (colored orange in all panels). The β strands that make up the three FnIII domains (NTD, D2, and D3) and the N and C termini are labeled. The cysteine residues in GMRα are represented as yellow spheres, and the bound sugars at N116 and N176 of GMRα are shown as sticks. (B) A close-up view highlighting key interactions between GM-CSF and the GMRα NTD (site 1b). Hydrogen bonds are shown as black dotted lines in all panels. (C) A close-up view highlighting key interactions between GM-CSF and the GMRα D2-D3 linker region (site 1a). (D) A close-up view highlighting key interactions between GM-CSF and the GMRα D3 (site 1a). A crystallographic water molecule involved in site 1a D3 interactions is shown as a small red sphere. See also Figures S1–S3; Table S1. Structure  , DOI: ( /j.str ) Copyright © 2016 Elsevier Ltd Terms and Conditions

4 Figure 2 Comparison of the GM-CSF Binary and Ternary Complexes
(A) The binary and ternary complexes have been superimposed via their α subunit CRMs and shown as cartoons. In the binary complex, GM-CSF and GMRα have the same coloring as Figure 1. In the GM-CSF:GMRα:βc ternary complex, GM-CSF is colored green, GMRα pink, and the βc dimer dark and light blue (for clarity only part of the dimer is shown). Hydrogen bonds are shown as black dotted lines in all panels. (B) Comparison of the binary and ternary complexes at the GMRα D2-D3 and D3 linker region (site 1a). (C) Interactions between GMRα D3:βc D4 (site 3, βc D4 is shown as surface) showing the contrast in the GMRα D3 D strand conformation between the binary (gray) and ternary (pink) complexes. (D) Interactions involving GM-CSF and the GMRα NTD (site 1b) in the ternary complex (TC). (E) Docking of the binary complex (BC) to the apo-βc dimer (PDB: 2GYS) in silico to illustrate the residue movements that occur upon assembly of the ternary complex. (F) Interactions between GM-CSF and D1 and D4 of the βc dimer (site 2) in the ternary complex (TC). (G) Illustration of the movement in the βc D4 BC loop (Y365 on the BC loop is shown, apo in yellow and ternary complex in blue), R24 and E21 in GM-CSF that occur upon assembly of the ternary complex (TC). The steric clash between GM-CSF R24 in the ternary complex and apo-βc Y365 is depicted (for clarity the clash between GM-CSF N17 in the ternary complex and apo-βc H367 is not shown). (H) Model of the GM-CSF E21R mutation at site 2 in the ternary complex (TC). Steric clashes are indicated by red disks. See also Figure S4; Tables S1 and S3. Structure  , DOI: ( /j.str ) Copyright © 2016 Elsevier Ltd Terms and Conditions

5 Figure 3 Functional Characterization of GMRα Site 1 Mutants and βc Site 2 Mutants (A) CTL-EN cells stably expressing βc and wild-type GMRα (gray ■) or the R170D (blue ○), D192R (black □), K195D (blue ⋄), R170D,K195D (blue ●), K194D (red ○), D231K (red □), or K194D,D231K (red ●) mutants, were stimulated with a titration of GM-CSF for 40 hr and cell proliferation determined by [3H]thymidine incorporation. Data are the mean of triplicate determinations from a representative experiment (n = 2) and error bars represent SD. (B) CTL-EN cells stably expressing GMRα and wild-type βc (gray ■) or the Y421A (white □), Y421F (black ■), Y39A (blue ○), F103A (blue ●), Y365A (white ⋄), E366A (black ♦), H367A (white △), I368A (black ▴), Y365A,E366A,H367A,I368A (red □), or Y365A,E366A,H367A,I368A,Y421A (red ■) mutants, were stimulated with a titration of GM-CSF for 40 hr and cell proliferation determined as for (A). (C) The functional parameters for cells expressing wild-type GM-CSF receptors (white □), GMRα site 1 mutants (blue ●), and βc site 2 mutants (red ●) are plotted by comparing GM-CSF functional activity (ED50, ng/ml) against GM-CSF high-affinity binding (KD, nM). The measurement limit for GMRα mutants in high-affinity binding experiments is defined as KD = 250 nM (blue dashed line) while for βc mutants the limit is defined by the GMRα affinity, KD = 20 nM (red dashed line). Data for selected GMRα and βc mutants are labeled: K194D (a), D231K (b), R170D,K195D (c), K194D,D231K (d), and Y365A,E366A,H367A,I368A,Y421A (e). (D) CTL-EN cells stably expressing GMRα and the same βc variants described in (B) were stimulated with a titration of GM-CSF E21R for 40 hr and cell proliferation determined as for (A). See also Figures S5 and S6; Table S2. Structure  , DOI: ( /j.str ) Copyright © 2016 Elsevier Ltd Terms and Conditions

6 Figure 4 Movement of Membrane Proximal Domains upon Higher-Order Assembly (A) Overlay of the binary (BC) and ternary (TC) complex via GM-CSF, indicating movement of the D3 domain of GMRα upon βc recruitment (GMRα colored pink [TC] and gray [BC], cytokine orange [BC] and green [TC], and βc blue and deep teal). (B) Overlay of the apo-βc (colored gray) and βc in the ternary complex (TC) indicating an accommodating shift of the βc D4 upon complex formation. Other colors as in (A). Domain movement is indicated by red arrows. See also Figure S4 and Table S3. Structure  , DOI: ( /j.str ) Copyright © 2016 Elsevier Ltd Terms and Conditions


Download ppt "Volume 24, Issue 8, Pages (August 2016)"

Similar presentations


Ads by Google