Volume 24, Issue 12, Pages 2043-2052 (December 2016) The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins  Laure Yatime, Cristine Betzer, Rasmus Kjeldsen Jensen, Sofia Mortensen, Poul Henning Jensen, Gregers Rom Andersen  Structure  Volume 24, Issue 12, Pages 2043-2052 (December 2016) DOI: 10.1016/j.str.2016.09.011 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 2043-2052DOI: (10.1016/j.str.2016.09.011) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Structure Determination of the hRAGE-VC1C2:S100A6 Complex (A) Overall structure of the hRAGE-VC1C2:mS100A6 complex as present in the asymmetric unit of the crystals. The RAGE ectodomain is colored in different shades of blue and the mS100A6 monomer is shown in beige. Divalent cations are indicated as spheres (green for Ca2+ and black for Zn2+). N-ter, N-terminus. (B) Omit electron density map calculated using simulated annealing in PHENIX.REFINE (Adams et al., 2002) from a model where residues 3–14 (first half of helix H1) and 51–89 (helices H3 and H4) from mS100A6 have been deleted. The 2mFo − DFc omit map (gray mesh) is contoured at 1σ. The final complete model is superimposed to show the quality of the fit. (C) Final model, 2mFo − DFc (blue mesh, 1σ) and mFo − DFc maps (green mesh, 3σ) centered on the RAGE C1 domain. See also Figure S1. Structure 2016 24, 2043-2052DOI: (10.1016/j.str.2016.09.011) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 The RAGE:S100A6 Interface in Complex 1 and Comparison with Other V Domain:S100 Complexes (A) Overview of the RAGE V domain:S100A6 interface in the complex 1 model derived from our crystal structures. The interacting region on S100A6 is shown in red. (B) Zoom-in to highlight the residues on both proteins present at the interface. (C) Same as (B) but with the structure of calcium-free S100A6 (Otterbein et al., 2002) in place of Ca-bound S100A6, revealing that the interface is preserved in the absence of calcium. (D) NMR model for the interaction between the RAGE V domain and S100A6 mutant C3S (Mohan et al., 2013). (E) NMR model for the interaction between the RAGE V domain and S100P (Penumutchu et al., 2014). (F) Crystal structure of the complex between S100B and RAGE V domain-derived peptide (Jensen et al., 2015). In (D) to (F), the S100 residues proposed to interact with the RAGE V domain are shown in red. Structure 2016 24, 2043-2052DOI: (10.1016/j.str.2016.09.011) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 The RAGE:S100A6 Interface in Complex 2 (A) Overall structure of the hRAGE-VC1C2:mS100A6 complex 2. The two RAGE ectodomains are colored in cyan and red and the mS100A6 subunits are shown in beige and purple. Divalent cations are indicated as spheres (green for Ca2+ and black for Zn2+) and the different zinc layers are indicated as gray transparent discs. (B) The first region of contact between hRAGE-C1 and the S100A6 homodimer centered on Zn3. The residues from both RAGE and S100A6 involved in stabilizing the interface are indicated as sticks and water molecules are displayed as red spheres. The hydrogen bonding network extends to Zn4, which bridges the two S100A6 monomers. (C) The second region of contact between hRAGE-C1 and mS100A6. The view is rotated by 90° compared with (B). (D) Electrostatic and water-mediated interactions between hRAGE-C2 and mS100A6 fixing the relative orientation of the C2 domains with respect to the VC1 tandem domain. (E) Superimposition of the hS100A6 structure obtained in the absence of calcium (in yellow [Otterbein et al., 2002]) with the RAGE:S100A6 complex 2 (S100A6 in purple). See also Figures S2 and S3 and Table S1. Structure 2016 24, 2043-2052DOI: (10.1016/j.str.2016.09.011) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 A Non-canonical Homodimeric Conformation for S100 Proteins (A) The mS100A6 dimer conformation in the hRAGE-VC1C2:mS100A6 complex shown in two different orientations. Zoomed-in regions displayed in (C) and (D) are indicated by dotted boxes. N-ter, N-terminus; C-ter, C-terminus. (B) Structure of the calcium-loaded hS100A6 homodimer (Otterbein et al., 2002) in the canonical dimer conformation. The hS100A6 monomer in red is displayed in the same orientation as the mS100A6 monomer in beige from (A). (C) Zoom-in on the interactions stabilizing the S100A6 dimeric conformation observed in our structures around H1 from one monomer and the H3-H4 cleft from the other monomer. Zinc coordination of Zn4 by residues from both monomers, as well as insertion of Leu5 into the H3-H4 hydrophobic cleft from the other monomer, stabilizes the non-canonical dimer interface. (D) Zoom-in on the interactions stabilizing the S100A6 dimeric conformation observed in our structures around the H4 C-terminus from one monomer and the H2-H2′ loop from the other monomer. Side chains of key residues involved in the new H4-H4 packing are also indicated. Structure 2016 24, 2043-2052DOI: (10.1016/j.str.2016.09.011) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Comparison of the RAGE Ectodomain Homodimerization Mode in Apo-VC1C2 and the VC1C2:S100A6 Complex and a Model for Signal Transduction by the RAGE:S100A6 Complex (A) Model for the RAGE ectodomain homodimer based on the structure of hRAGE ectodomain alone (Yatime and Andersen, 2013) showing an extended dimeric conformation formed through dimerization of two V domains with a maximum separation of 190 Å between the C-termini of the RAGE C2 domains. C-ter, C-termini. (B) The S100A6-mediated RAGE homodimeric conformation with the two C2 domains separated by 60 Å, which coincides with the distance between the two receptor binding loops from the TIR domains homodimer (bottom) of the adaptor Mal/TIRAP (Valkov et al., 2011) suggested to be involved in RAGE signal transduction (Sakaguchi et al., 2011). RAGE transmembrane (TM) helices (middle) may participate in signal transduction through various mechanisms: (1) dissociation from a helix:helix homodimer possibly formed via the GxxxG motif found in RAGE TM helices (Yatime and Andersen, 2013); (2) formation and/or changes in the orientation of the TM helix:helix homodimer; and (3) interaction with additional TM partners (another RAGE homodimer or yet unknown interacting partners). See also Figure S4. Structure 2016 24, 2043-2052DOI: (10.1016/j.str.2016.09.011) Copyright © 2016 Elsevier Ltd Terms and Conditions