The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins

Article

The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins Graphical Abstract

Authors Laure Yatime, Cristine Betzer, Rasmus Kjeldsen Jensen, Sofia Mortensen, Poul Henning Jensen, Gregers Rom Andersen

Correspondence [email protected] (L.Y.), [email protected] (G.R.A.)

In Brief Yatime et al. report the crystallographic structure of an S100-bound full-length RAGE ectodomain. The structure reveals a unique dimeric conformation of RAGE, which appears suited for signal transduction, and shows that the S100A6 protein adopts a non-canonical homodimeric arrangement.

Highlights d

The structure of RAGE:S100A6 complex with a full-length RAGE ectodomain is presented

d

Structural basis for S100A6-elicited signal transduction through the RAGE receptor

d

S100A6 forms a unique homodimeric conformation upon RAGE binding

Yatime et al., 2016, Structure 24, 1–10 December 6, 2016 ª 2016 Elsevier Ltd. http://dx.doi.org/10.1016/j.str.2016.09.011

Accession Numbers 4P2Y 4YBH

Please cite this article in press as: Yatime et al., The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.09.011

Structure

Article The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins Laure Yatime,1,3,4,* Cristine Betzer,2 Rasmus Kjeldsen Jensen,1 Sofia Mortensen,1 Poul Henning Jensen,2 and Gregers Rom Andersen1,* 1Department

of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus, Denmark of Biomedicine, Danish Research Institute of Translational Neuroscience - DANDRITE, Aarhus University, Ole Worms Alle´ 1170, 8000 Aarhus, Denmark 3Present address: DIMNP – UMR5235, University of Montpellier, Place Euge ` ne Bataillon, Baˆt. 24 cc107, 34095 Montpellier Cedex 5, France 4Lead Contact *Correspondence: [email protected] (L.Y.), [email protected] (G.R.A.) http://dx.doi.org/10.1016/j.str.2016.09.011 2Department

SUMMARY

S100 proteins are calcium-dependent regulators of homeostatic processes. Upon cellular response to stress, and notably during tumorigenesis, they relocalize to the extracellular environment where they induce pro-inflammatory signals by activating the receptor for advanced glycation end products (RAGE), thereby facilitating tumor growth and metastasis. Despite its importance in sustaining inflammation, the structural basis for RAGE-S100 crosstalk is still unknown. Here we report two crystal structures of the RAGE:S100A6 complex encompassing a full-length RAGE ectodomain. The structures, in combination with a comprehensive interaction analysis, suggest that the primary S100A6 binding site is formed by the RAGE C1 domain. Complex formation with S100A6 induces a unique dimeric conformation of RAGE that appears suited for signal transduction and intracellular effector recruitment. Intriguingly, S100A6 adopts a dimeric conformation radically different from all known S100 dimers. We discuss the physiological relevance of this non-canonical homodimeric form in vivo.

INTRODUCTION The receptor for advanced glycation end products (RAGE) belongs to the immunoglobulin (Ig) subfamily of cell surface receptors (Neeper et al., 1992). RAGE is a pattern recognition receptor that senses endogenous stress signals (Stern et al., 2002) and its broad ligand repertoire includes advanced glycation end products (Neeper et al., 1992), S100 proteins (Hofmann et al., 1999), high-mobility group box 1 protein (Hori et al., 1995), amyloid b oligomers (Yan et al., 1996), nucleic acids (Tian et al., 2007), phospholipids (Rai et al., 2012b; Yamamoto et al., 2011), and glycosaminoglycans (Mizumoto et al., 2012). These ligands

accumulate at inflammatory sites during the pathogenesis of various diseases, including diabetes, vascular complications, neurodegenerative disorders, and cancers (Kierdorf and Fritz, 2013; Salama et al., 2008; Ulloa and Messmer, 2006; Yan et al., 2003), and RAGE transduces their binding into pro-inflammatory responses, notably via nuclear factor kB-dependent cell activation (Xie et al., 2013). Ligand recognition by the RAGE ectodomain leads to receptor activation, i.e., signal transduction through the membrane, adaptor protein binding to RAGE cytoplasmic domain, and activation of downstream signaling cascades (Fritz, 2011; Sorci et al., 2013). More than ten members of the S100 protein family are now described as RAGE ligands (Leclerc et al., 2009). S100 proteins are EF-hand calcium-binding proteins exclusively expressed in vertebrates, in a tissue- and cell-specific manner (Donato et al., 2013; Marenholz et al., 2004). Under homeostatic conditions, they function intracellularly in calcium homeostasis, cell growth and differentiation, cytoskeleton dynamics, and energy metabolism, generally in a calcium-dependent manner. During cellular response to stress, they can be secreted or passively released in the extracellular matrix where they act as damageassociated molecular patterns and exert cytokine-like functions, promoting sustained inflammation, tissue damage, and disease progression, through RAGE-mediated signaling (Leclerc and Heizmann, 2011; Riehl et al., 2009; Sparvero et al., 2009). As they form homodimers or higher oligomers, S100 proteins are thought to require RAGE oligomerization for efficient binding and signal transduction (Fritz, 2011; Yatime and Andersen, 2013). The RAGE ectodomain is composed of three Ig domains (V, C1, and C2) and the V domain is believed to be the main ligand binding domain, although a few ligands have also been shown to interact with either the C1 or the C2 domains, including S100A6, S100A12, and S100A13 (Leclerc et al., 2007; Rani et al., 2014; Xie et al., 2007). A nuclear magnetic resonance (NMR) structure of the (RAGE-C2:S100A13)2 heterotetrameric complex was reported (Rani et al., 2014), and three models for the interaction of the RAGE V domain with S100P (Penumutchu et al., 2014), a mutated S100A6 (Mohan et al., 2013), and a mutated S100A9 (Chang et al., 2016) have also been proposed based on NMR chemical shift variations observed between the ligand-free and Structure 24, 1–10, December 6, 2016 ª 2016 Elsevier Ltd. 1

Please cite this article in press as: Yatime et al., The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.09.011

Table 1. Data Collection and Refinement Statistics of the Native Datasets for hRAGE-VC1C2:mS100A6 and hRAGE-VC1C2: hS100A6 Crystals hRAGE-VC1C2: mS100A6 Complex

hRAGE-VC1C2: hS100A6 Complex

Data Collection X-ray source

911-2 (MAX-lab)

ID23-2 (ESRF)

Wavelength (A˚)

1.041

0.8726

Space group

I222

I222

76.78, 113.39, 140.32

75.98, 112.53, 139.89

a, b, g ( )

90, 90, 90

90, 90, 90

Resolution (A˚)a

35–2.3 (2.36–2.3)

50–2.4 (2.5–2.4)

Rmeas (%)a

8.0 (46.9)

9.3 (81.9)

I/sIa

15.56 (4.12)

14.21 (2.06)

Completeness (%)a

99.7 (100)

99.6 (99.7)

Redundancya

7.0 (7.2)

4.6 (4.5)

Resolution (A˚)

27–2.3

47–2.4

No. of reflections

27,064

23,037

Rwork/Rfree

18.66/20.67

19.47/23.30

Cell dimensions a, b, c (A˚)

Refinement

No. of atoms

A two-step binding mode, with similar Kd values for each step (0.5 and 0.6 mM, respectively), has been proposed for the S100A6 interaction with the RAGE ectodomain based on surface plasmon resonance (SPR) measurements (Leclerc et al., 2007). Furthermore, S100A6 was shown to bind both the isolated V and C2 domains of RAGE with dissociation constants ranging from low micromolar (10
5–10
6) to medium nanomolar (10
8) (Leclerc et al., 2007; Mohan et al., 2013). The discrepancy in the reported Kd values between the isolated domains and the full-length ectodomain, however, renders it difficult to evaluate which interactions are relevant in vivo. To gain insights into S100A6 recognition by RAGE, we determined the crystal structures of the human RAGE ectodomain (hRAGE-VC1C2) in complex with mouse and human S100A6 (mS100A6 and hS100A6, respectively). Two possible 2:2 complexes encompassing one S100A6 homodimer and two RAGE molecules were identified in our crystal structures. A comprehensive interaction analysis using PISA indicates that the most plausible complex possesses a binding site for S100A6 located primarily in the RAGE C1 domain. This complex displays unique conformations for both the RAGE ectodomain and the S100A6 homodimer. We suggest that our structure of the S100A6-bound receptor represents an active conformation of RAGE capable of efficient signal transduction and intracellular effector recruitment. In addition, we discuss the physiological relevance of the non-canonical and very unique homodimeric conformation of S100A6 present in the crystal structures.

Protein

2,985

2,992

Ligand/ion

42

26

Water

186

208

RESULTS

Protein

53

53

Ligand/ion

57

62

Water

46

46

Crystal Structures of the RAGE:S100A6 Complex To investigate the recognition mode of S100A6 by the RAGE ectodomain, we crystallized the hRAGE-VC1C2:mS100A6 and hRAGE-VC1C2:hS100A6 complexes under similar conditions. The crystals diffracted X-rays to a maximal resolution of 2.3 and 2.4 A˚, respectively (Table 1, Figure 1), with an asymmetric unit composed of one molecule of RAGE and one molecule of S100A6 (Figure 1A). The two complexes are superimposable with an average root-mean-square deviation (RMSD) on Ca atoms between the two structures of 0.34 A˚ (over 776 Ca atoms). In agreement with all known structures of S100 proteins, S100A6 forms a homodimer in the crystals through a 2-fold crystallographic axis (Figures 1B and S1A). mS100A6 displays 96% sequence identity with hS100A6, with conservative mutations accounting for most of the differences. Only one of these divergent residues (Lys22 in mS100A6, Arg22 in hS100A6) is involved in RAGE binding, but similar electrostatic interactions are achieved by these residues in both complexes. As the RAGE:mS100A6 complex was solved at slightly higher resolution and displays better statistics, we will only describe this complex in the following. We identified two possible complexes encompassing a S100A6 dimer and two RAGE molecules in the crystals (Figures S1B and S1C). In complex 1 (Figure S1B), the S100A6 dimer interacts exclusively with the two RAGE V domains, each monomer in a symmetrical manner, and the interface is limited to the lateral side of the RAGE V domain and to the first Ca2+ EF-hand in S100A6. In complex 2 (Figure S1C), the S100A6 dimer is sandwiched between the C1 and C2 domains of the

B factors

RMSD Bond lengths (A˚)

0.004

0.006

Bond angles ( )

0.875

0.701

Ramachandran plot (%) Favored

99.2

97.7

Allowed

0.8

2.3

Outliers

0

0

a

Values in parentheses are for highest-resolution shell.

the S100-bound V domain. Finally, the crystal structure of a complex between S100B and a peptide derived from the RAGE V domain was also recently described (Jensen et al., 2015). These structural models all propose different binding modes to RAGE, thus complicating our understanding of the RAGE signal transduction mechanism. Furthermore, they only depict binding to isolated RAGE domains or shorter fragments. Whether these interactions can all be observed with the full-length, membranebound receptor thus remains to be clarified. The S100A6 protein is found at high levels in muscles, lungs, kidney, spleen, and brain (Kuznicki et al., 1989). Like many other S100 proteins (Salama et al., 2008), S100A6 is overexpressed in several cancers and may be involved in cell proliferation, cytoskeleton reorganization, and tumorigenesis (Lesniak et al., 2009). 2 Structure 24, 1–10, December 6, 2016

Please cite this article in press as: Yatime et al., The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.09.011

Figure 1. Structure Determination of the hRAGE-VC1C2:S100A6 Complex (A) Overall structure of the hRAGEVC1C2: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 1s. The final complete model is superimposed to show the quality of the fit. (C) Final model, 2mFo
DFc (blue mesh, 1s) and mFo
DFc maps (green mesh, 3s) centered on the RAGE C1 domain. See also Figure S1.

two RAGE molecules, interacting with both domains and their connecting linker. In each complex, the two S100A6 molecules are calcium loaded and the monomer is fairly similar to unbound Ca2+-S100A6 (Otterbein et al., 2002), with an RMSD on Ca atoms between the two structures of 0.97 A˚ (over 88 Ca atoms). The RAGE-bound S100A6 dimer, however, adopts a dramatically different conformation than the free Ca2+-S100A6 homodimer (see below). The relative orientation of the two RAGE molecules is also identical in both complexes. Thus, the only difference between the two complexes is the anchor point of the S100A6 homodimer on the RAGE molecules. Interestingly, the two RAGE ectodomains have no direct interaction with each other and they are brought in close proximity by the S100A6 homodimer. Regardless of the complex considered, the two resulting 1:1 VC1C2:S100A6 subcomplexes (Figures S1B and S1C) are perfectly interchangeable and related by a 2-fold rotational symmetry, suggesting a binding mechanism where each monomer from the S100A6 homodimer binds successively one RAGE molecule, a mechanism which agrees with the binding mode in two steps of equivalent affinities deduced from SPR measurements (Leclerc et al., 2007). In the following, we will describe both complexes in details and analyze which one may be more relevant in vivo. Complex 1, a Small Interface Diverging from Other V Domain:S100 Complexes Within complex 1, the RAGE:S100A6 interface covers a relatively small surface (Figure 2A), with a total buried area of 650 A˚2 for the two symmetrical binding regions according to PISA (Krissinel and Henrick, 2007). Only seven residues from each S100A6 monomer are present at the interface, corresponding mostly to residues from the first calcium EF-hand and Glu67 from the second calcium EF-hand, whereas on the RAGE V domains, only six residues on the lateral loop linking the two b sheets are in close proximity to S100A6 (Figure 2B). There is no direct interaction between the RAGE V domain and S100A6 except for Asp25 (S100A6) and Arg57 (RAGE), which are connected by watermediated hydrogen bonds. Thus the interface appears to be quite weak. S100A6 does not bind to the other RAGE domains, including the C2 domain, which can move freely as compared

with the VC1 fragment (Yatime and Andersen, 2013), and the orientation of the two RAGE molecules is not enforced by direct RAGE-RAGE contacts. This suggests that if the RAGE-S100A6 interface observed in complex 1 occurred in vivo, it would not be rigid. This renders it difficult to understand how formation of this interface would lead to efficient signal transduction, unless higher-order oligomers rigidifying the overall structure are formed in vivo. Furthermore, this complex fails to explain the calcium dependency of the interaction. Indeed, in calcium-free S100A6, helix H3 adopts a quite distinct orientation due to a partial unfolding of the second calcium EF-hand (Otterbein et al., 2002). However, these conformational changes do not concern the first calcium EF-hand and superimposition of Ca2+-free S100A6 onto our complex 1 shows that the interfacial residues described above are still in close proximity to the RAGE V domain (Figure 2C). This predicts that the RAGE:S100A6 interface observed in complex 1 is stable in the absence of calcium, in disagreement with the expected Ca2+ dependency of the interaction. Several structures of complexes between S100 proteins and the RAGE V domain have been reported in the literature. However, none of these structures involves the same binding epitopes, both on RAGE and on the S100 protein, as the S100A6:RAGE interface we observe in complex 1. In the recently described NMR-based model for the interaction of the RAGE V domain with S100A6 mutant C3S (Mohan et al., 2013), the RAGE:S100A6 interface is positioned in a similar region as the one we observe in our complex 1 (Figure 2D). However, there are no shared S100A6 residues between these two interfaces, and the relative position of the RAGE V domain as compared with S100A6 is radically different. In the NMR model for the interaction of S100P with RAGE (Penumutchu et al., 2014), the V domain is positioned on the opposite side of S100P as compared with S100A6, in the groove delineated by helix H4 and the loop connecting helices H2 and H3 (Figure 2E). Finally, the crystal structure of S100B with a V domain-derived peptide (Jensen et al., 2015) places the RAGE fragment in the cleft between helices H3 and H4 (Figure 2F). This region is not accessible in our RAGE:S100A6 complex due to the non-canonical conformation of the S100A6 homodimer. In conclusion, the Structure 24, 1–10, December 6, 2016 3

Please cite this article in press as: Yatime et al., The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.09.011

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 calciumfree 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.

RAGE:S100A6 interface presented in complex 1 does not resemble any of the previously reported interfaces between S100 proteins and the RAGE V domain. Furthermore, the relatively small size of the interface argues against it being responsible for the formation of a rigidified complex, and the absence of rationale for a calcium-dependent interaction renders the physiological significance of complex 1 questionable. Complex 2, a Larger Interface Stabilized by Divalent Cations Within complex 2, each S100A6 monomer engages with the two RAGE molecules through several surfaces (Figure 3A). The N-terminal helix H1, the loop following the first calcium EFhand, and the beginning of helix H4 interact with the C1 domain of one RAGE molecule (Figures 3B and 3C), and charged residues in helix H2 make electrostatic contacts with the adjacent C2 domain (Figure 3D), thereby fixing the C2 domain in a rigid orientation with respect to the RAGE VC1 fragment. Interactions with the C1 domain in the second RAGE molecule are mediated by residues from helix H3 and the loop preceding the second 4 Structure 24, 1–10, December 6, 2016

calcium EF-hand (Figure 3B). The total buried surface area at the interface is 2,025 A˚2 according to PISA (Krissinel and Henrick, 2007), suggesting a much more stable complex than complex 1. Besides Ca2+ ions, 12 other ions were identified and assigned as Zn2+ based on their anomalous scattering properties (Table S1, Figure S2). These Zn2+ ions occupy three distinct layers within the RAGE:S100A6 complex (Figure 3A). The second layer, encompassing four Zn2+ ions, is positioned between the S100A6 dimer and the RAGE C1 domains (Figure 3A) and appears to further stabilize the RAGE:S100A6 interface. The interaction between the first RAGE C1 domain and S100A6 is built on hydrophobic contacts engaging the central part of S100A6 helix H1 and is further maintained by p-p stacking interactions between Arg228 (RAGE) and Phe70 (S100A6), and between Trp230 (RAGE) and His17 (S100A6) (Figure 3C). At both extremities of helix H1, the interface is strengthened by polar interactions spreading around two zinc ions, Zn3 and Zn5. Zn3 is tetra-coordinated by the side chains of Asp160 and Asp201 from the RAGE C1 domain and two Cl
ions (Figure 3B). This site is further stabilized by a network of water molecules held in place by the side chains of Arg62, Cys3, and Asp6 from both S100A6 monomers and RAGE Arg203, the latter being engaged in a stacking interaction with S100A6 Arg62. At the other end of helix H1 (Figure 3C), Zn5 is tetra-coordinated by His17 and His27 from S100A6, Glu132 from the RAGE C1 domain, and a Cl
ion stabilized by RAGE Arg228. Although S100A6 is known to bind zinc with a 300-fold greater affinity than calcium (Kordowska et al., 1998), no data so far have reported a possible effect of zinc on the binding of S100A6 to RAGE. However, two other S100 proteins, S100A9

Please cite this article in press as: Yatime et al., The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.09.011

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.

and S100A12, were shown to require zinc for RAGE binding (Bjork et al., 2009; Liu et al., 2009), and several other S100 proteins bind zinc through a conserved site located at the interface between the two monomers of the S100 dimer (Brodersen et al., 1999; Lin et al., 2016; Moroz et al., 2009; Murray et al., 2012; Ostendorp et al., 2011) (Figure S3). Dual coordination of zinc by both RAGE and its S100 ligand, as observed in our RAGE:S100A6 complex 2, is therefore a plausible mechanism by which zinc could enhance complex formation for specific RAGE:S100 complexes. In contrast to complex 1, the conformation of helix H3 from calcium-free S100A6 (Otterbein et al., 2002) is incompatible with the recognition of the second RAGE molecule as observed in our RAGE:S100A6 complex 2. Indeed, in the Ca-free S100A6 conformation, residues Arg55, Asp59, and Arg62 would not reach the C1 domain from the second RAGE molecule due to the different positioning of helix H3 (Figure 3E). In the absence of calcium, each RAGE C1 domain would only be able to interact with the S100A6 N-terminus. This would render the RAGES100A6 interface much weaker since it would only rely on the set of interactions described in Figure 3C and the weak electrostatic interactions provided by the RAGE C2 domain (Figure 3D), whereas the stabilizing network of interactions described in Figure 3B would be disrupted. A tight and stable 2:2 RAGE:S100A6 complex 2 can therefore only be obtained when S100A6 is calcium loaded, leading to locking of each RAGE molecule in a rigid orientation due to the simultaneous interaction of each C1 domain with both S100A6 monomers. This is in agree-

ment with the Ca2+ dependency of the RAGE:S100 interaction observed for almost all S100 proteins. A Unique, Non-canonical Homodimeric Conformation for S100A6 Over 160 structures of 20 different S100 proteins have been reported and reveal a similar canonical homodimer, no matter whether they are in the apo-state, bound to Ca2+/Zn2+ ions, or in complex with other proteins. In contrast, the quaternary structure adopted by the RAGE-bound S100A6 homodimer (in both possible complexes) differs dramatically from this canonical S100 dimer. The orientation between the two monomers has been shifted by a rotation of approximately 90 (Figures 4A and 4B). As a result, in RAGE-bound S100A6, helices H1 and H4 from one monomer surround helix H4 in the other monomer as compared with H1 in the canonical Ca2+-S100A6 homodimer (Otterbein et al., 2002). This H1-H4-H4-H1 packing forms the hydrophobic core of the homodimer, which is further stabilized by hydrogen bond networks at the N and C termini of both monomers (Figures 4C and 4D). In the dimer conformation observed in our structures, the N-terminus of helix H1 from one S100A6 monomer inserts the side chain of Leu5 into the hydrophobic cleft formed between helices H3 and H4 from the opposite monomer (Figure 4C). The same cleft is frequently used by interacting proteins for binding S100 proteins in a calcium-dependent manner since the cleft is formed upon Ca2+ binding in the canonical dimer (Rezvanpour and Shaw, 2009). This is, for example, the case for the S100A6-interacting protein Siah-1 which inserts Structure 24, 1–10, December 6, 2016 5

Please cite this article in press as: Yatime et al., The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.09.011

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-H20 loop from the other monomer. Side chains of key residues involved in the new H4-H4 packing are also indicated.

two leucine side chains into this cleft (Lee et al., 2008) or for the RAGE-derived peptide interacting with S100B as shown in Figure 2F (Jensen et al., 2015). Thus, the second S100A6 monomer within the S100A6 homodimer we observe in our structures mimics a known binding mode used by ligands interacting with the canonical S100 dimer conformation. Analysis of the canonical S100A6 homodimer and the one observed in our structures with PISA (Krissinel and Henrick, 2007) shows that the monomer-monomer interfaces in the two S100A6 dimer conformations are almost equivalent both in terms of their intermolecular interaction areas and the strength of the interactions. The total surface area buried at the monomer-monomer interface in our S100A6 homodimer is 2,575 A˚2 compared with 2,758 A˚2 in the canonical S100A6 dimer. The patches of residues involved in stabilizing the packing of helices H1 and H4 from both monomers are also very similar in both conformations (Figure 4). Finally, 14 hydrogen bonds maintain the S100A6-S100A6 interaction in the canonical homodimer, whereas the new S100A6-S100A6 interface observed in the RAGE:S100A6 complex is stabilized by ten hydrogen bonds. Furthermore, a unique Zn2+ binding site, never encountered before in S100 proteins, bridges the two S100A6 molecules through Zn4 coordination by Cys3 from one monomer and Arg55 and Asp59 from the other monomer (Figure 4C). Thus, once formed the non-canonical S100A6 homodimer we describe 6 Structure 24, 1–10, December 6, 2016

is likely to have stability comparable with that of the canonical dimer and it is very unlikely to be a crystal-packing artifact. Zn2+ binding to S100A6 is believed to induce conformational changes that involve reorganization of hydrophobic patches on the S100A6 surface (Kordowska et al., 1998). This is consistent with the importance of Zn2+ for the formation of the non-canonical S100A6 homodimer and the required modification of the inter-monomer contacts between the highly hydrophobic helices H1 and H4. A Structural Basis for Signal Transduction In the absence of ligands, RAGE can already assemble into oligomers (Xie et al., 2008) that may be further stabilized by cell surface glycosaminoglycans (Xu et al., 2013). The apoRAGE ectodomain is prone to homodimerization through its V domains as seen in solution (Sarkany et al., 2011) and in the crystal structures of the RAGE ectodomain and its VC1 fragment (Koch et al., 2010; Xu et al., 2013; Yatime and Andersen, 2013) (Figure 5A). This V domain dimerization may represent a resting form of the receptor and serve as a starting point for further oligomerization required for signal transduction (Xu et al., 2013; Yatime and Andersen, 2013). In our complex 2 model, the interaction with S100A6 fixes the orientation between the VC1 tandem and the C2 domain, in contrast to what we observed for the unbound ectodomain (Yatime and Andersen, 2013).

Please cite this article in press as: Yatime et al., The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.09.011

Furthermore, the RAGE ectodomain adopts a different quaternary structure compared with all previously reported RAGE structures. The V domains have been released from their 2-fold symmetrical V-V interaction and the two RAGE molecules are now brought into proximity only through their interaction with the S100A6 dimer. In addition, the two C2 domains are now locked into fixed positions resulting in a distance of 60 A˚ between their C-termini (Figure 5B). This coincides with the 60 A˚ distance observed between the two C2 domains in the structure of the heterotetrameric (hRAGE-C2:hS100A13)2 complex (Rani et al., 2014) (Figure S4), suggesting that this is a general feature for at least some RAGE-ligand complexes. Interestingly, adaptors binding to RAGE cytoplasmic domain, such as Mal/TIRAP, may also form homodimers that reach across 60 A˚ (Sakaguchi et al., 2011; Valkov et al., 2011) (Figure 5B). Thus, binding of two distinct S100 proteins appears to induce a repositioning of the C2 domains in a geometry favorable to intracellular adaptor recruitment. This could contribute to signaling in addition to ligand-induced clustering of RAGE intracellular domains previously suggested to be important for RAGE signaling in general (Rai et al., 2012a). DISCUSSION

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 A˚ 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 A˚, 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)

Here we report a comprehensive analysis of the interaction between the human RAGE receptor and its ligand S100A6, based on crystallographic studies. Our data concur to the formation of a Ca2+-dependent complex encompassing two RAGE ectodomains and one S100A6 homodimer, with the RAGE:S100A6 interface being most probably centered on the RAGE C1 domain. S100A6 has previously been proposed to bind both the V and C2 domains of recombinant RAGE (Leclerc et al., 2007; Mohan et al., 2013), and a role for the C1 domain was also suggested by the inhibition of the RAGE:S100A6 interaction with anti-C1 antibodies (Leclerc et al., 2007). The data presented here now strongly suggest that the C1 and C2 domains and their connecting linker also form a relevant binding site for S100A6. Whether this binding mode can occur simultaneously with the one mediated by the RAGE V domain as observed by (Mohan et al., 2013) (for example, within large RAGE:S100A6 oligomers) or whether each binding mode occurs independently in a specific cellular context remains to be determined. The hRAGE-VC1C2:S100A6 structures presented here provide the description of a RAGE:ligand complex encompassing a full-length ectodomain (which has never been reported before) and a structure-based model for the active signaling complex can be proposed. Prior models have suggested that ligand-induced RAGE signaling is driven by receptor clustering as ligands stabilize and further oligomerize preformed RAGE oligomers in the membrane (Fritz, 2011; Rai et al., 2012a). This will create a locally high concentration of RAGE intracellular domains, which will promote the recruitment of adaptor proteins

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 24, 1–10, December 6, 2016 7

Please cite this article in press as: Yatime et al., The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.09.011

such as Diaphanous 1. Our study suggests that, in addition to simple clustering, ligand binding can induce substantial conformational changes in the receptor ectodomain and bring into proximity the intracellular domains from two RAGE molecules bound to the same dimeric S100 ligand. Whether S100A6 is unique in this aspect remains to be discovered. Interestingly, a rather different model of the RAGE signaling complex has been proposed recently for the RAGE:S100B interaction (Xue et al., 2016) where the S100B homodimer bridges two preformed, asymmetrical RAGE homodimers, leading to a 2:4 complex. In this model, the distance between the C-terminal regions of the most opposite RAGE C2 domains is 100 A˚, a value that coincides with the span of the membrane Diaphanous 1 protein, another adaptor protein transducing RAGE signaling (Xue et al., 2016). Thus, different types of signaling complexes may be formed depending on both the ligand bound and/or the downstream effector that needs to be recruited. The role of the RAGE transmembrane (TM) segment in signaling is still unclear. In particular, whether TM helices are associated as homodimers, as previously suggested (Yatime and Andersen, 2013), and whether signal transduction requires a reorientation or dissociation of such TM homodimers, remains to be determined (Figure 5B). A special feature of RAGE is the presence of a presumably unstructured linker region of conserved length between the membrane proximal C2 domain and the TM segment. This is in contrast to, e.g., the epidermal growth factor receptor, where the membrane proximal domain is directly connected to the TM segment. Here ligand binding is suggested to induce conformational changes in the dimerized TM segment and the intracellular region (Arkhipov et al., 2013). This would at first glance be an unlikely scenario for RAGE due to the unstructured C2-TM linker being apparently unsuitable for transmitting a power-stroke through the TM segment following ligand binding to the ectodomain. Thus, further rigidification of this linker, either through RAGE interaction with yet unknown additional binding partners, possibly membranebound, or through the formation of higher-order RAGE oligomers that would restrain the linker and ectodomain flexibility, may be required for efficient signal transduction. RAGE has also been proposed to form constitutive oligomers in vivo through the formation of intermolecular disulfide bridges between the C2 domains of two adjacent receptor molecules (Wei et al., 2012). The two cysteines (Cys259 and Cys301) forming this intermolecular disulfide bridge are buried within each C2 domain in our structures of both the isolated RAGE ectodomain (Yatime and Andersen, 2013) and the present S100A6 complex, and they engage in an intramolecular bond within the canonical Ig fold. A dimerization mode based on such an intermolecular disulfide bridge therefore implies a partial unfolding of the C2 domains to expose these two residues. Whether this reorganization would be compatible with the formation of the RAGE:S100A6 complex we describe and whether it would provide further rigidification of the complex remains to be determined. Our structural data also reveal a unique homodimerization mode for S100A6, never encountered before. Based on interface analysis, this dimer appears as stable as the canonical dimer which argues strongly against the possibility that this conformation is a crystallization artifact. But we cannot completely rule out that this non-canonical form was induced by the high zinc 8 Structure 24, 1–10, December 6, 2016

concentrations or the isopropanol present in the crystallization conditions and that RAGE then crystallized in a non-relevant complex with this non-canonical S100A6 dimer. Unfortunately we were not able to directly test the effect of high zinc concentrations on S100A6 in solution since the protein precipitated when mixed with more than 5 mM ZnCl2 at even 1 mg/mL. S100A6 is believed to belong to the ‘‘CYS-Zn binding’’ class of S100 proteins (Moroz et al., 2011) since the second epitope for ‘‘HIS-Zn binding’’ present on helix H4 in other S100 proteins is not conserved in S100A6 (Figure S3). A cysteine-dependent zinc binding mode is therefore expected for this class of S100 proteins, in agreement with what we observe in our RAGE-bound S100A6 structure. In the absence of a crystal structure of Zn2+bound S100A6 alone, i.e., without RAGE present, we cannot conclude whether zinc alone would induce the conformational rearrangements observed in our S100A6 homodimer or not. A second possibility is that a small fraction of our recombinant S100A6 protein, and possibly of the protein expressed in vivo, already displays the non-canonical dimeric conformation. As this homodimer is very similar, both in terms of overall shape and solvent-exposed surfaces, to the canonical one, it is extremely difficult to assess whether such form is present in small quantities in our protein samples. Finally, a third option is that the conformational rearrangements inducing the non-canonical S100A6 dimer are RAGE dependent, explaining why this conformation has not been observed previously. Further studies are therefore required to prove the existence of this form in vivo and its relevance for RAGE binding. EXPERIMENTAL PROCEDURES Protein Expression and Purification The complete preparation protocol is available in the Supplemental Experimental Procedures. In brief, the sequences coding for the human RAGE ectodomain (VC1C2 fragment) and for both mouse and human S100A6 were cloned into vector pETM11. The RAGE domain construct was transformed in Escherichia coli Shuffle T7 Express cells (New England Biolabs), whereas the S100A6 constructs were transformed in E. coli BL21 (DE3) cells (Stratagene). The resulting proteins, expressed as fusions with an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site, were purified using two-step Ni-column affinity chromatography, including removal of the affinity-tag by overnight incubation with TEV protease, followed by cation-exchange chromatography for the VC1C2 domain, or by size-exclusion chromatography for the S100A6 proteins. All purified proteins were flash frozen in liquid nitrogen and stored at
80 C until use. Crystallization, Data Collection, and Structure Determination RAGE VC1C2 (final concentration: 6 mg/mL) and m/hS100A6 were mixed in a 1:1 or 1:2 molar ratio. Crystals appeared overnight at 4 C over a reservoir containing 0.2 M Zn acetate, 0.1 M Na cacodylate (pH 6.5), 10% (v/v) isopropanol, only in the drops with a 1:2 molar ratio between VC1C2 and S100A6. For data collection, the crystals were cryoprotected by soaking into the reservoir solution supplemented with 35%–40% (v/v) isopropanol as final concentration followed by flash cooling in liquid nitrogen. Datasets for the hRAGE-VC1C2:mS100A6 complex extending to 2.3 A˚ resolution (Table 1) were collected at 100 K on the 911-2 beamline at MAXlab (Lund University, Sweden) while anomalous diffraction data (Table S1) were collected at the MAX-lab 911-3 beamline. The hRAGE-VC1C2:hS100A6 crystals diffracted to a maximum resolution of 2.4 A˚ (Table 1) and a complete dataset was collected at 100 K on the ID23-2 beamline at the European Synchrotron Radiation Facility (Grenoble, France). All datasets were processed with XDS (Kabsch, 1993). The hRAGE-VC1C2:mS100A6 crystals displayed an I222 symmetry with one molecule of RAGE and one molecule of S100A6

Please cite this article in press as: Yatime et al., The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.09.011

per asymmetric unit. The structure was solved by molecular replacement (MR) in PHASER (McCoy et al., 2005), using the VC1 moiety of our previously determined hRAGE-VC1 structure (Yatime and Andersen, 2013) and a homology model of the mouse S100A6 protein based on the crystal structure of human S100A6 (Otterbein et al., 2002). Automated rebuilding of the structure with PHENIX.AUTOBUILD (Adams et al., 2002) improved the electron density such that the C2 domain not present in the MR model could be placed manually. Refinement of the model was carried out by alternating cycles of manual rebuilding in COOT (Emsley and Cowtan, 2004) and cycles of positional refinement with PHENIX.REFINE (Adams et al., 2002) using individual isotropic atomic displacement parameters as well as translation-librationscrew parameterization. The final model yielded Rwork and Rfree values of 18.66% and 20.67%, respectively. The hRAGE-VC1C2:mS100A6 model was then used to solve the structure of the hRAGE-VC1C2:hS100A6 complex by MR. Refinement of the model was carried out using the same procedure as for the hRAGE-VC1C2:mS100A6 complex. The final model yielded Rwork and Rfree values of 19.47% and 23.30%, respectively. The quality of both models was assessed using MolProbity (Davis et al., 2007). All figures were made with the PyMOL Molecular Graphics System, version 0.99rc6 (DeLano Scientific). ACCESSION NUMBERS Coordinates and structure factors for the hRAGE-VC1C2:mS100A6 and the hRAGE-VC1C2:hS100A6 complexes are available at the PDB under accession codes PDB: 4P2Y and 4YBH, respectively. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, four figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.str.2016.09.011. AUTHOR CONTRIBUTIONS L.Y. expressed and purified all proteins, performed the crystallization experiments, collected and processed the data, determined and refined the structure, and analyzed all the data. C.B. and P.H.J. designed and performed initial cellular assays. R.K.J. performed binding measurements. S.M. performed initial binding experiments. G.R.A. secured funding. L.Y. and G.R.A. designed the experiments and wrote the paper with input and proofreading from all authors. The two corresponding authors, L.Y. and G.R.A., contributed equally to the conceptualization, methodology design, and supervision of the work as well as to the preparation of the manuscript text and figures. ACKNOWLEDGMENTS We would like to thank the beamline staffs at MAX-lab and ESRF for support during data collection and Dr. Takeshi Tomita for the mS100A6 construct. We thank the Lundbeck Foundation for supporting this work through the grant: Lundbeck Foundation Nanomedicine Center for Individualized Management of Tissue Damage and Regeneration, and for their support of DANDRITE. This project was also supported by DANSCATT, the Danish Cancer Society, and by the Novo-Nordisk Foundation through a Hallas-Møller Fellowship to G.R.A. Received: May 23, 2016 Revised: August 26, 2016 Accepted: October 5, 2016 Published: November 3, 2016 REFERENCES Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., Moriarty, N.W., Read, R.J., Sacchettini, J.C., Sauter, N.K., and Terwilliger, T.C. (2002). PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954.

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