1. Volume 25, Issue 12, Pages 1907-1915.e5 (December 2017)
Structural Evidence for a Role of the Multi-functional Human Glycoprotein Afamin in Wnt Transport 
Andreas Naschberger, Andrew Orry, Stefan Lechner, Matthew W. Bowler, Didier Nurizzo, Mislav Novokmet, Markus A. Keller, Gregor Oemer, Daniele Seppi, Martin Haslbeck, Kathrin Pansi, Hans Dieplinger, Bernhard Rupp 
Structure 
Volume 25, Issue 12, Pages e5 (December 2017)
DOI: /j.str
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2. Structure 2017 25, 1907-1915.e5DOI: (10.1016/j.str.2017.10.006)
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3. Figure 1
Superposition of the Two Afamin Models and Multi-conformer Models
(A) Superposition of the two afamin models. The main binding site is circled with a dashed blue line, where the fatty acid chain in the binding site is displayed as a yellow ball-and-stick model. Indicated are the positions of the four confirmed glycosylation sites, with modeled glycans displayed in ball-and-stick representation. The double arrows indicate the relative proximity of domains I and III in each of the models, respectively.
(B and C) A superposition of 25 multi-conformer model traces visualizes the distinct differences in dynamics between the two molecules in the asymmetric unit depending on the crystallographic packing environment. The N-terminal parts of domain IA (blue) and domain IIIB (orange, red) exhibit high conformational freedom in both molecules.
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4. Figure 2
Hydrophobic Binding Pocket of Afamin Modeled with Palmitoleic Acid
(A) 2mFo-DFc maximum-likelihood electron density contoured at 0.8σ (blue grid), superimposed with a Polder Fo-Fc difference omit map contoured at 4σ (green grid). In the palmitoleic acid (PAM) model, the cis-double bond between C9 and C10 is planar with correct geometry. The binding cleft, open toward solvent at the top of the panel, is presented with property-colored residue surfaces (white, aromatic/lipophilic; red, hydrogen-bonding acceptor potential; blue, hydrogen-bond donor potential).
(B) LigPlot presentation of hydrophobic interactions (red dashed lines) deep in the predominantly hydrophobic afamin binding pocket.
(C) Palmitic acid model viewed in the binding cleft in different orientation, with property-colored residue surfaces (red, hydrogen-bonding acceptor potential; blue, hydrogen-bond donor potential; white, aromatic lipophilic; green, non-aromatic lipophilic).
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5. Figure 3 The Predicted Complex between Wnt3a and Afamin
(A) Overview of the Wnt3a-afamin complex. The afamin crystal structure model is displayed as a white surface, and Wnt3a as a green surface. Shown as a ball-and-stick model is the N381 glycan of afamin (all other glycosylations are located on the back of the molecule), and the arrow indicates the location of potential glycosylation site N298 on Wnt3a. The other glycosylation consensus site, N87, is located on the reverse side of the palm and not visible.
(B) Same as (A), with Wnt3a represented as a green ribbon with shaded yellow emphasizing the contact surface with afamin.
(C) A closer view of the contacts between Wnt3a (green stick and ribbon) and afamin; the key contact residues are labeled in color (green, Wnt3a; black, afamin). A supplemental interactive iSee package, presenting details of the modeled complex, annotated contacts, and visualizing the accommodation of the palmitoleic tail in the deep hydrophobic binding cleft of afamin, is provided in STAR Methods.
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6. Figure 4 Superposition of Fatty Acids in the Afamin Binding Pocket
Green: the Wnt3a-afamin complex, with palmitoylated S209; light green: free PAM as observed in the afamin crystal structure. Palmitic acids (PLM 1007) in crystal structures 1e7h (red) and 4bke (blue). These PLMs sit deeper in the albumin binding cleft than the PAM in afamin and the modeled complex. In contrast to afamin, the albumin binding pocket is lined at its narrower entrance with polar and charged residues, likely adverse to accommodation of the acyl chain of S209. In yellow, the extended PAM acylation on S187 of XWnt8a in the XWnt8-Fz8-CRD complex structure. 1e7h and 4bke contain six more bound PLM molecules, which in afamin did not correspond to any significant or interpretable electron density.
Structure  , e5DOI: ( /j.str )
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