Recomendaciones

Figure 1. Schematic IgG structures

Heavy chains (identical polypeptide chains, but colored differently for illustrating domain swapping) are cyan and yellow, light chains are gray, and antigen binding sites are shown as starbursts. (A) Conventional IgG with flexible Fab arms and two antigen binding sites at the VH/VL interfaces. (B) 2G12 monomer with domain-swapped (Fab)2 unit and an

additional antigen binding site at the VH/VH’ interface. (C) 2G12 dimer with two domain-

swapped (Fab)2 units and six antigen binding sites. See also Figure S1.

Figure 2. Packing in 2G12 dimer crystals

(A) Asymmetric unit of a solvent flattened 8.0 Å resolution 2Fo-Fo electron density map

contoured at 1.5 σ. The asymmetric unit contained three half-dimers, i.e., three Fc regions and three (Fab)2 units from three distinct 2G12 dimers (A, B, and C). Crystal contacts

involved the antigen binding sites in the (Fab)2 units (green circles); see also Figure S2.

(B) Application of symmetry operators to generate the second half of each 2G12 dimer. Dimer A (cyan) was structurally distinct from Dimers B (magenta) and C (indigo), which exhibited the same conformation. (C) Non-crystallographic six-fold symmetry axis showing a hexamer of Fc regions coincident with the 61 screw axis along the

crystallographic c axis. The asymmetric unit is highlighted in yellow. (D) The three dimers in one layer of the crystal, represented by the cyan, magenta, and indigo dimers A, B, and C (panel B), as they fit into the Fc hexamer ring in panel C; see also Figure S2.

Figure 3. Native Patterson validation of packing in 2G12 dimer crystals

(A,B) Native Patterson maps. Comparison of experimental and calculated Harker sections. (C) Two layers of 2G12 dimer molecules as they are packed into the P6122

crystals. Left: top-down view showing the cyan (dimer A), magenta (dimer B), and indigo (dimer C) 2G12 dimers (Figure 2) in the same layer. The magenta and indigo dimers are conformationally identical and the cyan dimer is distinct. The layer below contains three additional dimers (light green, light pink, and light blue), formed via a rotation and translation along the crystallographic 61 axis. Symmetry mates are indicated

as a darker and lighter version of the same color. Right: a 90º rotation showing hexamer of Fc regions in the middle layer. See also Figure S3.

Figure 4. Structures of the three distinct 2G12 dimers from two independent crystal structures

Dimer structures are shown as 25 Å electron density envelopes calculated from (Fab)2

and Fc coordinates located by molecular replacement. The approximate distance between the tips of the (Fab)2 units of each dimer is shown on the left. The symmetry of two of the

2G12 dimer conformations, dimer A and the dimer in the 2G12/2G12.1 peptide structure (panels A and C), was point group C2 (the dimer two-fold symmetry axis was coincident with a crystallographic two-fold axis). The dimer B/C conformation (panel B) represented a distortion from C2 symmetry. A 2G12 dimer could also have point group D2 symmetry with the (Fab)2 units in a similar position as in a C2 dimer except with their

internal two-fold axes coincident, and with the Fcs above and below the plane of the (Fab)2 units with the Fc two-fold axes coincident with each other and perpendicular to the

(Fab)2 two-fold axes. Although the D2 conformation appeared plausible, it was not

observed in our structures. To interconvert these forms would require one (Fab)2 unit to

rotate 180˚. (A) Dimer A, one of two conformationally-distinct 2G12 dimers in the P6122

2G12 dimer structure. Middle and Right: Heavy chains are cyan and yellow, light chains are transparent gray. Middle: Top-down view of the 2G12 dimer with Fc regions pointing into the page. Right: Two rotated views showing the large distance between (Fab)2 units

and Fc regions and an angle of 180º between (Fab)2 arms. (B) Dimers B and C, the

second of the two distinct 2G12 dimers in the P6122 2G12 dimer structure. Middle and

Right: Heavy chains are magenta, indigo, light pink, or light blue; light chains are transparent gray. Middle: Top-down view of the 2G12 dimer molecule with Fc regions pointing into the page. Right: Two rotated views showing an obtuse angle between (Fab)2

units. (C) 2G12 dimer in crystals of 2G12 bound to the 2G12.1 peptide. Middle and Right: Heavy chains are orange, green, light orange, or light green; light chains are light gray. Middle: Top-down view. The two (Fab)2 units are extended at 180° with respect to

each other, as are the two Fc regions (not seen in this view). Right: Two rotated views showing the Fc regions under the (Fab)2 units. See also Figure S4.

Figure 5. Negative stain EM of 2G12 oligomers

Representative two-dimensional class averages (left) and false-colored images (right) of 2G12 (Fab)2, 2G12 monomer, 2G12 dimer, and 2G12 trimer showing the orientations of

the domain-exchanged (Fab)2 units and the Fc regions. Fabs are colored in cyan and Fcs

in yellow. Bar = 10 nm. (B-D) 2D class averages of 2G12 oligomers analyzed by negative stain EM. Bar = 10 nm. Panels of 64 class averages of the (B) 2G12 monomer,

(C) 2G12 dimer, and (D) 2G12 trimer generated using reference-free Xmipp Clustering 2D alignment. (E) Back projections sampled at 20˚ Euler angles of the low-pass filtered 2G12 Dimer A crystal structure. Bar = 10 nm. Many of the back projections resembled the reference-free class averages of the 2G12 IgG dimer in panel C. See also Figure S5 for SAXS data supporting the EM and crystallographic results.

Figure 6. Surface plasmon resonance-based binding studies

Sensorgrams were derived from binding experiments in which 2G12 monomer, 2G12 dimer or a conventional monomeric anti-gp120 IgG was injected over immobilized gp120 from HIV-1 strain SF162. Experimental data (colored lines) were fit to a 1:1 binding model (black lines). Residual plots are shown in Figure S6. (A) 2G12 monomer, 2G12 dimer, and a conventional anti-gp120 IgG (NIH45-46G54W) injected over gp120 immobilized at high coupling density (~100 RU). 2G12 monomer, but not 2G12 dimer or the conventional IgG, could be fit to a 1:1 binding model. (B) 2G12 monomer, 2G12 dimer, and a conventional anti-gp120 IgG (NIH45-46G54W) injected over gp120 immobilized at medium coupling density (~50 RU). 2G12 monomer, but not 2G12 dimer or the conventional IgG, could be fit to a 1:1 binding model. (C) 2G12 monomer, 2G12 dimer, and a conventional anti-gp120 IgG (2909) injected over gp120 immobilized at low coupling density (<10 RU). All could be fit to a 1:1 binding model.

Figure 7. Equilibrium gel filtration analyses of FcRn-2G12 complexes

(A) FcRn was incubated with 2.5 µM 2G12 monomer in the presence of 0, 1, 2, or 3 (additional) equivalents of FcRn in a buffer containing 5 µM FcRn (equilibration buffer).

Samples were then injected onto a column equilibrated in the equilibration buffer. The peak that eluted first corresponded to an FcRn-2G12 monomer complex. The second peak or trough was at the elution volume of free FcRn. (B) FcRn was incubated with 1.25 µM 2G12 dimer in the presence of 0, 2, 4, or 6 (additional) equivalents of FcRn in a buffer containing 5 µM FcRn (equilibration buffer). Samples were then injected onto a column equilibrated in the equilibration buffer. The peak that eluted first corresponds to an FcRn-2G12 dimer complex. The second peak or trough was at the elution volume of free FcRn.

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