Figure 1.
Structures of ligands and scFv6H4 sequence.
(a) Structures of METH and metabolites AMP and p-OH-METH (b) Amino acid sequence of anti-METH scFv6H4. The variable heavy (H) and light (L) chains are numbered according to the Kabat numbering convention [29]. The location of the framework regions (FR), complementarity determining regions (CDRs), linker, and His-tag are indicated graphically above the amino acids. The antigen binding sites of antibodies are made up of three CDRs from the heavy chain and three from the light chain.
Figure 2.
A comparison of the free form of scfv6H4 with the METH bound form.
(a) A stereo-view depicting the superposition of the Cα atoms of the free form (pink) with the METH complex (purple). The largest deviations are all in the CDR loops. The METH is shown in green. The CDR H1, CDR H2 and CDR H3 are labeled as H1, H2 and H3. Likewise, CDR L1, CDR L2 and CDR L3 are labeled as L1, L2 and L3. Residues Gly H26, Asp H27, Ser H97 and Met H100B are labeled as 26, 27, 97 and 100B. The light chains are shown in lighter colors.
(b) Trimer formation. Left panel: A lateral of a view cartoon representation of the trimer. The three molecules assemble around the 3-fold axis to give it a light bulb-like shape. The Ni2+ ion is shown as a sphere (in the stem region on the top) and the sulfate moiety (towards the bottom of the sphere) is shown as a CPK model. Right panel: A view from the top along the 3-fold axis. The three molecules are labeled as Mol-A (red), Mol-B (green) and Mol-C (blue). The light chains are shown in lighter colors.
Figure 3.
(a) A stereo view of the composite β-sheet: One of the β-sheets of the VH consists of five β-strands and it joins together with identical sheets from the symmetry related molecules to form a composite β-sheet with a 3-fold symmetry. The outer strand (Tyr H100 to Ser H112) is the longest, with a significant bend which separates it into two segments. The two segments interact with separate molecules joining the three sheets together. The residues Tyr H100, Gln H105 and Ser H112 are labeled as 100, 105 and 112 respectively. The location of the CDR H3 loop is labeled as H3.
(b) Interactions of the sulfate moiety: The sulfur atom and one of the oxygen atoms sits on the crystallographic 3-fold axis. The oxygen atom on the three fold interacts with three Gln residues (Gln H77 of the three VH) related by symmetry. The other oxygen atoms interact with Ser residues (Ser H23 of the three VH). (c) A view of the electron density of the His-tag residues. This diagram depicts the 2Fo-Fc electron density map of the His-tag residues. The contours are drawn at 1.2 σ level. (d) Ni-coordination: The Ni 2+ ion sits on the crystallographic 3-fold symmetry axis coordinating with 6 Histidine residues in octahedral geometry. Each of the symmetry related chains contributes two histidine residues for coordination. The bound nickel is shown as a sphere and histidine residues are represented as stick models. The three symmetry related molecules are shown in green, cyan and pink.
Figure 4.
Conformational changes upon trimer formation.
(a) CDR H3 loop: A superposition of the Cα atoms of residues Thr H89-Thr H107 in the apo structure (magenta) with the METH bound structure (yellow). Six residues belonging to the CDR H3 loop (Ser H97 to Met H100B) show significant displacements. In the apo crystal, the loop residues take up extended conformations elongating the two anti-parallel β-strands to have a sharp turn connection in the place of a long loop. In the METH-bound structure, the CDR H3 region assumes a more spread out conformation to create the environment for METH binding. (b) The shapes of the binding pockets in the free and the antigen bound states: The left panel (apo form) and the right (METH complex) show identical cross sections. The entrance of the binding pocket is broader in apo structure, compared to the METH complex. Color code: Left panel: heavy chain -magenta and light chain -light pink. Right panel: heavy chain- blue light chain-light blue.
Figure 5.
(a) Electron density of the ligands: Left panel: A view showing the electron density (2Fo-Fc) of AMP. Right panel: A 2Fo-Fc map of p-OH-METH. The contours are drawn at 1.2 σ level. (b) A comparison of the binding modes of the two metabolites with METH. Left panel: A superposition of AMP (pink) with METH (blue). The antibody binds both AMP and METH in a similar manner, but there is a shift in the position of the nitrogen atom which forms hydrogen bonds with Glu H101 of the VH chain and His H89 of the VL chain. The hydrogen bond distances indicate that in the AMP structure they are much weaker. Right panel: p-OH-METH: The geometry of the binding pocket is the same in the METH structure and the hydrogen bonds are of comparable strength as in the METH structure.