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Figure 1.
Figure 1. Structural Analysis of Apu2.16 and Apu3.A8 Anti-K63
Linkage Fabs Bound to K63-Linked Diubiquitin
(A) The
complex between K63-linked diubiquitin (orange) and the Apu2.16
Fab fragment (heavy chain: green, light chain: magenta). Heavy
chain CDR3 (H3) contacts both ubiquitins on either side of the
isopeptide linkage. H3 side chains within 4.2 Å of
diubiquitin and ubiquitin side chains within 4.2 Å of H3
are shown as sticks. Residues mentioned in the text are in bold
for ubiquitin and italics for the Fab. K63 in the acceptor
ubiquitin is shown as a sphere.
(B) Comparison of K63-
(top) and K48-linked (bottom) diubiquitin. Lysine donor
ubiquitins are light orange and acceptor ubiquitins are bright
orange. K48-linked diubiquitin forms a more compact shape with
the chain extending perpendicular to the ubiquitin dimer, while
the K63-linked diubiquitin chain will extend in a more elongated
manner.
(C) Superposition of Apu2.16 (colored as in A) and
Apu3.A8 (heavy chain: light green, light chain: pink) showing
the location of the two changes in L2 (S52R) and H3 (S52T)
introduced in the affinity maturation process to create Apu3.A8.
The structural differences in L1 in the two Fabs are likely due
to crystal packing. L1 (as well as the N terminus) are in a
noncanonical conformation likely due to interactions with L3,
which has sequence and structural differences relative to the
phage library parent sequence (Figure S2).
(D) Charge
complementarity between Apu3.A8 (transparent) and diubiquitin
(solid). Electrostatic surfaces were calculated with PyMol.
Regions of positive potential are blue; regions with negative
potential are red. In the Apu3.A8 light chain, R52 (which is
introduced in Apu3.A8) and R66 contribute to a positive region
that is close to a negatively charged region on the ubiquitin
surface, created in part by residues D21, D58, and E18 from the
K63 acceptor ubiquitin.
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