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Rationally Designed Ligands for Use in Affinity Chromatography 99
3. Methods
3.1. Design of PpL Mimic Ligands(15)
1. Study of the complex between PpL and Fab: The complex structure is asymmetric
because a single PpL domain contacts similar V L regions of two Fab molecules
via independent interfaces; the PpL domain is, in effect, sandwiched between two
antibody Fab molecules (see Fig. 2 ). In the first interface, there are six hydrogen
bonds joining the -sheets of the PpL domain and the V L domain into a unique
sheet, through a -zipper type of interaction. In total, there are 13 residues from
the Fab involved in the interaction with the C* PpL domain. There are 12 residues
from the PpL domain (strand 2 and helix) involved in the interaction: Lys24,
Ile34, Gln35, Thr36, Ala37, Glu38, Phe39, Lys40, Glu49, Arg52, Tyr53 and
Leu56. Residues in bold are critical residues in the interaction with the light
chains, not only by being conserved in different PpL domains, but also by being
largely buried upon complex formation. The second binding interface involves
15 residues from the V L domain, 10 of them in common with the first binding
interface. None of the PpL domain residues that contribute significantly for the
second binding interface are involved in the first one. Arg52 is a common residue
to both interfaces, although this position is not conserved amongst different Igbinding
domains from PpL (it is replaced by an Ala). The 14 PpL domain residues
involved in the second interaction are located in strand 3 and helix (Phe43,
Glu44, Thr47, Ala48, Tyr51, Arg52, Asp55, Tyr64, Thr65, Ala66, Asp67, Leu68,
Fig. 2. Basic structure of immunoglobulins (Ig) (a) showing the main components
of IgG: the Fab fragments contain the antigen-binding sites of the molecule whereas the
Fc fragment comprise of the C H 2 and C H 3 domains as well as the carbohydrate portion.
The hinge region is responsible for the flexibility of the Ig molecules, particularly
conferring a wide range of movements to the Fab portions. The X-ray crystallographic
structure of the complex formed between two human Fab fragments and one Protein L
(PpL) domain is shown on part (b) of the figure (1HEZ.pdb). The structural information
inferred from this biological interaction was used as the basis for the de novo design
of PpL biomimetic ligands.