PDBsum entry 2ap2

Immune system

PDB id: 2ap2

Contents

Protein chains

239 a.a. *

12 a.a. *

14 a.a. *

Waters ×64

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Antibody c219 recognizes an alpha-Helical epitope on p-Glycoprotein.

Authors

J.M.Van den elsen, D.A.Kuntz, F.J.Hoedemaeker, D.R.Rose.

Ref.

Proc Natl Acad Sci U S A, 1999, 96, 13679-13684. [DOI no: 10.1073/pnas.96.24.13679]

PubMed id

10570132

Abstract

The ABC transporter, P-glycoprotein, is an integral membrane protein that mediates the ATP-driven efflux of drugs from multidrug-resistant cancer and HIV-infected cells. Anti-P-glycoprotein antibody C219 binds to both of the ATP-binding regions of P-glycoprotein and has been shown to inhibit its ATPase activity and drug binding capacity. C219 has been widely used in a clinical setting as a tumor marker, but recent observations of cross-reactivity with other proteins, including the c-erbB2 protein in breast cancer cells, impose potential limitations in detecting P-glycoprotein. We have determined the crystal structure at a resolution of 2.4 A of the variable fragment of C219 in complex with an epitope peptide derived from the nucleotide binding domain of P-glycoprotein. The 14-residue peptide adopts an amphipathic alpha-helical conformation, a secondary structure not previously observed in structures of antibody-peptide complexes. Together with available biochemical data, the crystal structure of the C219-peptide complex indicates the molecular basis of the cross-reactivity of C219 with non-multidrug resistance-associated proteins. Alignment of the C219 epitope with the recent crystal structure of the ATP-binding subunit of histidine permease suggests a structural basis for the inhibition of the ATP and drug binding capacity of P-glycoprotein by C219. The results provide a rationale for the development of C219 mutants with improved specificity and affinity that could be useful in antibody-based P-glycoprotein detection and therapy in multidrug resistant cancers.

Figure 2.
Fig. 2. Molecular surface representation of the scFv C219 (molecule II) binding site with the bound -helical P-glycoprotein epitope peptide. The molecular surface is colored for electrostatic potential (red for negative charge, blue for positive charge). Peptide residues and the approximate locations of C219 heavy (H) and light chain (L) hypervariable loops are indicated. Fig. 2 was produced with the program GRASP (32).

Figure 3.
Fig. 3. Interactions between the -helical peptide and the C219 binding site. (A) Two-dimensional LIGPLOT (33) representation of the interactions between residues of the minimal NBD-epitope peptide (P), C219 heavy (H) and light chain (L) residues, and solvent molecules (S), as seen in molecule I. The residues that form van der Waals contacts with the peptide are depicted as labeled arcs with radial spokes pointing toward the peptide atoms with which they interact. C219 residues that form hydrogen bonds are shown in a ball-and-stick representation, and the hydrogen bonds are presented as dashed lines. Of all of the intrapeptide hydrogen bonds present in the structure, only the bonds between Gln 3P and Asp 7P are shown. (B) Stereoplot of the Fv-peptide interactions seen in molecule II. (C) Comparison of the bound NBD-epitope peptide in molecule I and II. In B and C, light (L) and heavy chain (H) residues and backbone positions of the scFv C219 are shown in green and magenta. Peptide backbone and side chains are shown in khaki for molecule I and in gold for molecule II. Positions of water molecules are indicated as red spheres. Different positions of binding site residues and water molecules in molecule I are also colored khaki. B and C were generated by using MOLSCRIPT (34) and RASTER3D (35).

Secondary reference #1

Title

A single chain fv fragment of p-Glycoprotein-Specific monoclonal antibody c219. Design, Expression, And crystal structure at 2.4 a resolution.

Authors

F.J.Hoedemaeker, T.Signorelli, K.Johns, D.A.Kuntz, D.R.Rose.

Ref.

J Biol Chem, 1997, 272, 29784-29789. [DOI no: 10.1074/jbc.272.47.29784]

PubMed id

9368049

Figure 4.
Fig. 4. Detail of the electron density at the V[L]-V[H] interface, contoured at 1 . View is "up" toward the CDRs along the pseudo-2-fold^ axis. Two short-range hydrogen bonds between glutamines in the^ light and heavy chains are indicated in green. Figs. 4 and 5A^ were produced with SETOR (49).

Figure 6.
Fig. 6. Helical wheel plot of the peptide epitope. Hydrophobic residues are indicated with boldface lettering; residues that are potentially important for binding are boxed (13). The epitope^ sequence is extended by one helix turn (VVQEALDKARE) to show the^ continuous amphiphilicity.

The above figures are reproduced from the cited reference with permission from the ASBMB