PDBsum entry 3i9g

Immune system

PDB id: 3i9g

Contents

Protein chains

222 a.a. *

213 a.a. *

Ligands

S1P

Metals

_CA ×2

_MG ×5

Waters ×283

* Residue conservation analysis

PDB id:

3i9g

Name:

Immune system

Title:

Crystal structure of the lt1009 (sonepcizumab) antibody fab fragment in complex with sphingosine-1-phosphate

Structure:

Sonepcizumab antibody fab fragment, heavy chain. Chain: h. Fragment: fab fragment. Engineered: yes. Sonepcizumab antibody fab fragment, light chain. Chain: l. Fragment: fab fragment. Engineered: yes

Source:

Mus musculus. Organism_taxid: 10090. Gene: igg1k. Expressed in: cricetulus griseus. Expression_system_taxid: 10029. Expression_system_cell_line: cho cells. Expression_system_cell_line: cho cells

Resolution:

1.90Å R-factor: 0.192 R-free: 0.219

Authors:

T.Huxford

Key ref:

J.M.Wojciak et al. (2009). The crystal structure of sphingosine-1-phosphate in complex with a Fab fragment reveals metal bridging of an antibody and its antigen. Proc Natl Acad Sci U S A, 106, 17717-17722. PubMed id: 19815502 DOI: 10.1073/pnas.0906153106

Date:

10-Jul-09 Release date: 29-Sep-09

Protein chain

No UniProt id for this chain

Struc: 222 a.a.

Protein chain

No UniProt id for this chain

Struc: 213 a.a.

DOI no: 10.1073/pnas.0906153106

Proc Natl Acad Sci U S A 106:17717-17722 (2009)

PubMed id: 19815502

The crystal structure of sphingosine-1-phosphate in complex with a Fab fragment reveals metal bridging of an antibody and its antigen.

J.M.Wojciak, N.Zhu, K.T.Schuerenberg, K.Moreno, W.S.Shestowsky, M.Hiraiwa, R.Sabbadini, T.Huxford.

ABSTRACT

The pleiotropic signaling lipid sphingosine-1-phosphate (S1P) plays significant roles in angiogenesis, heart disease, and cancer. LT1009 (also known as sonepcizumab) is a humanized monoclonal antibody that binds S1P with high affinity and specificity. Because the antibody is currently in clinical trials, it is important to confirm by structural and biochemical analyses that it binds its target in a predictable manner. Therefore, we determined the structure of a complex between the LT1009 antibody Fab fragment and S1P refined to 1.90 A resolution. The antibody employs unique and diverse strategies to recognize its antigen. Two metal ions bridge complementarity determining regions from the antibody light chain and S1P. The coordination geometry, inductively coupled plasma spectroscopy, surface plasmon resonance spectroscopy, and biochemical assays suggest that these are Ca(2+). The amino alcohol head group of the sphingosine backbone is recognized through hydrogen bonding interactions from 1 aa side chain and polypeptide backbone atoms of the antibody light and heavy chains. The S1P hydrophobic tail is almost completely enclosed within a hydrophobic channel formed primarily by the heavy chain. Both treatment of the complex with metal chelators and mutation of amino acids in the light chain that coordinate the metal atoms or directly contact the polar head group abrogate binding, while mutations within the hydrophobic cavity also decrease S1P binding affinity. The structure suggests mechanistic details for recognition of a signaling lipid by a therapeutic antibody candidate. Moreover, this study provides direct structural evidence that antibodies are capable of using metals to bridge antigen:antibody complexes.

Selected figure(s)

Figure 2.
X-ray crystal structure of the LT1009 Fab:S1P complex. (A) A ribbon diagram representation of the antibody Fab fragment. The light chain is colored gold and the heavy chain is brown. S1P is depicted as a ball-and-stick model with cpk coloring and the 2 Ca^2+ ions are represented as light gray spheres. (B) Close-up view of the S1P binding site. Electron density from a 2 F[O] - F[C] refined map contoured at 1.1 σ is displayed for the region of the model within 1.9 Å of each atom in S1P. (C) A semitransparent surface representation of the Fab reveals the extent to which S1P, represented as cpk spheres, is buried upon binding to LT1009.

Figure 5.
(A) Overlay of LT1009 and Q425 anti-CD4 antibody Fab crystal structures illustrates their overall homology and different Ca^2+ binding sites. LT1009 is colored as in previous figures while the Q425 light chain is cyan and the heavy chain fragment is blue. For the sake of clarity, S1P has been removed from the LT1009 Fab model. (B) A close up view of Ca^2+ binding sites in the 2 models reveals conservation of metal coordinating side chains and the different conformations of CDR-H3. (C) Schematic representation of a possible mechanism of S1P binding by LT1009. In its unbound conformation, the light chain (labeled “L” in the figure) binds one Ca^2+ at a site similar to that observed in the Q425 antibody structures. S1P binding introduces conformational changes that disrupt the original Ca^2+ binding motif as well as potentially introducing one additional Ca^2+. The phosphate group of S1P then combines with 2 Ca^2+ to produce the extremely stable Ca^2+ coordination complex observed in the S1P:LT1009 Fab complex crystal structure.

Figures were selected by an automated process.