Abstract
The complement system is an important part of the innate immune response to infection but may also cause severe complications during inflammation. Small molecule antagonists to complement receptor 3 (CR3) have been widely sought, but a structural basis for their mode of action is not available. We report here on the structure of the human CR3 ligand-binding I domain in complex with simvastatin. Simvastatin targets the metal ion-dependent adhesion site of the open, ligand-binding conformation of the CR3 I domain by direct contact with the chelated Mg(2+) ion. Simvastatin antagonizes I domain binding to the complement fragments iC3b and C3d but not to intercellular adhesion molecule-1. By virtue of the I domain's wide distribution in binding kinetics to ligands, it was possible to identify ligand binding kinetics as discriminator for simvastatin antagonism. In static cellular experiments, 15-25 μm simvastatin reduced adhesion by K562 cells expressing recombinant CR3 and by primary human monocytes, with an endogenous expression of this receptor. Application of force to adhering monocytes potentiated the effects of simvastatin where only a 50-100 nm concentration of the drug reduced the adhesion by 20-40% compared with untreated cells. The ability of simvastatin to target CR3 in its ligand binding-activated conformation is a novel mechanism to explain the known anti-inflammatory effects of this compound, in particular because this CR3 conformation is found in pro-inflammatory environments. Our report points to new designs of CR3 antagonists and opens new perspectives and identifies druggable receptors from characterization of the ligand binding kinetics in the presence of antagonists.
Highlights
Tiple infectious agents and in guiding the adaptive immune effector and memory responses
Using x-ray crystallography and molecular dynamics (MD) simulations, we reveal that the carboxylate in the hydroxy acid form of simvastatin engages in a ligand receptor-like complex with the metal ion-dependent adhesion site (MIDAS) of the ␣MI
The initial electron density indicated that one molecule of simvastatin in its hydroxy acid form (Fig. 1A) acted as a ligand to the MIDAS Mg2ϩ ion, using its carboxylate group to complete the coordination sphere of the Mg2ϩ ion (Fig. 1, B and C)
Summary
The Crystal Structure of the ␣MI-Simvastatin Complex Reveals the Molecular Basis for Simvastatin Binding—To examine the molecular basis for the effect of simvastatin on CR3, we determined the crystal structure of the CR3 I domain bound to simvastatin at 2.0 Å resolution (Fig. 1 and Table 1). As in the crystal structure, Ser-142, Ser-144, and Thr-209 formed hydrogen bonds to the simvastatin carboxylate group These were maintained as stable contacts during the simulation, with minimal fluctuations in bond length (Fig. 2B). We extracted the binding kinetics for ␣MI binding to its ligands from the single sensorgrams shown, D–F These were produced with 10 M ␣MI and a range of simvastatin concentrations from 0 to 100 M. As shown from the inclusion of the raw signals Fcoated and Funcoated (Fig. 8B), the simvastatin concentration significantly impacted the background binding in uncoated wells This creates a difficulty in comparing the cell binding through Equation 1, because there is a non-linear change in CA with changes in Funcoated, ѨCA ѨFuncoated ϭ
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