Littérature scientifique sur le sujet « AtGLR3.3 ligand-binding domain structure »

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 Mg2+ 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.

Abstract The αIIbβ3 headpiece (αIIb, 1–621; β3, 1–472) was expressed in CHO cells, purified, digested with chymotrypsin, mixed with either mAb 10E5 Fab (form A) or without (form B), repurified, digested with carboxypeptidase (leaving αIIb, 1–452 and β3, 1–440) and crystallized with PEG, Mg acetate, and Na cacodylate at 4°C. Cocrystallization of αIIbβ3/10E5 (A) with eptifibatide or tirofiban was with imidazole instead of cacodylate. Crystals were diffracted at APS and CHESS and analyzed by HKL2000, AMoRe, O, CNS, and CCP4 software. Crystal forms A and B contain 1 and 3 molecules/asymmetric unit (2.7–3.1 and 2.9 Å resolution), respectively. Ca2+ was assigned at the 4 αIIb β-hairpin sites in blades 4–7, and I-like (βA) LIMBS and ADMIDAS; Mg2+ was assigned to MIDAS. The major findings are: 1) As compared to unliganded αVβ3, αIIbβ3 has a ~62° outward pivot of the β3 hybrid domain from the I-like (βA) domain, indicating adoption of an open, high affinity conformation driven by cacodylate or the Asp (D) carboxyl of the drugs binding to MIDAS and acting as activating ligand equivalents. 2) The αIIb ligand binding cleft is rigid and includes αIIb D224 [end-on H bond to ligand Lys (K) or Arg (R)] and hydrophobic residues F160, Y190, and F231, accounting for the selective binding to αIIbβ3 (vs αVβ3) of KGD and homoarginine-GDW peptides, fibrinogen γ-chain peptide, eptifibatide, and tirofiban. 3) 10E5 Fab interacts with a unique “cap” subdomain in αIIb formed by 4 insertions in β-propeller loops in blades 1–3 that form a β-sheet and α-helix structure involved in ligand binding. 4) Comparison of unliganded αVβ3 and liganded αIIbβ3 indicates that receptor activation and ligand binding involves: extensive movement of β3 subunit β1-α1 loop and α1 helix, and β6-α7 loop and α7 helix; alterations in the coordinating residues at the ADMIDAS, MIDAS, and LIMBS; and breaking the ADMIDAS Ca2+ coordination by the M335 backbone carbonyl (providing a mechanism by which Mn2+, which competes with Ca2+ at the ADMIDAS but has a lower propensity for carbonyl coordination than Ca2+, activates integrins). The 62° pivot results from a one-turn piston-like displacement of the α7 helix involving a hydrophobic ratchet of the β6-α7 loop; a ratchet motion of the α1-helix in which L134 moves to the space previously occupied by V340; and complete remodeling at the interface between the β3 I-like (βA) and hybrid domains. 5) The structure of the β3 PSI domain reveals that the long range disulfide is between β3 C13 (rather than C5) and C435, and comparison to the PSI of semaphorin 4D demonstrates that C435 is an integral part of the PSI domain fold. Thus, the I-like (βA) domain appears to be inserted in the hybrid domain, which is inserted in the PSI domain. 6) The structure reveals the location of the Leu/Pro-33 PSI polymorphism responsible for the HPA1 alloantigen. At a rigid interface with the hybrid domain, polymorphism of Arg93 demonstrates the requirement of the hybrid/PSI interface for alloantigenicity at Leu-33. Overall, the structure reveals how allostery regulates ligand binding affinity of αIIbβ3, and how the outward swing of the lever-like hybrid and PSI domains communicates the conformation of the ligand binding site to the α and β leg domains, and to the membrane and cytosol.

Natural killer (NK) cells, large granular lymphocytes, play an important role in the innate immune response against viruses, parasites and tumour cells. NK cells use a wide repertoire of surface receptors to modulate their activity [1]. The family of NKR-P1 surface receptors of NK cells belong to proteins with C-type lectin-like (CTL) fold. The overall architecture of other known CTL receptors (e.g. members of Ly49 family, NKG2D, CD94, mouse CLRg) is conserved [2]. The mechanism of ligand binding has been revealed by the crystal structure of Nkp65 bound to its keratinocyte ligand [3]. However, observation of domain swapping in crystal structure of mouse (m) NKR-P1A represents an unusual structural feature that might be involved in a new mechanism of ligand binding that would be specific for some members of NKR-P1 family. Nevertheless, our crystal structure of mNKR-P1A represents a unique structural observation that demands careful analysis. Even the latest structural studies do not answer the question of function or role of swapped domain of the receptor in potential ligand binding. We have generated new variants of mNKR-P1A of varied chain length that undergo biochemical and structural analysis including mass spectrometry.

Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). Here, the first crystal structure of an ANT(3′′)(9) adenyltransferase, AadA fromSalmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5 Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis.In vivoresistance andin vitrobinding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.