Arginines 97 and 108 in CYP2C9 are important determinants of the catalytic function.

Human cytochrome P450 2C9 (CYP2C9) is one of the major drug metabolising enzymes which exhibits a broad substrate specificity. The B-C loop is located in the active-site but has been difficult to model, owing to its diverse and flexible structure. To elucidate the function of the B-C loop we used homology modelling based on the Cyp102 structure in combination with functional studies of mutants using diclofenac as a model substrate for CYP2C9. The study shows the importance of the conserved arginine in position 97 and the arginine in position 108 for the catalytic function. The R97A mutant had a 13-fold higher K(m) value while the V(max) was in the same order as the wild type. The R108 mutant had a 100-fold lower activity with diclofenac compared to the wild-type enzyme. The other six mutants (S95A, F100A, L102A, E104A, R105A, and N107A) had kinetic parameters similar to the CYP2C9 wild-type. Our homology model based on the CYP102 structure as template indicates that R97, L102, and R105 are directed into the active site, whereas R108 is not. The change in catalytic function when arginine 97 was replaced with alanine and the orientation of this amino acid in our homology model indicates its importance for substrate interaction.

A new cytokine-receptor binding mode revealed by the crystal structure of the IL-1 receptor with an antagonist.

Inflammation, regardless of whether it is provoked by infection or by tissue damage, starts with the activation of macrophages which initiate a cascade of inflammatory responses by producing the cytokines interleukin-1 (IL-1) and tumour necrosis factor-alpha (ref. 1). Three naturally occurring ligands for the IL-1 receptor (IL1R) exist: the agonists IL-1alpha and IL-1beta and the IL-1-receptor antagonist IL1RA (ref. 2). IL-1 is the only cytokine for which a naturally occurring antagonist is known. Here we describe the crystal structure at 2.7 A resolution of the soluble extracellular part of type-I IL1R complexed with IL1RA. The receptor consists of three immunoglobulin-like domains. Domains 1 and 2 are tightly linked, but domain three is completely separate and connected by a flexible linker. Residues of all three domains contact the antagonist and include the five critical IL1RA residues which were identified by site-directed mutagenesis. A region that is important for biological function in IL-1beta, the 'receptor trigger site' is not in direct contact with the receptor in the IL1RA complex. Modelling studies suggest that this IL-1beta trigger site might induce a movement of domain 3.