Hoare Sam R J, Brown Brock T, Santos Mark A, Malany Siobhan, Betz Stephen F, Grigoriadis Dimitri E
Department of Discovery Biology, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA.
Biochem Pharmacol. 2006 Jul 14;72(2):244-55. doi: 10.1016/j.bcp.2006.04.007. Epub 2006 Apr 25.
The molecular interactions between non-peptide antagonists and the corticotropin-releasing factor type 1 (CRF1) receptor are poorly understood. A CRF1 receptor mutation has been identified that reduces binding affinity of the non-peptide antagonist NBI 27914 (M276I in transmembrane domain 5). We have investigated the mechanism of the mutation's effect using a combination of peptide and non-peptide ligands and receptor mutations. The M276I mutation reduced binding affinity of standard non-peptide antagonists 5-75-fold while having no effect on peptide ligand binding. We hypothesized that the side chain of isoleucine, beta-branched and so rotationally constrained when within an alpha-helix, introduces a barrier to non-peptide antagonist binding. In agreement with this hypothesis, mutation of M276 to the rotationally constrained valine produced similar reductions of affinity as M276I mutation, whereas mutation to leucine (with an unbranched beta-carbon) minimally affected non-peptide antagonist affinity. Mutation to alanine did not appreciably affect non-peptide antagonist affinity, implying the methionine side chain does not contribute directly to binding. Three observations suggested M276I/V mutations interfere with binding of the heterocyclic core of the compounds: (1) all compounds affected by M276I/V mutations possess a planar heterocyclic core. (2) None of the M276 mutations affected binding of an acylic compound. (3) The mutations differentially affected affinity of two compounds that differ only by core methylation. These findings imply that non-peptide antagonists, and specifically the heterocyclic core of such molecules, bind in the vicinity of M276 of the CRF1 receptor. M276 mutations did not affect peptide ligand binding and this residue is distant from determinants of peptide binding (predominantly in the extracellular regions), providing molecular evidence for non-overlapping (allosteric) binding sites for peptide and non-peptide ligands within the CRF1 receptor.
非肽类拮抗剂与促肾上腺皮质激素释放因子1型（CRF1）受体之间的分子相互作用目前了解甚少。已鉴定出一种CRF1受体突变，该突变降低了非肽类拮抗剂NBI 27914的结合亲和力（跨膜结构域5中的M276I）。我们使用肽类和非肽类配体以及受体突变的组合来研究该突变效应的机制。M276I突变使标准非肽类拮抗剂的结合亲和力降低了5至75倍，而对肽类配体的结合没有影响。我们推测，异亮氨酸的侧链在α螺旋中呈β分支且旋转受限，会给非肽类拮抗剂的结合带来障碍。与该假设一致，将M276突变为旋转受限的缬氨酸会产生与M276I突变相似的亲和力降低，而突变为亮氨酸（β碳无分支）对非肽类拮抗剂亲和力的影响最小。突变为丙氨酸对非肽类拮抗剂亲和力没有明显影响，这意味着甲硫氨酸侧链对结合没有直接贡献。三项观察结果表明M276I/V突变会干扰化合物杂环核心的结合：（1）所有受M276I/V突变影响的化合物都具有平面杂环核心。（2）M276突变均未影响无环化合物的结合。（3）这些突变对仅因核心甲基化而不同的两种化合物的亲和力有不同影响。这些发现表明，非肽类拮抗剂，特别是此类分子的杂环核心，在CRF1受体的M276附近结合。M276突变不影响肽类配体的结合，并且该残基与肽类结合的决定因素（主要在细胞外区域）距离较远，这为CRF1受体内肽类和非肽类配体的非重叠（变构）结合位点提供了分子证据。
