Department of Chemistry, Virginia Tech, Blacksburg, VA 24061, USA; Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA.
Anal Biochem. 2010 Jul 1;402(1):77-82. doi: 10.1016/j.ab.2010.03.018. Epub 2010 Mar 15.
Peptidyl prolyl cis-trans isomerase (PPIase) interacting with NIMA-1 (Pin1) catalyzes the cis-trans isomerization of pSer/pThr-Pro amide bonds. Pin1 is a two-domain protein that represents a promising target for the treatment of cancer. Both domains of Pin1 bind the pSer/pThr-Pro motif; PPIase enzymatic activity occurs in the catalytic domain, and the WW domain acts as a recognition module for the pSer/pThr-Pro motif. An assay we call an enzyme-linked enzyme-binding assay (ELEBA) was developed to measure the K(d) of ligands that bind selectively to the WW domain. A ligand specific for the WW domain of Pin1 was covalently immobilized in a 96-well plate. Commercially available Pin1 conjugated to horseradish peroxidase was used for chemiluminescent detection of ligands that block the association of the WW domain with immobilized ligand. The peptide ligands were derived from the cell cycle regulatory phosphatase, Cdc25c, residues 45-50. The K(d) values for Fmoc-VPRpTPVGGGK-NH2 and Ac-VPRpTPV-NH2 were determined to be 36+/-4 and 110+/-30 microM, respectively. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain, even in the presence of the catalytic domain. This method may be applied to any dual specificity, multidomain protein.
肽基脯氨酰顺反异构酶（PPIase）与 NIMA-1（Pin1）相互作用，催化 pSer/pThr-Pro 酰胺键的顺反异构化。Pin1 是一种具有两个结构域的蛋白质，是治疗癌症的有希望的靶标。Pin1 的两个结构域都与 pSer/pThr-Pro 基序结合；PPIase 酶活性发生在催化结构域中，而 WW 结构域作为 pSer/pThr-Pro 基序的识别模块。我们开发了一种称为酶联酶结合测定法（ELEBA）的测定法来测量选择性结合 WW 结构域的配体的 K(d)。Pin1 的 WW 结构域的特异性配体被共价固定在 96 孔板中。商购的与辣根过氧化物酶偶联的 Pin1 用于化学发光检测阻止 WW 结构域与固定配体结合的配体。肽配体衍生自细胞周期调节磷酸酶 Cdc25c，残基 45-50。Fmoc-VPRpTPVGGGK-NH2 和 Ac-VPRpTPV-NH2 的 K(d)值分别确定为 36±4 和 110±30 μM。ELEBA 提供了一种选择性方法来检测与 Pin1 WW 结构域结合的配体，即使在存在催化结构域的情况下也是如此。该方法可应用于任何双特异性、多结构域蛋白。
