Susan F Foley, Herman W T van Vlijmen, Raymond E Boynton, Heather B Adkins, Anne E Cheung, Juswinder Singh, Michele Sanicola, Carmen N Young, Dingyi Wen
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引用次数: 20
摘要
人类CRIPTO蛋白的CRIPTO/FRL-1/CRYPTIC (CFC)结构域的二硫结构是通过酶促和化学裂解相结合的方法确定的,随后对片段进行色谱分离,并通过质谱和n端测序进行表征。这些研究表明,Cys115与Cys133、Cys128与Cys149、Cys131与Cys140形成二硫键。蛋白质数据库检索和分子模型显示,Cripto的CFC结构域的二硫键模式与丝氨酸蛋白酶抑制剂PARS interbralis major Peptide C (PMP-C)中的二硫键模式相同,并且预计Cripto的EGF-CFC结构域在结构上与Jagged 1和Jagged 2的C端胞外部分的EGF-VWFC结构域同源。通过荧光活化细胞分选分析对ALK4与Cripto的CFC结构域相互作用的生化研究表明,CFC结构域独立于EGF结构域与ALK4结合。基于PMP-C的核磁共振结构,构建了Cripto的CFC结构域分子模型。该模型揭示了在假定的ALK4结合位点的相反区域有一个疏水斑块。这种疏水斑块可能对分子内或分子间复合物的形成具有重要的功能。
The CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto. Functional and structural insights through disulfide structure analysis.
The disulfide structure of the CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto protein was determined by a combination of enzymatic and chemical fragmentation, followed by chromatographic separation of the fragments, and characterization by mass spectrometry and N-terminal sequencing. These studies showed that Cys115 forms a disulfide bond with Cys133, Cys128 with Cys149, and Cys131 with Cys140. Protein database searching and molecular modeling revealed that the pattern of disulfide linkages in the CFC domain of Cripto is the same as that in PARS intercerebralis major Peptide C (PMP-C), a serine protease inhibitor, and that the EGF-CFC domains of Cripto are predicted to be structurally homologous to the EGF-VWFC domains of the C-terminal extracellular portions of Jagged 1 and Jagged 2. Biochemical studies of the interactions of ALK4 with the CFC domain of Cripto by fluorescence-activated cell sorter analysis indicate that the CFC domain binds to ALK4 independent of the EGF domain. A molecular model of the CFC domain of Cripto was constructed based on the nuclear magnetic resonance structure of PMP-C. This model reveals a hydrophobic patch in the domain opposite to the presumed ALK4 binding site. This hydrophobic patch may be functionally important for the formation of intra or intermolecular complexes.