The N domain of human angiotensin-I-converting enzyme: The role of N-glycosylation and the crystal structure in complex with an N domain-specific phosphinic inhibitor, RXP407
Anthony, C. S., Corradi, H. R., Schwager, S. L. U., Redelinghuys, P., Georgiadis, D., Dive, V., Acharya, K. R. and Sturrock, E. D., 2010. The N domain of human angiotensin-I-converting enzyme: The role of N-glycosylation and the crystal structure in complex with an N domain-specific phosphinic inhibitor, RXP407. Journal of Biological Chemistry, 285 (46), pp. 35685-35693.
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Angiotensin-I-converting enzyme (ACE) plays a critical role in the regulation of blood pressure through its central role in the renin-angiotensin and kallikrein-kinin systems. ACE contains two domains, the N and C domains, both of which are heavily glycosylated. Structural studies of ACE have been fraught with severe difficulties because of surface glycosylation of the protein. In order to investigate the role of glycosylation in the N domain and to create suitable forms for crystallization, we have investigated the importance of the 10 potential N-linked glycan sites using enzymatic deglycosylation, limited proteolysis, and mass spectrometry. A number of glycosylation mutants were generated via site-directed mutagenesis, expressed in CHO cells, and analyzed for enzymatic activity and thermal stability. At least eight of 10 of the potential glycan sites are glycosylated; three C-terminal sites were sufficient for expression of active N domain, whereas two N-terminal sites are important for its thermal stability. The minimally glycosylated Ndom389 construct was highly suitable for crystallization studies. The structure in the presence of an N domain-selective phosphinic inhibitor RXP407 was determined to 2.0 A resolution. The Ndom389 structure revealed a hinge region that may contribute to the breathing motion proposed for substrate binding.
|Creators||Anthony, C. S., Corradi, H. R., Schwager, S. L. U., Redelinghuys, P., Georgiadis, D., Dive, V., Acharya, K. R. and Sturrock, E. D.|
|Departments||Faculty of Science > Biology & Biochemistry|
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