Structure of testis ACE glycosylation mutants and evidence for conserved domain movement
Journal Article
2006
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Biochemistry
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American Chemical Society
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University of Cape Town
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Abstract
Human angiotensin-converting enzyme is an important drug target for which little structural information has been available until recent years. The slow progress in obtaining a crystal structure was due to the problem of surface glycosylation, a difficulty that has thus far been overcome by the use of a glucosidase-1 inhibitor in the tissue culture medium. However, the prohibitive cost of these inhibitors and incomplete glucosidase inhibition makes alternative routes to minimizing the N-glycan heterogeneity desirable. Here, glycosylation in the testis isoform (tACE) has been reduced by Asn-Gln point mutations at N-glycosylation sites, and the crystal structures of mutants having two and four intact sites have been solved to 2.0 Å and 2.8 Å, respectively. Both mutants show close structural identity with the wild-type. A hinge mechanism is proposed for substrate entry into the active cleft, based on homology to human ACE2 at the levels of sequence and flexibility. This is supported by normal-mode analysis that reveals intrinsic flexibility about the active site of tACE. Subdomain II, containing bound chloride and zinc ions, is found to have greater stability than subdomain I in the structures of three ACE homologues. Crystallizable glycosylation mutants open up new possibilities for cocrystallization studies to aid the design of novel ACE inhibitors.
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Watermeyer, J. M., Sewell, B. T., Schwager, S. L., Natesh, R., Corradi, H. R., Acharya, K. R., & Sturrock, E. D. (2006). Structure of testis ACE glycosylation mutants and evidence for conserved domain movement. Biochemistry, 45(42), 12654-12663.