title: Structure of testis ACE glycosylation mutants and evidence for conserved domain movement.
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 A and 2.8 A, 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.
authors.affiliation: Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa.
authors.forename: Jean M
authors.initials: JM
authors.lastname: Watermeyer
authors.forename: B Trevor
authors.initials: BT
authors.lastname: Sewell
authors.forename: Sylva L
authors.initials: SL
authors.lastname: Schwager
authors.forename: Ramanathan
authors.initials: R
authors.lastname: Natesh
authors.forename: Hazel R
authors.initials: HR
authors.lastname: Corradi
authors.forename: K Ravi
authors.initials: KR
authors.lastname: Acharya
authors.forename: Edward D
authors.initials: ED
authors.lastname: Sturrock
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D017124:Conserved Sequence; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013737:Testis; D014162:Transfection
chemical_list: D007527:Isoenzymes; D007703:Peptidyl-Dipeptidase A