Human testis angiotensin-converting enzyme: crystal structure of a glycosylation mutant and investigation of a putative hinge-mechanism by normal mode analysis
Watermeyer, Jean Margaret
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Human angiotensin-converting enzyme (ACE) is a key enzyme in the regulation of blood pressure via the renin-angiotensin and kallikrein-kinin systems. A number of orally active drugs have been developed over the years that target somatic ACE, for the treatment of hypertension, myocardial infarction and congestive heart failure. Protein structural information about ACE is an important key for the understanding of the mechanism and substrate-specificity of the enzyme. However, this information has only begun to be elucidated in the past year, with the solution of crystal structures of human testis ACE (tACE), and homologues Drosophila AnCE and human ACE2. tACE is identical to the C-terminal domain of somatic ACE, which consists of two homologous domains, each having a slightly different substrate-specificity. This thesis describes the purification, crystallisation and X-ray crystal structure-determination of a glycosylation-deficient mutant of tACE, tACEG1,3, to 2.9 Å. The structure of tACE-G1,3 aligns closely with that of native tACE, indicating that the mutations did not alter the conformation. The ability to achieve minimal glycosylation of tACE for crystallisation purposes via mutation, rather than using expensive glycosidase inhibitors, iii should prove advantageous for further structural studies, such as the study of the binding of novel inhibitors. In all of the tACE structures thus far observed, the active site is closed off from the external medium in a deep cleft, so that it is unclear how a large substrate molecule could gain access. However, a hinge motion that opens this cleft has been observed in the structures of ACE2. Temperature factor and sequence comparison between tACE, tACE-G1,3, AnCE and ACE2 suggests the functional conservation of three flexible loop regions, as well as the sequence conservation of three constrained regions, involved in the hinge. Normal mode analysis reveals the intrinsic flexibility of tACE, and further suggests that a putative open form of tACE would behave similarly to the open form of ACE2. Based on these indications, a conservation of the ACE2 hinge-bending mechanism is proposed. Temperature factor analysis also reveals that subdomain II, containing bound chloride ions, is more structurally rigid than subdomain I, in all structures considered. Based on these results, lines of investigation are suggested that should yield insight into the mechanisms of action of ACE and its association with various substrates and inhibitors, ideally aiding in the development of novel drugs for the treatment of cardiac disease.