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 2.0Å and 2.8Å, 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.Crystallisable glycosylation mutants open up new possibilities for co-crystallisation studies to aid the design of novel ACE inhibitors. Keywordsangiotensin-converting enzyme; X-ray crystal structure; testis ACE; normal mode; hinge-bending Since its isolation in 1956 as "hypertensin-converting enzyme", human angiotensinconverting enzyme (ACE 1 ) has been known to play a key role in the regulation of blood † This work was supported by the Carnegie Corporation of New York, the University of Cape Town, the South Afican National Research Foundation and the Wellcome Trust (UK) grants 070060 and 071047. # The atomic coordinates and structure factors for glycosylation mutants tACE-G13 (codes 2iul and r2iulsf) and tACE-G1234 (codes 2iux and r2iuxsf) have been deposited in the RCSB Protein Data Bank, www.pdb.org. 1 Abbreviations: ACE, angiotensin converting enzyme 1; sACE, somatic ACE; tACE, testis ACE; NBDNJ, N-butyldeoxynojirimycin; Ndom, N-terminal domain of sACE; ACE2, human angiotensin-converting enzyme 2; NMA, normal mode analysis; tACE-G13, mutant of human tACE lacking the N-terminal 36 residues and all but the first and third N-glycosylation sites; tACE-G1234, mutant of human tACE lacking the N-terminal 36 residues and all but the first four N-glycosylation sites; ACE2o, open unliganded structure of ACE2; ACE2c, closed liganded structure of ACE2; tACEo, model of human tACE based on ACE2o; tACEc, model of human tACE based on ACE2c; NAG, N-acetyl glucosamine.

Angiotensin-Converting Enzyme - New Insights into Structure, Biological Significance and Prospects for Domain-Selective Inhibitors

“…Similar hinging could be predicted for unliganded ACE2 and a modelled open form of ACE by normal mode analysis and is thus probably an intrinsic feature of this type of fold [79]. Also consistent with a closing active site model for ACE are crystallographic temperature factors [79], the preferential binding of monoclonal antibodies to liganded, over unliganded, N domain [80], and calorimetric studies that revealed a large entropic component to substrate binding [81].…”

“…Moreover, a human ACE homologue, ACE2, has been discovered and shown to be important for the binding of the severe acute respiratory syndrome (SARS) coronavirus (reviewed by [11,12]). The solution of crystal structures of both ACE domains (N and C), as well as co-crystal struc-*Address correspondence to this author at the ITN-UCSF, 185 Berry Street, Suite 3515, San Francisco, CA 94107, USA; Tel: (415) 353-4402; E-mail mehlers@immunetolerance.org tures with a number of inhibitors, has allowed the formation of a clearer understanding of the functioning of the active site and of the reasons for some of the differences between the domains [13][14][15][16][17][18][19]. This wealth of knowledge has begun to inform new developments in ACE inhibitor design, which aim to improve on the safety and efficacy of ACE inhibitor therapy.…”

Simulated Interactions between Angiotensin-Converting Enzyme and Substrate Gonadotropin-Releasing Hormone: Novel Insights into Domain Selectivity

“…Normal-mode analysis revealed intrinsic flexibility about the active site of tACE so that six hinge regions could be identified: 98-125, 296-297, 400-409, 434-439, 535-537, and 569-578 (tACE numbering). In particular, region 98-125 lies close to the surface between the R2 lid helix and R4; region 569-578 lies between the conserved HEMGH motif and E411 that binds zinc so that hinging about this region results in opening up the catalytic site (52). The mobility of the simulated ACE-GnRH complexes was investigated by calculating the B-factors of the backbone CR atoms from their root-mean square fluctuations during the 2-ns MD simulation (Figure 4).…”

Angiotensin I–Converting Enzyme Inhibitors Are Allosteric Enhancers of Kinin B1 and B2 Receptor Function

“…The two active domains of ACE have different specificities64 and exhibit negative cooperativity 65; binding of ACE inhibitor to one domain alters the conformation of the second domain, rendering it inaccessible to a second inhibitor. The crystal structures of the C- or N- domain revealed a deep active site cleft closed to exterior by a “lid”; consequently, a conformational change would be required for the access of substrate or inhibitor to the enzyme 61, 66, 67. Modeling of the ACE structure indicates intrinsic flexibility around the active site, suggesting a hinge mechanism to open it for substrate/inhibitor binding 67.…”