Crystal structure of the N domain of human somatic angiotensin I-converting enzyme provides a structural basis for domain-specific inhibitor design

Human somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of "domain-specific" second-generation ACE inhibitors.     

The role of glycosylation and domain interactions in the thermal stability of human angiotensin-converting enzyme

Abstract The N and C domains of somatic angiotensin-converting enzyme (sACE) differ in terms of their substrate specificity, inhibitor profiling, chloride dependency and thermal stability. The C domain is thermally less stable than sACE or the N domain. Since both domains are heavily glycosylated, the effect of glycosylation on their thermal stability was investigated by assessing their catalytic and physicochemical properties. Testis ACE (tACE) expressed in mammalian cells, mammalian cells in the presence of a glucosidase inhibitor and insect cells yielded proteins with altered catalytic and physicochemical properties, indicating that the more complex glycans confer greater thermal stabilization. Furthermore, a decrease in tACE and N-domain N-glycans using site-directed mutagenesis decreased their thermal stability, suggesting that certain N-glycans have an important effect on the protein's thermodynamic properties. Evaluation of the thermal stability of sACE domain swopover and domain duplication mutants, together with sACE expressed in insect cells, showed that the C domain contained in sACE is less dependent on glycosylation for thermal stabilization than a single C domain, indicating that stabilizing interactions between the two domains contribute to the thermal stability of sACE and are decreased in a C-domain-duplicating mutant.     

Roots‐eye view: Using microdialysis and microCT to non‐destructively map root nutrient depletion and accumulation zones

Improvement in fertilizer use efficiency is a key aspect for achieving sustainable agriculture in order to minimize costs, greenhouse gas emissions, and pollution from nutrient run‐off. To optimize root architecture for nutrient uptake and efficiency, we need to understand what the roots encounter in their environment. Traditional methods of nutrient sampling, such as salt extractions can only be done at the end of an experiment, are impractical for sampling locations precisely and give total nutrient values that can overestimate the nutrients available to the roots. In contrast, microdialysis provides a non‐invasive, continuous method for sampling available nutrients in the soil. Here, for the first time, we have used microCT imaging to position microdialysis probes at known distances from the roots and then measured the available nitrate and ammonium. We found that nitrate accumulated close to roots whereas ammonium was depleted demonstrating that this combination of complementary techniques provides a unique ability to measure root‐available nutrients non‐destructively and in almost real time.     

Cryo‐EM reveals mechanisms of angiotensin I‐converting enzyme allostery and dimerization

Hypertension (high blood pressure) is a major risk factor for cardiovascular disease, which is the leading cause of death worldwide. The somatic isoform of angiotensin I‐converting enzyme (sACE) plays a critical role in blood pressure regulation, and ACE inhibitors are thus widely used to treat hypertension and cardiovascular disease. Our current understanding of sACE structure, dynamics, function, and inhibition has been limited because truncated, minimally glycosylated forms of sACE are typically used for X‐ray crystallography and molecular dynamics simulations. Here, we report the first cryo‐EM structures of full‐length, glycosylated, soluble sACE (sACE^S1211). Both monomeric and dimeric forms of the highly flexible apo enzyme were reconstructed from a single dataset. The N‐ and C‐terminal domains of monomeric sACE^S1211 were resolved at 3.7 and 4.1 Å, respectively, while the interacting N‐terminal domains responsible for dimer formation were resolved at 3.8 Å. Mechanisms are proposed for intradomain hinging, cooperativity, and homodimerization. Furthermore, the observation that both domains were in the open conformation has implications for the design of sACE modulators.     

Are combined orthopaedic and rheumatology clinics worth while?

The practice of holding combined orthopaedic and rheumatology clinics is widespread, but no attempt has been made to assess their usefulness. In a one year prospective study patients were randomised either to a combined clinic with a rheumatologist and an orthopaedic surgeon or to a clinic with an orthopaedic surgeon alone. The details of the patients'' disease, the interview, the surgeons'' and rheumatologists'' responses, and the patients'' opinions were recorded on questionnaires. Interviews were appreciably longer in the combined clinic, and more referrals for surgery were made. The rheumatologist correctly predicted the need for surgery in 95% of cases, and his presence in the clinic was considered desirable by the surgeon, principally when the referral letter was inadequate. It is concluded that most patients with rheumatic diseases can be seen more efficiently in routine orthopaedic clinics provided a good quality letter of referral is sent.     

Loss of phylogenetic information in chorion gene families of Bombyx mori gene conversion

The silkmoth chorion has provided a stimulating model for the study of evolution and developmental regulation of gene families. Previous attempts at inferring relationships among chorion sequences have been based on pairwise comparisons of overall similarity, a potentially problematic approach. To remedy this, we identified the alignable regions of low sequence variability and then analyzed this restricted database by parsimony and neighbor-joining methods. At the deepest level, the chorion sequence tree is split into two branches, called "alpha" and "beta." Within each branch, early- and late-expressing genes each constitute monophyletic groups, while the situation with middle-expressing genes remains uncertain. The HcB gene family appears to be the most basal beta-branch group, but this conclusion is qualified because the effect of gene conversion on branching order is unknown. Previous studies by Eickbush and colleagues have strongly suggested that ErA, HcA, and HcB families undergo gene conversion within a gene family, whereas the ErB family does not. The occurrence of conversion correlates with a particular tree structure; namely, branch lengths are much greater at the base of the family than at higher internodes and terminal branches. These observations raise the possibility that chorion gene families are defined by gene conversion events (reticulate evolution) rather than by descent with modification (synapomorphy).