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Human somatic angiotensin-converting enzyme (ACE) is a key zinc metallopeptidase that plays a pivotal role in the renin–angiotensin–aldosterone system (RAAS) by regulating blood pressure and electrolyte balance. Inhibition of ACE is a cornerstone in the management of hypertension, cardiovascular diseases, and renal disorders. Recent advances in structural biology techniques have provided invaluable insights into the molecular mechanisms underlying ACE inhibition, facilitating the design and development of more effective therapeutic agents. This review focuses on the latest advancements in elucidating the structural basis for ACE inhibition. High-resolution crystallographic studies of minimally glycosylated individual domains of ACE have revealed intricate molecular details of the ACE catalytic N- and C-domains, and their detailed interactions with clinically relevant and newly designed domain-specific inhibitors. In addition, the recently elucidated structure of the glycosylated form of full-length ACE by cryo-electron microscopy (cryo-EM) has shed light on the mechanism of ACE dimerization and revealed continuous conformational changes which occur prior to ligand binding. In addition to these experimental techniques, computational approaches have also played a pivotal role in elucidating the structural basis for ACE inhibition. Molecular dynamics simulations and computational docking studies have provided atomic details of inhibitor binding kinetics and energetics, facilitating the rational design of novel ACE inhibitors with improved potency and selectivity. Furthermore, computational analysis of the motions observed by cryo-EM allowed the identification of allosteric binding sites on ACE. This affords new opportunities for the development of next-generation allosteric inhibitors with enhanced pharmacological properties. Overall, the insights highlighted in this review could enable the rational design of novel ACE inhibitors with improved efficacy and safety profiles, ultimately leading to better therapeutic outcomes for patients with hypertension and cardiovascular diseases.

Plastic pollution has emerged as a significant environmental concern in recent years and has prompted the exploration of innovative biotechnological solutions to mitigate plastic’s negative impact. The discovery of enzymes capable of degrading specific types of plastics holds promise as a potential solution. However, challenges with efficiency, industrial scalability, and the diverse range of the plastic waste in question, have hindered their widespread application. Structural biology provides valuable insights into the intricate interactions between enzymes and plastic materials at an atomic level, and a deeper understanding of their underlying mechanisms is essential to harness their potential to address the mounting plastic waste crisis. This review article examines the current biochemical and biophysical methods that may facilitate the development of enzymes capable of degrading polyethylene terephthalate (PET), one of the most extensively used plastics. It also discusses the challenges that must be addressed before substantial advancements can be achieved in using these enzymes as a solution to the plastic pollution problem.

Botulinum neurotoxins (BoNTs) cause flaccid neuromuscular paralysis by cleaving one of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. BoNTs display high affinity and specificity for neuromuscular junctions, making them one of the most potent neurotoxins known to date. There are seven serologically distinct BoNTs (serotypes BoNT/A to BoNT/G) which can be further divided into subtypes (e.g., BoNT/A1, BoNT/A2…) based on small changes in their amino acid sequence. Of these, BoNT/A1 and BoNT/B1 have been utilised to treat various diseases associated with spasticity and hypersecretion. There are potentially many more BoNT variants with differing toxicological profiles that may display other therapeutic benefits. This review is focused on the structural analysis of the cell-binding domain from BoNT/A1 to BoNT/A6 subtypes (HC/A1 to HC/A6), including features such as a ganglioside binding site (GBS), a dynamic loop, a synaptic vesicle glycoprotein 2 (SV2) binding site, a possible Lys–Cys/Cys–Cys bridge, and a hinge motion between the HCN and HCC subdomains. Characterising structural features across subtypes provides a better understanding of how the cell-binding domain functions and may aid the development of novel therapeutics.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two late onset neurodegenerative diseases, have been shown to share overlapping cellular pathologies and genetic origins. Studies suggest that a hexanucleotide repeat expansion in the first intron of the C9orf72 gene is the most common cause of familial FTD and ALS pathology. The C9orf72 protein is predicted to be a differentially expressed in normal and neoplastic cells domain protein implying that C9orf72 functions as a guanine nucleotide exchange factor (GEF) to regulate specific Rab GTPases. Reported studies thus far point to a putative role for C9orf72 in lysosome biogenesis, vesicular trafficking, autophagy and mechanistic target of rapamycin complex1 (mTORC1) signaling. Here we report the expression, purification and biochemical characterization of C9orf72 protein. We conclusively show that C9orf72 is a GEF. The distinctive presence of both Rab- and Rho-GTPase GEF activities suggests that C9orf72 may function as a dual exchange factor coupling physiological functions such as cytoskeleton modulation and autophagy with endocytosis.

Botulinum neurotoxins (BoNTs) are categorised into immunologically distinct serotypes BoNT/A to /G). Each serotype can also be further divided into subtypes based on differences in amino acid sequence. BoNTs are ~150 kDa proteins comprised of three major functional domains: an N-terminal zinc metalloprotease light chain (LC), a translocation domain (HN), and a binding domain (HC). The HC is responsible for targeting the BoNT to the neuronal cell membrane, and each serotype has evolved to bind via different mechanisms to different target receptors. Most structural characterisations to date have focussed on the first identified subtype within each serotype (e.g., BoNT/A1). Subtype differences within BoNT serotypes can affect intoxication, displaying different botulism symptoms in vivo, and less emphasis has been placed on investigating these variants. This review outlines the receptors for each BoNT serotype and describes the basis for the highly specific targeting of neuronal cell membranes. Understanding receptor binding is of vital importance, not only for the generation of novel therapeutics but also for understanding how best to protect from intoxication.

Angiotensin-converting enzyme (ACE) has a critical role in cardiovascular function by cleaving the carboxy terminal His-Leu dipeptide from angiotensin I to produce a potent vasopressor octapeptide, angiotensin II. Inhibitors of ACE are a first line of therapy for hypertension, heart failure, myocardial infarction and diabetic nephropathy. Notably, these inhibitors were developed without knowledge of the structure of human ACE, but were instead designed on the basis of an assumed mechanistic homology with carboxypeptidase A 1 . Here we present the X-ray structure of human testicular ACE and its complex with one of the most widely used inhibitors, lisinopril ( N 2 -[(S)-1-carboxy-3-phenylpropyl]- l -lysyl- l -proline; also known as Prinivil or Zestril), at 2.0 Å resolution. Analysis of the three-dimensional structure of ACE shows that it bears little similarity to that of carboxypeptidase A, but instead resembles neurolysin 2 and Pyrococcus furiosus carboxypeptidase 3 —zinc metallopeptidases with no detectable sequence similarity to ACE. The structure provides an opportunity to design domain-selective ACE inhibitors that may exhibit new pharmacological profiles.

Botulinum neurotoxins (BoNT) are a group of clostridial toxins that cause the potentially fatal neuroparalytic disease botulism. Although highly toxic, BoNTs are utilized as therapeutics to treat a range of neuromuscular conditions. Several serotypes (BoNT/A-/G, /X) have been identified with vastly differing toxicological profiles. Each serotype can be further sub-categorised into subtypes due to subtle variations in their protein sequence. These minor changes have been attributed to differences in both the duration of action and potency for BoNT/A subtypes. BoNTs are composed of three domains—a cell-binding domain, a translocation domain, and a catalytic domain. In this paper, we present the crystal structures of the botulinum neurotoxin A2 cell binding domain, both alone and in complex with its receptor ganglioside GD1a at 1.63 and 2.10 Å, respectively. The analysis of these structures reveals a potential redox-dependent Lys-O-Cys bridge close to the ganglioside binding site and a hinge motion between the HCN and HCC subdomains. Furthermore, we make a detailed comparison with the previously reported HC/A2:SV2C structure for a comprehensive structural analysis of HC/A2 receptor binding.

We recently identified angiogenin ( ANG ) as a candidate susceptibility gene for amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by adult-onset loss of motor neurons. We now report the finding of seven missense mutations in 15 individuals, of whom four had familial ALS and 11 apparently 'sporadic' ALS. Our findings provide further evidence that variations in hypoxia-inducible genes have an important role in motor neuron degeneration.

Human angiotensin-I-converting enzyme (ACE) is involved in vasoregulation, inflammation, and neurodegenerative disorders. The enzyme is formed of two domains; the C-domain (cACE) is primarily involved in blood pressure regulation, whereas the N-domain (nACE) is strongly linked to fibrosis; hence, designing domain-specific inhibitors could make a difference between treating one condition without having a negative effect on another. AnCE (a close homologue of ACE) is derived from Drosophila melanogaster and has a high similarity specifically to cACE. Due to high similarity and ease of crystallisation, AnCE has been chosen as a model protein for ACE studies and for the design of ACE inhibitors. In this study, enzyme kinetic assays and X-ray crystallography techniques revealed the significance of using dipeptides as selective inhibitors for AnCE and how this knowledge could be applied to cACE and nACE. All the dipeptides tested in this study were shown to bind AnCE in two distinct locations, i.e., the non-prime and prime subsites. It was found that a hydrophobic residue at the S1 and S1′ subsites, with a tryptophan at the S2 and S2′ subsites, showed highest affinity towards AnCE. It was also observed that a key pocket within the S2′ subsite had a major influence on the binding orientation within the prime subsites and could potentially explain ACE’s dipeptidyl carboxypeptidase activity. Importantly these dipeptides are found in functional foods, making them potentially available from diets. Knowledge of the dipeptide binding presented here could aid in the development of ACE domain-specific inhibitors.

Angiogenin ( hANG ), a member of the Ribonuclease A superfamily has angiogenic, neurotrophic and neuroprotective activities. Mutations in hANG have been found in patients with Amyotrophic lateral sclerosis (ALS). The zebrafish ( Danio rerio ) rnasel-1 , 2 and 3 are orthologues of hANG and of these only Rnasel-1 and Rnasel-2 have been shown to be angiogenic. Herein we show that NCI-65828, a potent and specific small molecule inhibitor of hANG inhibits Rnasel-1 to a similar extent. Treatment of early zebrafish embryos with NCI-65828, or with terrein, a fungal metabolite which prevents the secretion of hANG, resulted in spinal neuron aberrations as well defects in trunk vasculature. Our detailed expression analysis and inhibitor studies suggest that Rnasel-1 plays important roles in neuronal migration and pathfinding as well as in angiogenesis in zebrafish. Our studies suggest the usefulness of the zebrafish as a model to dissect the molecular consequences of the ANG ALS variants.