K. Ravi Acharya

Botulinum and tetanus neurotoxins: structure, function and therapeutic utility

The toxic products of the anaerobic bacteria Clostridium botulinum, Clostridium butyricum, Clostridium barati and Clostridium tetani are the causative agents of botulism and tetanus. The ability of botulinum neurotoxins to disrupt neurotransmission, often for prolonged periods, has been exploited for use in several medical applications and the toxins, as licensed pharmaceutical products, now represent the therapeutics of choice for the treatment for several neuromuscular conditions. Research into the structures and activities of botulinum and tetanus toxins has revealed features of these proteins that might be useful in the design of improved vaccines, effective inhibitors and novel biopharmaceuticals. Here, we discuss the relationships between structure, mechanism of action and therapeutic use.

The CCN family of proteins: structure–function relationships

The CCN proteins are key signalling and regulatory molecules involved in many vital biological functions, including cell proliferation, angiogenesis, tumourigenesis and wound healing. How these proteins influence such a range of functions remains incompletely understood but is probably related to their discrete modular nature and a complex array of intra- and inter-molecular interactions with a variety of regulatory proteins and ligands. Although certain aspects of their biology can be attributed to the four individual modules that constitute the CCN proteins, recent results suggest that some of their biological functions require cooperation between modules. Indeed, the modular structure of CCN proteins provides important insight into their structure–function relationships.

Ace revisited: A new target for structure-based drug design

Current-generation angiotensin-converting enzyme (ACE) inhibitors are widely used for cardiovascular diseases, including high blood pressure, heart failure, heart attack and kidney failure, and have combined annual sales in excess of US

Crystal structure of human α-lactalbumin at 1.7 Å resolution

6 billion. However, the use of these ACE inhibitors, which were developed in the late 1970s and early 1980s, is hampered by common side effects. Moreover, we now know that ACE actually consists of two parts (called the N- and C-domains) that have different functions. Therefore, the design of specific domain-selective ACE inhibitors is expected to produce next-generation drugs that might be safer and more effective. Here we discuss the structural features of current inhibitors and outline how next-generation ACE inhibitors could be designed by using the three-dimensional molecular structure of human testis ACE. The ACE structure provides a unique opportunity for rational drug design, based on a combination of in silico modelling using existing inhibitors as scaffolds and iterative lead optimization to drive the synthetic chemistry.

Eosinophil granule proteins: form and function.

Abstract The three-dimensional X-ray structure of human α-lactalbumin, an important component of milk, has been determined at 1·7 A (0·17 nm) resolution by the method of molecular replacement, using the refined structure of baboon α-lactalbumin as the model structure. The two proteins are known to have more than 90% amino acid sequence identity and crystallize in the same orthorhombic space group, P21212. The crystallographic refinement of the structure using the simulated annealing method, resulted in a crystallographic R-factor of 0·209 for the 11,373 observed reflections (F ≥ 2σ(F)) between 8 and 1·7 A resolution. The model comprises 983 protein atoms, 90 solvent atoms and a bound calcium ion. In the final model, the root-mean-square deviations from ideality are 0·013 A for covalent bond distances and 2·9° for bond angles. Superposition of the human and baboon α-lactalbumin structures yields a root-mean-square difference of 0·67 A for the 123 structurally equivalent Cα atoms. The C terminus is flexible in the human α-lactalbumin molecule. The striking structural resemblance between α-lactalbumins and C-type lysozymes emphasizes the homologous evolutionary relationship between these two classes of proteins.

Crystal structure of human Charcot-Leyden crystal protein, an eosinophil lysophospholipase, identifies it as a new member of the carbohydrate-binding family of galectins.

Experimental and clinical data strongly support a role for the eosinophil in the pathogenesis of asthma, allergic and parasitic diseases, and hypereosinophilic syndromes, in addition to more recently identified immunomodulatory roles in shaping innate host defense, adaptive immunity, tissue repair/remodeling, and maintenance of normal tissue homeostasis. A seminal finding was the dependence of allergic airway inflammation on eosinophil-induced recruitment of Th2-polarized effector T-cells to the lung, providing a missing link between these innate immune effectors (eosinophils) and adaptive T-cell responses. Eosinophils come equipped with preformed enzymatic and nonenzymatic cationic proteins, stored in and selectively secreted from their large secondary (specific) granules. These proteins contribute to the functions of the eosinophil in airway inflammation, tissue damage, and remodeling in the asthmatic diathesis. Studies using eosinophil-deficient mouse models, including eosinophil-derived granule protein double knock-out mice (major basic protein-1/eosinophil peroxidase dual gene deletion) show that eosinophils are required for all major hallmarks of asthma pathophysiology: airway epithelial damage and hyperreactivity, and airway remodeling including smooth muscle hyperplasia and subepithelial fibrosis. Here we review key molecular aspects of these eosinophil-derived granule proteins in terms of structure-function relationships to advance understanding of their roles in eosinophil cell biology, molecular biology, and immunobiology in health and disease.

Microbial superantigens: from structure to function.

BACKGROUND The Charcot-Leyden crystal (CLC) protein is a major autocrystallizing constituent of human eosinophils and basophils, comprising approximately 10% of the total cellular protein in these granulocytes. Identification of the distinctive hexagonal bipyramidal crystals of CLC protein in body fluids and secretions has long been considered a hallmark of eosinophil-associated allergic inflammation. Although CLC protein possesses lysophospholipase activity, its role(s) in eosinophil or basophil function or associated inflammatory responses has remained speculative. RESULTS The crystal structure of the CLC protein has been determined at 1.8 A resolution using X-ray crystallography. The overall structural fold of CLC protein is highly similar to that of galectins -1 and -2, members of an animal lectin family formerly classified as S-type or S-Lac (soluble lactose-binding) lectins. This is the first structure of an eosinophil protein to be determined and the highest resolution structure so far determined for any member of the galectin family. CONCLUSIONS The CLC protein structure possesses a carbohydrate-recognition domain comprising most, but not all, of the carbohydrate-binding residues that are conserved among the galectins. The protein exhibits specific (albeit weak) carbohydrate-binding activity for simple saccharides including N-acetyl-D-glucosamine and lactose. Despite CLC protein having no significant sequence or structural similarities to other lysophospholipase catalytic triad has also been identified within the CLC structure, making it a unique dual-function polypeptide. These structural findings suggest a potential intracellular and/or extracellular role(s) for the galectin-associated activities of CLC protein in eosinophil and basophil function in allergic diseases and inflammation.

Cwp84, a Surface-associated Cysteine Protease, Plays a Role in the Maturation of the Surface Layer of Clostridium difficile

Superantigens are highly potent immune stimulators with a unique ability to interact simultaneously with MHC class II molecules and T cell receptors, forming a trimolecular complex that induces profound T-cell proliferation and massive cytokine production. Recent structural studies have provided a wealth of information regarding these complex interactions, and it is now emerging that, despite their common 3-D architecture, superantigens are able to crosslink MHC class II molecules and T cell receptors in a variety of ways.

Charcot-Leyden crystal protein (galectin-10) is not a dual function galectin with lysophospholipase activity but binds a lysophospholipase inhibitor in a novel structural fashion.

Clostridium difficile is a major and growing problem as a hospital-associated infection that can cause severe, recurrent diarrhea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood but undoubtedly involves protein components within the surface layer (S-layer), which play a role in adhesion. In C. difficile, the S-layer is composed of two principal components, the high and low molecular weight S-layer proteins, which are formed from the post-translational cleavage of a single precursor, SlpA. In the present study, we demonstrate that a recently characterized cysteine protease, Cwp84 plays a role in maturation of SlpA. Using a gene knock-out approach, we show that inactivation of the Cwp84 gene in C. difficile 630ΔErm results in a bacterial phenotype in which only immature, single chain SlpA comprises the S-layer. The Cwp84 knock-out mutants (CDΔCwp84) displayed significantly different colony morphology compared with the wild-type strain and grew more slowly in liquid medium. SlpA extracted from CDΔCwp84 was readily cleaved into its mature subunits by trypsin treatment. Addition of trypsin to the growth medium also cleaved SlpA on CDΔCwp84 and increased the growth rate of the bacterium in a dose-dependent manner. Using the hamster model for C. difficile infection, CDΔCwp84 was found to be competent at causing disease with a similar pathology to the wild-type strain. The data show that whereas Cwp84 plays a role in the cleavage of SlpA, it is not an essential virulence factor and that bacteria expressing immature SlpA are able to cause disease.

Superantigens: structure-function relationships.

Charcot-Leyden crystal (CLC) protein, initially reported to possess weak lysophospholipase activity, is still considered to be the eosinophils lysophospholipase, but it shows no sequence similarities to any known lysophospholipases. In contrast, CLC protein has moderate sequence similarity, conserved genomic organization, and near structural identity to members of the galectin superfamily, and it has been designated galectin-10. To definitively determine whether or not CLC protein is a lysophospholipase, we reassessed its enzymatic activity in peripheral blood eosinophils and an eosinophil myelocyte cell line (AML14.3D10). Antibody affinity chromatography was used to fully deplete CLC protein from eosinophil lysates. The CLC-depleted lysates retained their full lysophospholipase activity, and this activity could be blocked by sulfhydryl group-reactive inhibitors, N-ethylmaleimide andp-chloromercuribenzenesulfonate, previously reported to inhibit the eosinophil enzyme. In contrast, the affinity-purified CLC protein lacked significant lysophospholipase activity. X-ray crystallographic structures of CLC protein in complex with the inhibitors showed that p-chloromercuribenzenesulfonate bound CLC protein via disulfide bonds with Cys29 and with Cys57 near the carbohydrate recognition domain (CRD), whereas N-ethylmaleimide bound to the galectin-10 CRD via ring stacking interactions with Trp72, in a manner highly analogous to mannose binding to this CRD. Antibodies to rat pancreatic lysophospholipase identified a protein in eosinophil and AML14.3D10 cell lysates, comparable in size with human pancreatic lysophospholipase, which co-purifies in small quantities with CLC protein. Ligand blotting of human and murine eosinophil lysates with CLC protein as probe showed that it binds proteins also recognized by antibodies to pancreatic lysophospholipase. Our results definitively show that CLC protein is not one of the eosinophils lysophospholipases but that it does interact with eosinophil lysophospholipases and known inhibitors of this lipolytic activity.