Asiah Osman

Natural Product-Based Phenols as Novel Probes for Mycobacterial and Fungal Carbonic Anhydrases

In order to discover novel probes that may help in the investigation and control of infectious diseases through a new mechanism of action, we have evaluated a library of phenol-based natural products (NPs) for enzyme inhibition against four recently characterized pathogen β-family carbonic anhydrases (CAs). These include CAs from Mycobacterium tuberculosis, Candida albicans, and Cryptococcus neoformans as well as α-family human CA I and CA II for comparison. Many of the NPs selectively inhibited the mycobacterial and fungal β-CAs, with the two best performing compounds displaying submicromolar inhibition with a preference for fungal over human CA inhibition of more than 2 orders of magnitude. These compounds provide the first example of non-sulfonamide inhibitors that display β over α CA enzyme selectivity. Structural characterization of the library compounds in complex with human CA II revealed a novel binding mode whereby a methyl ester interacts via a water molecule with the active site zinc.

Hookworm SCP/TAPS protein structure: a key to understanding host–parasite interactions and developing new interventions

SCP/TAPS proteins are a diverse family of molecules in eukaryotes, including parasites. Despite their abundant occurrence in parasite secretomes, very little is known about their functions in parasitic nematodes, including blood-feeding hookworms. Current information indicates that SCP/TAPS proteins (called Ancylostoma-secreted proteins, ASPs) of the canine hookworm, Ancylostoma caninum, represent at least three distinct groups of proteins. This information, combined with comparative modelling, indicates that all known ASPs have an equatorial groove that binds extended structures, such as peptides or glycans. To elucidate structure-function relationships, we explored the three-dimensional crystal structure of an ASP (called Ac-ASP-7), which is highly up-regulated in expression in the transition of A. caninum larvae from a free-living to a parasitic stage. The topology of the N-terminal domain is consistent with pathogenesis-related proteins, and the C-terminal extension that resembles the fold of the Hinge domain. By anomalous diffraction, we identified a new metal binding site in the C-terminal extension of the protein. Ac-ASP-7 is in a monomer-dimer equilibrium, and crystal-packing analysis identified a dimeric structure which might resemble the homo-dimer in solution. The dimer interaction interface includes a novel binding site for divalent metal ions, and is proposed to serve as a binding site for proteins involved in the parasite-host interplay at the molecular level. Understanding this interplay and the integration of structural and functional data could lead to the design of new approaches for the control of parasitic diseases, with biotechnological outcomes.

Parasite annexins--new molecules with potential for drug and vaccine development.

In the last few years, annexins have been discovered in several nematodes and other parasites, and distinct differences between the parasite annexins and those of the hosts make them potentially attractive targets for anti‐parasite therapeutics. Annexins are ubiquitous proteins found in almost all organisms across all kingdoms. Here, we present an overview of novel annexins from parasitic organisms, and summarize their phylogenetic and biochemical properties, with a view to using them as drug or vaccine targets. Building on structural and biological information that has been accumulated for mammalian and plant annexins, we describe a predicted additional secondary structure element found in many parasite annexins that may confer unique functional properties, and present a specific antigenic epitope for use as a vaccine.

The Crystal Structure of Calcium-bound Annexin Gh1 from Gossypium hirsutum and Its Implications for Membrane Binding Mechanisms of Plant Annexins

Plant annexins show distinct differences in comparison with their animal orthologues. In particular, the endonexin sequence, which is responsible for coordination of calcium ions in type II binding sites, is only partially conserved in plant annexins. The crystal structure of calcium-bound cotton annexin Gh1 was solved at 2.5Å resolution and shows three metal ions coordinated in the first and fourth repeat in types II and III binding sites. Although the protein has no detectable affinity for calcium in solution, in the presence of phospholipid vesicles, we determined a stoichiometry of four calcium ions per protein molecule using isothermal titration calorimetry. Further analysis of the crystal structure showed that binding of a fourth calcium ion is structurally possible in the DE loop of the first repeat. Data from this study are in agreement with the canonical membrane binding of annexins, which is facilitated by the convex surface associating with the phospholipid bilayer by a calcium bridging mechanism. In annexin Gh1, this membrane-binding state is characterized by four calcium bridges in the I/IV module of the protein and by direct interactions of several surface-exposed basic and hydrophobic residues with the phospholipid membrane. Analysis of the protein fold stability revealed that the presence of calcium lowers the thermal stability of plant annexins. Furthermore, an additional unfolding step was detected at lower temperatures, which can be explained by the anchoring of the N-terminal domain to the C-terminal core by two conserved hydrogen bonds.

Phosphorylation of CRN2 by CK2 regulates F-actin and Arp2/3 interaction and inhibits cell migration

CRN2 (synonyms: coronin 1C, coronin 3) functions in the re-organization of the actin network and is implicated in cellular processes like protrusion formation, secretion, migration and invasion. We demonstrate that CRN2 is a binding partner and substrate of protein kinase CK2, which phosphorylates CRN2 at S463 in its C-terminal coiled coil domain. Phosphomimetic S463D CRN2 loses the wild-type CRN2 ability to inhibit actin polymerization, to bundle F-actin, and to bind to the Arp2/3 complex. As a consequence, S463D mutant CRN2 changes the morphology of the F-actin network in the front of lamellipodia. Our data imply that CK2-dependent phosphorylation of CRN2 is involved in the modulation of the local morphology of complex actin structures and thereby inhibits cell migration.

Crystal structure and immunological properties of the first annexin from Schistosoma mansoni: insights into the structural integrity of the schistosomal tegument

Schistosomiasis is a major parasitic disease of humans, second only to malaria in its global impact. The disease is caused by digenean trematodes that infest the vasculature of their human hosts. These flukes are limited externally by a body wall composed of a syncytial epithelium, the apical surface membrane of which is a parasitism‐adapted dual membrane complex. Annexins are thought to be of integral importance for the stability of this apical membrane system. Here, we present the first structural and immunobiochemical characterization of an annexin from Schistosoma mansoni. The crystal structure of annexin B22 confirms the presence of the previously predicted α‐helical segment in the II/III linker and reveals a covalently linked head‐to‐head dimer. From the calcium‐bound crystal structure of this protein, canonical type II, type III and B site positions are occupied, and a novel binding site has been identified. The dimer arrangement observed in the crystal structure suggests the presence of two prominent features, a potential non‐canonical membrane binding site and a potential binding groove opposite to the former. Results from transcriptional profiling during development show that annexin B22 expression is correlated with life stages of the parasite that possess the syncytial tegument layer, and ultrastructural localization by immuno‐electron microscopy confirms the occurrence of annexins in the tegument of S. mansoni. Data from membrane binding and aggregation assays indicate the presence of differential molecular mechanisms and support the hypothesis of annexin B22 providing structural integrity in the tegument.

Alpha-1 giardin is an annexin with highly unusual calcium-regulated mechanisms

Alpha-giardins constitute the annexin proteome (group E annexins) in the intestinal protozoan parasite Giardia and, as such, represent the evolutionary oldest eukaryotic annexins. The dominance of alpha-giardins in the cytoskeleton of Giardia with its greatly reduced actin content emphasises the importance of the alpha-giardins for the structural integrity of the parasite, which is particularly critical in the transformation stage between cyst and trophozoite. In this study, we report the crystal structures of the apo- and calcium-bound forms of α1-giardin, a protein localised to the plasma membrane of Giardia trophozoites that has recently been identified as a vaccine target. The calcium-bound crystal structure of α1-giardin revealed the presence of a type III site in the first repeat as known from other annexin structures, as well as a novel calcium binding site situated between repeats I and IV. By means of comparison, the crystal structures of three different alpha-giardins known to date indicate that these proteins engage different calcium coordination schemes, among each other, as well as compared to annexins of groups A-D. Evaluation of the calcium-dependent binding to acidic phosphoplipid membranes revealed that this process is not only mediated but also regulated by the environmental calcium concentration. Uniquely within the large family of annexins, α1-giardin disengages from the phospholipid membrane at high calcium concentrations possibly due to formation of a dimeric species. The observed behaviour is in line with changing calcium levels experienced by the parasite during excystation and may thus provide first insights into the molecular mechanisms underpinning the transformation and survival of the parasite in the host.

Insights into the membrane interactions of the saposin-like proteins Na-SLP-1 and Ac-SLP-1 from human and dog hookworm

Saposin-like proteins (SAPLIPs) from soil-transmitted helminths play pivotal roles in host-pathogen interactions and have a high potential as targets for vaccination against parasitic diseases. We have identified two non-orthologous SAPLIPs from human and dog hookworm, Na-SLP-1 and Ac-SLP-1, and solved their three-dimensional crystal structures. Both proteins share the property of membrane binding as monitored by liposome co-pelleting assays and monolayer adsorption. Neither SAPLIP displayed any significant haemolytic or bactericidal activity. Based on the structural information, as well as the results from monolayer adsorption, we propose models of membrane interactions for both SAPLIPs. Initial membrane contact of the monomeric Na-SLP-1 is most likely by electrostatic interactions between the membrane surface and a prominent basic surface patch. In case of the dimeric Ac-SLP-1, membrane interactions are most likely initiated by a unique tryptophan residue that has previously been implicated in membrane interactions in other SAPLIPs.