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Salmon, A.J., Williams, M.L., Hofmann, A., Poulsen, S.A. (2012)
Chem. Commun. 48, 2328-2330
Abstract
We have determined the protein X-ray crystal structures of four organometallic inhibitors in complex with their target enzyme carbonic anhydrase II. The barrel shaped hydrophobic ferrocene and ruthenocene moieties of the inhibitors provided a structure-based avenue to better occupy the hydrophobic binding patch within the enzyme active site. | | | PubMed | DOI |
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Cantacessi, C., Hofmann, A., Young, N.D., Broder, U., Hall, R.S., Loukas, A., Gasser, R.B. (2012)
PLoS ONE, in press
Abstract
Background: SCP/TAPS proteins of parasitic helminths have been proposed to play key roles in fundamental biological processes linked to the invasion of and establishment in their mammalian host animals, such as the transition from free-living to parasitic stages and the modulation of host immune responses. Despite the evidence that SCP/TAPS proteins of parasitic nematodes are involved in host-parasite interactions, there is a paucity of information on this protein family for parasitic trematodes of socio-economic importance.
Methodology/Principal Findings: We conducted the first large-scale study of SCP/TAPS proteins of a range of parasitic trematodes of both human and veterinary importance (including the liver flukes Clonorchis sinensis, Opisthorchis viverrini, Fasciola hepatica and F. gigantica as well as the blood flukes Schistosoma mansoni, S. japonicum and S. haematobium). We mined all current transcriptomic and/or genomic sequence datasets from public databases, predicted secondary structures of full-length protein sequences, undertook systematic phylogenetic analyses and investigated the differential transcription of SCP/TAPS genes in O. viverrini and F. hepatica, with an emphasis on those that are up-regulated in the developmental stages infecting the mammalian host.
Conclusions: This work, which sheds new light on SCP/TAPS proteins, guides future structural and functional explorations of key SCP/TAPS molecules associated with diseases caused by flatworms. Future fundamental investigations of these molecules in parasites and the integration of structural and functional data could lead to new approaches for the control of parasitic diseases. | | |
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Wang, C.K., Broder, U., Weeratunga, S.K., Gasser, R.B., Loukas, A., Hofmann, A. (2012)
Bioinformatics, in press
Abstract
Both alignment generation and visualisation are important processes for producing biologically meaningful sequence alignments. Computational tools that combine reliable, automated and semi-automated approaches to produce secondary structure-based alignments with an appropriate visualisation of the results are rare. We have developed SBAL, a tool to generate and edit secondary structure-based sequence alignments. It is easy to install and provides a user-friendly interface. Sequence alignments are displayed, with secondary structure assignments mapped to their corresponding regions in the sequence by using a simple colour scheme. The algorithm implemented for automated and semi-automated secondary structure-based alignment calculations shows a comparable performance to existing software. | | |
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Bauer, M., Georgousakis, M., Vu, T., Henningham, A., Hofmann, A., Rettel, M., Hafner, L., Sriprakash, S., McMillan, D. (2012)
Vaccine, in press
Abstract
A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified the most common variant sequences in the C-repeat region (CRR) of 77 different M-types, and have incorporated them into three constructs, SV1, SV2 and SV3. Each unique sequence was into the polyvalent constructs in a manner that produced an alpha-helical conformation, a conformational characteristic of the CRR. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against one construct, SV1, containing five variant sequences, kill S. pyogenes 88-30 and ES61 in in vitro bactericidal assays. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates. | | | PubMed | DOI |
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Young, N.D., Jex, A.R., Li, B., Liu, S., Yang, L., Xiong, Z., Li, Y., Cantacessi, C., Hall, R.S., Xu, X., Chen, F., Wu, X., Zerlotini, A., Oliveira, G., Hofmann, A., Zhang, G., Fang, X., Kang, Y., Campbell, B.E., Loukas, A., Ranganathan, S., Rollinson, D., Rinaldi, G., Brindley, P.J., Yang, H., Wang, J., Wang, J., Gasser, R.B. (2012)
Nat. Genet., in press
Abstract
Schistosomiasis is a neglected tropical disease affecting 200 million people worldwide and causing 300,000 deaths each year. The intestinal and urinary forms of this debilitating disease are caused by chronic infections with Schistosoma species (blood flukes or schistosomes). No vaccines are available, and treatment relies on the drug praziquantel. Of the three main species that infect people, Schistosoma haematobium has come into the spotlight because of its high prevalence (> 110 million people infected), as a cause of urogenital disease, its proven link to malignant bladder cancer and as a predisposing factor for HIV/AIDS in endemic countries. Through a complex two-host life cycle, S. haematobium is transmitted from a freshwater snail (Bulinus spp.) to humans. Adult schistosomes dwell as pairs in the blood vessels of the urinary bladder, where female worms release eggs that become embedded in the bladder wall to elicit chronic immune-mediated disease6 and serious complications, including squamous cell carcinoma. Here, we report the 385 million-base (Mb) draft genome of S. haematobium and compare it with sequences from related parasites. This genome provides an unprecedented resource for fundamental genomic, genetic, evolutionary, biological and epidemiological research and exciting prospects for the design of new interventions against a largely unknown disease complex of humans. | | | PubMed | DOI |
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Gasser, R.B., Cantacessi, C., Campbell, B.E., Hofmann, A., Otranto, D. (2011)
Parasit. Vectors 4, 53
Abstract
Canine vector-borne diseases (CVBDs) are of major socioeconomic importance worldwide. Although many studies have provided insights into CVBDs, there has been limited exploration of fundamental molecular aspects of most pathogens, their vectors, pathogen-host relationships and disease and drug resistance using advanced, ‘omic technologies. The aim of the present account is to take a prospective view of the impact that next-generation, ‘omics technologies could have, with an emphasis on describing the principles of transcriptomic/genomic sequencing as well as bioinformatic technologies and their implications in both fundamental and applied areas of CVBD research. Tackling key biological questions employing these technologies will provide a ‘systems biology’ context and could lead to radically new intervention and management strategies against CVBDs. | | | DOI | PDF | Publisher |
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Xavier, C.P., Rastetter, R.H., Blömacher, M., Stumpf, M., Himmel, M., Morgan, R.O., Fernandez, M.P., Wang, C., Osman, A., Miyata, Y., Gjerset, R.A., Eichinger, L., Hofmann, A., Linder, S., Noegel, A.A., Clemen, C.S. (2011)
Sci. Rep., in press
Abstract
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. | | | DOI |
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Osman, A., Wang, C.K., Winter, A., Loukas, A., Tribolet, L., Gasser, R.B., Hofmann, A. (2011)
Biotechnol. Advances, in press
Abstract
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 upregulated 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. | | | PubMed | DOI |
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Kori, L.D., Hofmann, A., Patel, B.K.C. (2012)
Acta Crystallogr. F 68, 240-243
Abstract
A ribokinase (rbk) from the anaerobic halothermophilic bacterium Halothermothrix orenii was cloned and over-expressed in Escherichia coli. The recombinant protein (Ho-rbk) was purified using immobilised metal ion affinity chromatography and crystals were obtained using the sitting drop method. Diffraction data was collected to a resolution of 3.1 Å using synchrotron radiation. The crystals belong to the orthorhombic space group P212121 with unit cell parameters a= 45.6, b= 61.1, c= 220.2, and contain two molecules per asymmetric unit. A molecular replacement solution has been found and attempts are currently under way to build a model of the ribokinase, as well as ways to improve crystal quality. | | | PubMed |
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Wang, C.K., Simon, A., Jessen, C.M., Oliveira, C.L.P., Mack, L., Braunewell, K., Ames, J.B., Pedersen, J.S., Hofmann, A. (2011)
PLoS ONE 6, e26793
Abstract
The NCS protein Visinin-like Protein 1 (VILIP-1) transduces calcium signals in the brain and serves as an effector of the non-retinal receptor guanylyl cyclases (GCs) GC-A and GC-B, and nicotinic acetyl choline receptors (nAchR). Analysis of the quaternary structure of VILIP-1 in solution reveals the existence of monomeric and dimeric species, the relative contents of which are affected but not exclusively regulated by divalent metal ions and Redox conditions. Using small-angle X-ray scattering, we have investigated the low resolution structure of the calcium-bound VILIP-1 dimer under reducing conditions. Scattering profiles for samples with high monomeric and dimeric contents have been obtained. The dimerization interface involves residues from EF-hand regions EF3 and EF4.
Using monolayer adsorption experiments, we show that myristoylated and unmyristoylated VILIP-1 can bind lipid membranes. The presence of calcium only marginally improves binding of the protein to the monolayer, suggesting that charged residues at the protein surface may play a role in the binding process.
In the presence of calcium, VILIP-1 undergoes a conformational re-arrangement, exposing previously hidden surfaces for interaction with protein partners. We hypothesise a working model where dimeric VILIP-1 interacts with the membrane where it binds membrane-bound receptors in a calcium-dependent manner. | | | PubMed | DOI |
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Willis, C., Wang, C.K., Osman, A., Simon, A., Pickering, D., Mulvenna, J., Riboldi-Tunicliffe, A., Jones, M.K., Loukas, A., Hofmann, A. (2011)
PLoS ONE 6, e25369
Abstract
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. | | | PubMed | DOI |
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Kellenberger, E., Hofmann, A., Quinn, R. (2011)
Nat. Prod. Reports 28, 1483-1492
Abstract
Natural products are made by nature through interacting with biosynthetic enzymes. Natural products also exert their effect as drugs by interaction with proteins. Does the recognition of the natural product by biosynthetic enzymes translate to recognition of the therapeutic target? Molecular modelling of flavonoid biosynthetic enzymes and protein kinases with a series of natural product kinase inhibitors led to the development of the concept of Protein Fold Topology (PFT). PFT describes cavity recognition points unrelated to protein fold similarity. The topology or spatial properties are preserved even though there is deformation of the protein elements that participate in the protein-ligand interactions. We observe helices or beta-strands as equivalent in providing the invariant topology for protein-ligand interaction. In this review, we explore: Will PFT aid Drug Discovery or is it the reason natural products have drug properties? | | | PubMed | DOI |
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Wang, C.K., Weeratunga, S.K., Pacheco, C.M., Hofmann, A. (2012)
Bioinformatics 28, 439-440
Abstract
Differential scanning fluorimetry (DSF) is a rapid technique that can be used in structural biology to study protein-ligand interactions. We have developed DMAN, a novel tool to analyse multi-well plate data obtained in DSF experiments. DMAN is easy to install and provides a user-friendly interface. Multi-well plate layouts can be designed by the user and experimental data can be annotated and analysed by DMAN according to the specified plate layout. Statistical tests for significance are performed automatically, and graphical tools are also provided to assist in data analysis. The modular concept of this software will allow easy development of other multi-well plate analysis applications in the future. | | | PubMed | DOI |
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Konopka-Postupolska, D., Clark, G., Hofmann, A. (2011)
Plant Sci. 181, 230-241
Abstract
Knowledge accumulated over the past 15 years on plant annexins clearly indicates that this disparate group of proteins builds on the common annexin function of membrane association, but possesses divergent molecular mechanisms. Functionally, the current literature agrees on a key role of plant annexins in stress response processes such as wound healing and drought tolerance. This is contrasted by only few established details of the molecular level mechanisms that are driving these activities.
In this review, we appraise the current knowledge of plant annexin molecular, functional and structural properties with a special emphasis on topics of less coverage in recent past overviews. In particular, plant annexin post-translational modification, roles in polar growth and membrane stabilisation processes are discussed. | | | PubMed | DOI |
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Campbell, B.E., Boag, P.R., Hofmann, A., Cantacessi, C., Wang, C.K., Taylor, P., Hu, M., Sindhu, Z., Loukas, A., Sternberg, P.W., Gasser, R.B. (2011)
Biotechnol. Advances 29, 338-350
Abstract
Almost nothing is known about atypical kinases in multicellular organisms, including parasites. Supported by information and data available for the free-living nematode, Caenorhabditis elegans, and other eukaryotes, the present article describes three RIO kinase genes, riok-1, riok-2 and riok-3, from Haemonchus contortus, one of the most important parasitic nematodes of small ruminants. Analyses of these genes and their products predict that they each play critical roles in developmental pathways of parasitic nematodes. The findings of this review indicate prospects for functional studies of these genes in C. elegans (as a surrogate) and opportunities for the design of a novel class of nematode-specific inhibitors of RIO kinases. The latter aspect is of paramount importance, given the serious problems linked to anthelmintic resistance in parasitic nematode populations of livestock. | | | PubMed | DOI |
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Davis, R.A., Hofmann, A., Osman, A., Hall, R.A., Mühlschlegel, F.A., Vullo, D., Innocenti, A., Supuran, C.T., Poulsen, S.-A. (2011)
J. Med. Chem. 54, 1682-1692
Abstract
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 beta-family carbonic anhydrases (CAs). These include CAs from Mycobacterium tuberculosis, Candida albicans and Cryptococcus neoformans as well as alpha-family human CA I and II, for comparison. Many of the NPs selectively inhibited the mycobacterial and fungal beta-CAs, with the two best performing compounds displaying submicromolar inhibition with a preference of fungal over human CA inhibition of more than two orders of magnitude. These compounds provide the first example of non-sulfonamide inhibitors that display beta- over alpha-CA enzyme selectivity. Structural characterisation 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. | | | PubMed | DOI |
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Kori, L.D., Hofmann, A., Patel, B.K.C. (2011)
Acta Crystallogr. F 67, 111-113
Abstract
The β-glucosidase A gene (BglA) has been cloned from the halothermophilic bacterium Halothermothrix orenii and the recombinant enzyme (BglA, EC. 3.2.1.21) has been bacterially expressed, purified by metal ion affinity chromatography and was subsequently crystallized. Orthorhombic crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystal structure with two molecules in the asymmetric unit was solved by molecular replacement using a library of known glucosidase structures. Attempts to collect higher resolution diffraction data from crystals grown under different conditions and structure refinement are currently in progress. | | | PubMed | DOI |
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Campbell, B.E., McCluskey, A., Hofmann, A., Gasser, R.B. (2011)
Biotechnol. Advances 29, 28-39
Abstract
Little is known about the fundamental biology of parasitic nematodes (= roundworms) that cause serious diseases, affecting literally billions of animals and humans worldwide. Unlocking the biology of these neglected pathogens using modern technologies will yield crucial and profound knowledge of their molecular biology, and could lead to new treatment and control strategies. Supported by studies in the free-living nematode, Caenorhabditis elegans, some recent investigations have provided improved insights into selected protein phosphatases (PPs) of economically important parasitic nematodes (Strongylida). In the present article, we review this progress and assess the potential of serine/threonine phosphatase (STP) genes and/or their products as targets for new nematocidal drugs. Current information indicates that some small molecules, known to specifically inhibit PPs, might be developed as nematocides. For instance, some cantharidin analogues are known to display exquisite PP-inhibitor activity, which indicates that some of them could be designed and tailored to specifically inhibit STPs of nematodes. This information provides prospects for the discovery of an entirely novel class of nematocides, which is of paramount importance, given the serious problems linked to anthelmintic resistance in parasitic nematode populations of livestock, and has the potential to lead to significant biotechnological outcomes. | | | PubMed | DOI |
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Hofmann, A., Wang, C.K., Osman, A., Camp, D. (2010)
Struct. Chem. 21, 1117-1129
Abstract
This review introduces the Structural Chemistry Program at Griffith University's Eskitis Institute, and provides a brief overview over its current and future research portfolio. Capitalising on the co-location with a unique library collection of small molecules, the Queensland Compound Library, our laboratory investigates the structure and function of proteins with the aim of learning about their molecular mechanisms. Consequently, these studies also feed into drug discovery and design.
The thematic focus of our Program is on proteins involved in infection, inflammation and neurological diseases, and this review highlights a few of our recent research efforts in this area. | | | DOI | Publisher |
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Hofmann, A., Osman, A., Leow, C.Y., Driguez, P., McManus, D.P., Jones, M.K. (2010)
BioEssays 32, 967-976
Abstract
In recent 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 summarise 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 unique antigenic epitope for use as a vaccine. | | | PubMed | DOI | PDF | Publisher |
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Nolan, M.J., Hofmann, A., Jex, A.R., Gasser, R.B. (2010)
Mol. Cell. Probes 24, 281-285
Abstract
Predicting how point mutations in genes alter the tertiary and quarternary structure of proteins is central to a number of areas of molecular biology and has implications in relation to the function and evolution of molecules. In the present study, we theoretically assessed the effects of 20 point mutations detected previously in a region of the triose-phosphate isomerase gene (tpi) of the protozoan Giardia duodenalis on the three-dimensional structure of the ‘wild-type’ protein (TPI). Amino acid substitutions arising from codon variations were mainly located at surface-accessible sites or in hydrophobic pockets of TPI. None of the substitutions was predicted to exert a significant change to the fold or functionality of the enzyme, with the exception of one alteration (Arg100). Almost all substitutions were either conservative or semi-conservative, and retained or even improved the expected stability of the fold. Overall, the findings provide support for the “neutral theory”, which contends that evolution at the molecular level is not solely shaped by “Darwinian selection but also by random drift of selectively neutral or nearly neutral mutants”. | | | PubMed | DOI |
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Clemen, C.S, Tangavelou, K., Strucksberg, K.-H., Just, S., Gaertner, L., Regus-Leidig, H., Stumpf, M., Reimann, J., Coras, R., Morgan, R.O., Fernandez, M.-P., Hofmann, A., Müller, S., Schoser, B., Hanisch, F.-G., Rottbauer, W., Blümcke, I., von Hörsten, S., Eichinger, L., Schröder, R. (2010)
Brain 133, 2920-2941
Abstract
Mutations of the human VCP gene cause autosomal-dominant inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD). We identified strumpellin as a novel VCP binding partner. Strumpellin mutations have been shown to cause hereditary spastic paraplegia. We demonstrate
that strumpellin is a ubiquitously expressed dual compartment protein present in cytosolic and endoplasmic reticulum fractions. Over-expression or ablation of the wild-type strumpellin in wound healing assays caused significantly reduced wound closure velocities, whereas over-expression of the disease causing strumpellin
N471D mutant showed no functional effect. Strumpellin knock-down experiments in human neuroblastoma cells resulted in a dramatic reduction of axonal outgrowth.
Knock-down studies in zebrafish revealed severe cardiac contractile dysfunction, tail curvature and impaired motility. The latter phenotype is due to a loss of central and peripheral motoneuron formation. These data imply a strumpellin loss-of-function pathogenesis in hereditary spastic paraplegia. In the human central nervous system strumpellin shows a pre-synaptic localization. We further identified strumpellin in
pathological protein aggregates in IBMPFD, various myofibrillar myopathies, and in cortical neurons of a Huntington disease mouse model. Beyond hereditary spastic paraplegia, our findings imply that strumpellin along with VCP may have a concerted pathogenic role in various protein aggregate diseases. | | | PubMed | DOI |
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Campbell, B.E., Rabelo, E.M., Hofmann, A., Hu, M., Gasser, R.B. (2010)
Mol. Cell. Probes 24, 178-189
Abstract
Full-length complementary DNA (cDNA) sequence (designated Hc-stp-1) encoding a serine/threonine phosphatase (Hc-STP-1) was isolated from Haemonchus contortus, a strongylid nematode parasite of small ruminants. Hc-stp-1 was shown to be transcribed in males of the adult and fourth larval stage, but not in females, early larval stages or eggs. The full-length gene (2854 bp) contained ten exons and nine introns, and encoded a cDNA of 951 bp. Comparisons of the conceptually translated protein (317 amino acids, estimated at ~35 kDa) with serine/threonine phosphatases (STPs) from other organisms revealed the presence of the conserved motif LRGNHE. Structural analysis, by comparative modelling, confirmed strict conservation of residues and features involved in catalytic activity, and variation in the ligand-binding interface. Phylogenetic analysis of amino acid sequence data revealed that Hc-STP-1 clustered with STPs from other nematodes (including Caenorhabditis elegans, Trichostrongylus vitrinus, Oesophagostomum dentatum, Ascaris suum and Brugia malayi) to the exclusion of STPs from other organisms. The protein was inferred to be most closely related to the PP1 class of STPs of C. elegans, within a group containing STPs encoded, amongst others, by the genes gsp-3 and gsp-4 in this free-living nematode. The functions of proteins GSP-3 and GSP-4 are known to be central to spermatogenesis and other male-specific processes in C. elegans. The findings from the present and previous studies support the proposal that Hc-stp-1 and its product play a significant role in reproductive and/or developmental processes in maturing or adult male H. contortus. | | | PubMed | DOI |
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Lopez, M., Paul, B., Hofmann, A., Morizzi, J., Wu, Q.K., Charman, S.A., Innocenti, A., Vullo, D., Supuran, C.T., Poulsen, S.-A. (2009)
J. Med. Chem. 52, 6421-6432
Abstract
In this paper we present a new class of carbonic anhydrase (CA) inhibitor that was designed to selectively target the extracellular domains of the cancer-relevant CA isozymes. The aromatic moiety of the classical zinc binding sulfonamide CA inhibitors is absent from these compounds and instead they incorporate a hydrophilic mono- or disaccharide fragment directly attached to the sulfonamide group to give S-glycosyl primary sulfonamides (1-10). The inhibition properties of these compounds at the physiologically abundant human CA isozymes I and II and cancer-associated IX and XII were determined and all compounds had moderate potency with Kis in the micromolar range. We present the crystal structures of anomeric sulfonamides 4, 7 and 10 and the sugar sulfamate drug topiramate in complex with human recombinant CA II. From these structures we have obtained valuable insights into ligand-protein interactions of these novel carbohydrate-based sulfonamides with CA. | | | PubMed | DOI |
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Georgousakis, M., Hofmann, A., McMillan, D., Batzloff, M., Sriprakash, S. (2009)
Vaccine 27, 6799-6806
Abstract
A conformationally restricted B-cell epitope has been identified from the major group A streptococcal virulence factor, the M protein, as a potential safe vaccine candidate. To maintain alpha-helical secondary structure, the minimal epitope is flanked with heterologous sequences to produce the chimeric vaccine candidate J14. As a strategy towards developing an affordable multivalent GAS vaccine, we have expressed J14 recombinantly with a second GAS protective antigen H12 (rJ14H12). When administered to mice sub-cutaneously, the fusion protein stimulated a strong serum IgG response to the H12 component, but J14 was poorly immunogenic. To increase the immunogenicity of J14 when expressed with the model fusion partner, amino acid modifications were made to the initial recombinant construct to produce rJJo. These changes stabilised the alpha-helical conformation of the recombinant antigen as assessed by circular dichroism. Mice immunised with rJJoH12, the fusion protein incorporating JJo, effectively stimulated a humoral response to both of the included antigens. This data supports the feasibility of developing a multivalent vaccine incorporating the conformationally restricted protective antigen J14. | | | PubMed | DOI |
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Braunewell, K.-H., Paul, B., Altarche-Xifro, W., Noack, C., Lange, K., Hofmann, A. (2010)
Aust. J. Chem. 63, 350-356
Abstract
The subcellular membrane localisation of neuronal calcium sensor (NCS) proteins in living cells, such as VILIP-1 and VILIP-3, differs substantially. We have followed the hypothesis that the differential localisation might be due to specific binding capabilities of individual VILIPs for phosphatidylinositol phosphates (PIPs). Several highly conserved lysine residues in the N-terminal region could provide favourable electrostatic interactions.
Molecular modelling results support a binding site for phospho-inositides in the N-terminal area of VILIP-1, and the involvement of the conserved N-terminal lysine residues in binding the phospho-inositol head group. Experimentally, the binding of VILIP-1 to inositol derivatives was tested by a PIP strip assay, which showed the requirement of phosphorylation of the inositol group for the interaction of the protein with PIPs. Monolayer adsorption measurements showed a preference of VILIP-1 binding to PI(4,5)P2 over PI(3,4,5)P3. The co-localisation of VILIP-1 with PI(4,5)P2 at the cell surface membrane in hippocampal neurons further supports the idea of direct interactions of VILIP-1 with PIPs in living cells. | | | DOI |
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Xavier, C.P., Rastetter, R.H., Stumpf, M., Rosentreter, A., Müller, R., Reimann, J., Cornfine, S., Linder, S., van Vliet, V., Hofmann, A., Morgan, R.O., Fernandez, M.P., Schröder, R., Noegel, A.A., Clemen, C.S. (2009)
J. Mol. Biol. 393, 287-299
Abstract
Coronin-1C (synonyms: coronin-3, CRN2), a WD40 repeat containing protein involved in cellular actin dynamics, is ubiquitously expressed in human tissues. Here we report on the identification and functional characterization of two novel coronin-1C isoforms, referred to as CRN2i2 and CRN2i3. These isoforms, which arise from an alternative first exon identified in intron 1 of the coronin-1C gene, associate with F-actin. Gel filtration assays suggest that the largest isoform 3 exists as a monomer,
whereas the conventional isoform 1 and isoform 2 both form trimers. Structural
modelling predicts that this difference of the oligomerization state results from an interaction of the elongated N-terminus of CRN2i3 with its C-terminal extension.
Analyses of the coronin-1C gene disclosed a single promoter containing binding sites for myogenic regulatory factors. CRN2i3 is expressed in mature skeletal muscle tissue and in differentiated myogenic cell lines. In human skeletal muscle CRN2i3 is a structural component of postsynaptic neuromuscular junctions and thin filaments of myofibrils. In addition, CRN2i1 and i2 are enriched within the F-actin core structure of podosomes. Our findings postulate a role of CRN2 isoforms in the structural and
functional organization of F-actin in highly ordered protein complexes.
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Cantacessi, C., Campbell, B.E., Visser, A., Geldhof, P., Nolan, M.J., Nisbet, A.J., Matthews, J.B., Loukas, A., Hofmann, A., Otranto, D., Sternberg, P.W., Gasser, R.B. (2009)
Biotechnol. Advances 27, 376-388
Abstract
A wide range of proteins belonging to the SCP/TAPS “family” has been described for various eukaryotic organisms, including plants and animals (vertebrates and invertebrates, such as helminths). Although SCP/TAPS proteins have been proposed to play key roles in a number of fundamental biological processes, such as host-pathogen interactions and defence mechanisms, there is a paucity of information on their genetic relationships, structures and functions and there is no standardised nomenclature for these proteins. A detailed analysis of the relationships of members of the SCP/TAPS family of proteins, based on key protein signatures, could provide a foundation for investigating these areas. In this article, we review the current state of knowledge of key SCP/TAPS proteins of eukaryotes, with an emphasis on those from parasitic helminths, undertake a comprehensive, systematic phylogenetic analysis of currently available full-length protein sequence data (considering characteristic protein signatures or motifs) to infer relationships and provide a framework (based on statistical support) for the naming of these proteins. This framework is intended to guide genomic and molecular biological explorations of key SCP/TAPS molecules associated with infectious diseases of plants and animals. In particular, fundamental investigations of these molecules in parasites and the integration of structural and functional data could lead to new and innovative approaches for the control of parasitic diseases, with important biotechnological outcomes. | | | PubMed | DOI |
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Laohavisit, A., Mortimer, J.C., Demidchik, V., Coxon, K.M., Stancombe, M.A., Macpherson, N., Brownlee, C., Hofmann, A., Webb, A.A.R., Miedema, H., Battey, N.H., Davies, J.M. (2009)
Plant Cell 21, 479-493
Abstract
Regulation of reactive oxygen species (ROS) and cytosolic free calcium ([Ca2+]cyt) is central
to plant function. Annexins are small proteins capable of Ca2+-dependent membrane-binding
or insertion. They possess structural motifs that could support both peroxidase activity and
calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca2+]cyt when
added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological
profile was consistent with annexin activation (at the extracellular plasma membrane face) of
Arabidopsis Ca2+-permeable non-selective cation channels. Secreted annexins could therefore
modulate Ca2+ influx. As maize annexins occur in the cytosol and plasma membrane, they
were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma
membrane. Here they generated an instantaneously-activating Ca2+-permeable conductance at
mildly acidic pH, sensitive to verapamil and Gd3+ with a Ca2+ to K+ permeability ratio of 0.36.
These results suggest that cytosolic annexins create a Ca2+ influx pathway directly,
particularly during stress responses involving acidosis. Maize annexin preparation also
demonstrated in vitro peroxidase activity which appeared independent of heme association. In
conclusion, this study has demonstrated that plant annexins create Ca2+-permeable transport
pathways, regulate [Ca2+]cyt and in vitro may function as peroxidases. | | | PubMed | DOI | Publisher |
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Wang, Y.-Q., Feechan, A., Yun, B.-W., Shafiei, M., Hofmann, A., Taylor, P., Xue, P., Yang, F.-Q., Xie, Z.-S., Pallas, J.A., Chu, C.-C., Loake, G. (2009)
J. Biol. Chem. 284, 2131-2137
Abstract
Changes in cellular redox status are a well established response across phyla following pathogen challenge. In this context, the synthesis of nitric oxide (NO) is a conspicuous feature of plants responding to attempted microbial infection and this RedOx-based regulator underpins the development of plant immunity. However, the associated molecular mechanism(s) have not been defined. Here we show that NO accretion during the nitrosative burst promotes increasing S-nitrosylation of the Arabidopsis thaliana salicylic acid binding protein 3 (AtSABP3) at cysteine (C)280, suppressing both binding of the immune activator, salicylic acid (SA) and the carbonic anhydrase (CA) activity of this protein. The CA function of AtSABP3 is required for the expression of resistance in the host against attempted pathogen infection. Therefore, inhibition of AtSBAP3 CA function by S-nitrosylation could contribute to a negative feedback loop that modulates the plant defense response. Thus, AtSABP3 is one of the first targets for S-nitrosylation in plants for which the biological function of this redox-based post-translational modification has been uncovered. These data provide a molecular connection between the changes in NO levels triggered by attempted pathogen infection and the expression of disease resistance. | | | PubMed | DOI | PDF |
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Hofmann, A. (2009)
J. Appl. Crystallogr. 42, 137-139
Abstract
ACDP is a Java application for processing protein circular dichroism (CD) spectra either individually or within a series. Data processing includes spectrum subtraction ('baseline correction'), conversion of the raw CD into units of mean residue ellipticity, wavelength monitoring and graphical inspection. Three different algorithms for secondary structure deconvolution have been implemented, and spectra can be analysed without the need for data reformatting using a neural network approach, variable selection or linear combination of prototype spectra. The application is written entirely in Java and is thus portable to a wide variety of platforms, requiring only the Java Runtime Environment. | | | DOI | More |
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Hu, N.-J., Yusof, A. M., Winter, A., Osman, A., Reeve, A. K., Hofmann, A. (2008)
J. Biol. Chem. 283, 18314-18322
Abstract
Plant annexins show distinct differences when compared to 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 2.5 Å resolution, and shows three metal ions coordinated in the first and fourth repeat in type II and III binding sites. While 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 shows 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 characterised by four calcium bridges in the I/IV module of the protein, and direct interactions of several surface-exposed basic and hydrophobic residues with the phospholipid membrane.
Analysis of the protein fold stability reveals 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. | | | PubMed | DOI | PDF | Publisher |
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Hu, N.-J., Bradshaw, J., Lauter, H., Buckingham, J., Solito, E., Hofmann, A. (2008)
Biophys. J. 94, 1773-1781
Abstract
Annexins constitute a family of calcium-dependent membrane binding proteins, and can be classified into two groups, depending on the length of the N-terminal domain unique for each individual annexin. The N-terminal domain of annexin A1 can adopt an α-helical conformation, and has been implicated in mediating the membrane aggregation behaviour of this protein. While the calcium-independent interaction of the annexin A1 N-terminal domain has been known for some time, there was no structural information about the membrane interaction of this secondary membrane binding site of annexin A1.
The present study used circular diochroism spectroscopy to show that a rat annexin A1 N-terminal peptide possess random coil structure in aqueous buffer, but an α-helical structure in the presence of small unilamellar vesicles. The binding of peptides to membranes was confirmed by surface pressure (Langmuir film balance) measurements using phosphatidylcholine/phosphatidylserine monolayers, which show a significant increase after injection of rat annexin A1 N-terminal peptides. Lamellar neutron diffraction with human and rat annexin A1 N-terminal peptides reveal an intercalation of the helical peptides with the phospholipid bilayer, with the helix axis lying parallel to the surface of membrane.
Our findings confirm that phospholipid membranes assist the folding of the N-terminal peptides into α-helical structures, and that this conformation enables favourable direct interactions with the membrane. The results are consistent with the hypothesis that the N-terminal domain of annexin A1 can serve as a secondary membrane binding site in the process of membrane aggregation by providing a peripheral membrane anchor. | | | PubMed | DOI | PDF | Publisher |
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McArdle, B., Hofmann, A. (2008)
The Coronin Family of Proteins , 56-71
Abstract
Until recently, structural information about coronins was scarce and the earlier identification of five WD40 repeats gave rise to a structural prediction of a five-bladed beta propeller for the N-terminal domain of these proteins. More detailed analyses revealed the presence of seven WD40 repeats and the hypothesis of a seven-bladed beta propeller structure. This model has recently been validated due to structural information from crystal structures of C-terminally truncated coronin 1, as well as its C-terminal coiled coil domain. Further structural information is available only indirectly from binding and functional studies.
Phosphorylation at distinct serine and tyrosine residues seems to be a common theme for various coronins. There are indications that this modification regulates the quaternary structure of coronin 3, and thus has implications for the cellular localisation and the general link between signalling and cytoskeletal remodelling. Similarly, phosphorylation-dependent sorting sequences recently discovered on coronin 7 might prove important for the molecular mechanisms of the longer coronins.
A matter that will require further clarification is the localisation of protein binding sites on coronins. While earlier reports presented a rather diverse map of actin binding sites, more recent studies, including the crystal structure of the coronin 1 N-terminal domain, deliver more detailed information in this respect. Interaction sites for other target proteins, such as Arp2/3, remain to be identified. Also, while membrane binding is a known feature of coronins, further details as to the binding sites and molecular level events remain to be elucidated. The N-terminal WD40 repeat domain seems to be the membrane-interacting domain, but other domains might provide regulatory effects, most likely by post-translational modification, in a fashion that is specific for each coronin.
In this chapter, we provide a structural overview of coronins 1, 2, 3 and 7, and also present results of our recent efforts to obtain structural models of coronins 3 and 7. Possible implications of these models on the function of these proteins are discussed. | | | PubMed | PDF | Publisher |
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Winter, A., Hofmann, A. (2008)
Curr. Chem. Biol. 2, 11-19
Abstract
Prohibitins comprise a family of highly conserved ubiquitous eukaryotic proteins that localise to different compartments of the cell. They have been implicated in important cellular processes such as cellular signalling and transcriptional control, apoptosis, cellular senescence, early development of Caenorhabditis elegans and mitochondrial biogenesis.
In yeast, mammals and C. elegans there exist at least two homologous prohibitin proteins (yeast: PHB1, PHB2; human: BAP32, BAP37), which assemble into high molecular weight complexes of about 1.2 MDa in the inner mitochondrial membrane. Experimentally determined structural information about these proteins has been elusive for a long time. Recently, however, the biogenesis and architecture of the yeast prohibitin complex has been analysed and yielded ring-shaped structures as visualised by single particle electron microscopy. Structural details at atomic level remain to be determined, but a first step into this direction is provided by modelling approaches. Prohibitins consist of three domains, an N-terminal transmembrane helix, a middle (PHB) domain and a C-terminal coiled coil domain. The PHB domain is the landmark feature within the super-family of SPFH (stomatin/prohibitin/flotillin/HflK/C) domain proteins. The recently determined NMR structure of mouse flotillin-2 provides a first access to structural details of prohibitins.
While the first functional role attributed to prohibitins was the regulation of cellular senescence, DNA transcription and tumour cell growth, there is recent evidence that they also can act as markers for adipose tissue. In a mouse model, an apoptotic peptide targeted at prohibitin was successful in reversing obesity. An extracellular complex containing both BAP32 and BAP37 was found to bind to the Vi capsular polysaccharide, first identified as a virulence antigen of Salmonella typhi, suggesting a key role for both proteins in infection with S. typhi. Furthermore, the interaction of prohibitin with compounds activating melanin production has placed these proteins at a central position in melanogenesis, and further implicates mitochondria in signalling pathways of the pigmentation process. Accumulating evidence suggests that prohibitins are implicated in mitochondrial, age and oxidative stress-related diseases, as well as in immunity and inflammation, cancer and cancer-like diseases, obesity, and drug resistance.
The complementary interplay between structural and chemical biology will provide important insights into the molecular mechanisms of prohibitins and, more generally, the functions of mitochondria in living cells. This review discusses the current state of knowledge about prohibitins, and provides a vision for further developments in the field of these eminently important proteins.
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Rosentreter, A., Hofmann, A., Xavier, C.-P., Stumpf, M., Noegel, A.A., Clemen, C.S. (2007)
Exp. Cell Res. 313, 878-895
Abstract
The actin interaction of coronin 3 has been mainly documented by in vitro experiments. Here, we discuss coronin 3 properties in the light of new structural information and focus on assays that reflect in vivo roles of coronin 3 and its impact on F-actin associated functions. Using GFP-tagged coronin 3 fusion proteins and RNAi silencing we show that coronin 3 has roles in wound healing, protrusion formation, cell proliferation, cytokinesis, endocytosis, axonal growth, and secretion. During formation of cell protrusions actin accumulation precedes the focal enrichment of coronin 3 suggesting a role for coronin 3 in events that follow the initial F-actin assembly. Moreover, we show that coronin 3 similar to other coronins interacts with the Arp2/3-complex and cofilin indicating that this family in general is involved in regulating Arp2/3 mediated events. | | | PubMed | DOI | PDF |
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Winter, A., Kämäräinen, O., Hofmann, A. (2007)
Proteins 68, 353-362
Abstract
Prohibitins comprise a family of highly conserved ubiquitous eukaryotic proteins that mainly localize to the mitochondria. They have been implicated in important cellular processes such as cellular signalling and transcriptional control, apoptosis, cellular senescence, and mitochondrial biogenesis.
Using molecular modelling techniques, we have generated structural models of human prohibitins BAP32 and BAP37 which have previously been shown to exist as large ring-like oligomers in the membrane-bound state. The middle domain of prohibitins is evolutionary conserved in the family of SPFH (PHB) domain proteins. Based on the known structure of flotillin-2, another member of the SPFH domain family, we have generated homology models for BAP32 and BAP37, and elucidated the implications for formation of high molecular weight oligomers. A model for the dimeric building block of BAP32:BAP37 for such assemblies was generated and its stability scrutinized by molecular dynamics simulations. The model of BAP32 was also analyzed as to potential ligand binding sites and the previously identified ligand melanogenin was docked into a membrane-proximal cavity.
The results are discussed in the context of prohibitin interactions with mitochondrial AAA-proteases and we suggest two possible interaction interfaces between the BAP32:BAP37 building block and the protease.
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