ORCID Profile
0000-0002-4756-911X
Current Organisation
University of Queensland
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Publisher: Elsevier BV
Date: 06-2021
Publisher: Elsevier BV
Date: 08-2020
Publisher: American Chemical Society (ACS)
Date: 21-01-2010
DOI: 10.1021/BI9015204
Publisher: American Chemical Society (ACS)
Date: 17-07-2009
DOI: 10.1021/BI900561A
Publisher: American Chemical Society (ACS)
Date: 10-2007
DOI: 10.1021/BI7013135
Abstract: Among the 118 genes upregulated by Pseudomonas aeruginosa in response to iron starvation [Ochsner, U. A., Wilderman, P. J., Vasil, A. I., and Vasil, M. L. (2002) Mol. Microbiol. 45, 1277-1287], we focused on the products of the two genes encoding electron transfer proteins, as a means of identifying the redox partners of the heme oxygenase (pa-HO) expressed under low-iron stress conditions. Biochemical and spectroscopic investigations demonstrated that the bfd gene encodes a 73-amino acid protein (pa-Bfd) that incorporates a [2Fe-2S]2+/+ center, whereas the fpr gene encodes a 258-residue NADPH-dependent ferredoxin reductase (pa-FPR) that utilizes FAD as a cofactor. In vitro reconstitution of pa-HO catalytic activity with the newly characterized proteins led to the surprising observation that pa-FPR efficiently supports the catalytic cycle of pa-HO, without the need of a ferredoxin. In comparison, electron transfer from pa-Bfd to pa-HO is sluggish, which strongly argues against the possibility that the seven electrons needed by pa-HO to degrade biliverdin are transferred from NADPH to pa-HO in a ferredoxin (Bfd)-dependent manner. Given that pa-HO functions to release iron from exogenous heme acquired under iron-starvation conditions, the use of a flavoenzyme rather than an iron-sulfur center-containing protein to support heme degradation is an efficient use of resources in the cell. The crystal structure of pa-FPR (1.6 A resolution) showed that its fold is comparable that of the superfamily of ferredoxin reductases and most similar to the structure of Azotobacter vinelandii FPR and Escherichia coli flavodoxin reductase. The latter two enzymes interact with distinct redox partners, a ferredoxin and a flavodoxin, respectively. Hence, findings reported herein extend the range of redox partners recognized by the fold of pa-FPR to include a heme oxygenase (pa-HO).
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1016/J.JMB.2012.06.041
Abstract: 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.
Publisher: Cold Spring Harbor Laboratory
Date: 12-2021
DOI: 10.1101/2021.11.30.470681
Abstract: Recent advances in protein structure prediction using machine learning such as AlphaFold2 and RosettaFold presage a revolution in structural biology. Genome-wide predictions of protein structures are providing unprecedented insights into their architecture and intradomain interactions, and applications have already progressed towards assessing protein complex formation. Here we present detailed analyses of the sorting nexin proteins that contain regulator of G-protein signalling domains (SNX-RGS proteins), providing a key ex le of the ability of AlphaFold2 to reveal novel structures with previously unsuspected biological functions. These large proteins are conserved in most eukaryotes and are known to associate with lipid droplets (LDs) and sites of LD-membrane contacts, with key roles in regulating lipid metabolism. They possess five domains, including an N-terminal transmembrane domain that anchors them to the endoplasmic reticulum, an RGS domain, a lipid interacting phox homology (PX) domain and two additional domains named the PXA and PXC domains of unknown structure and function. Here we report the crystal structure of the RGS domain of sorting nexin 25 (SNX25) and show that the AlphaFold2 prediction closely matches the experimental structure. Analysing the full-length SNX-RGS proteins across multiple homologues and species we find that the distant PXA and PXC domains in fact fold into a single unique structure that notably features a large and conserved hydrophobic pocket. The nature of this pocket strongly suggests a role in lipid or fatty acid binding, and we propose that these molecules represent a new class of conserved lipid transfer proteins.
Publisher: Cold Spring Harbor Laboratory
Date: 21-02-2023
DOI: 10.1101/2023.02.20.529329
Abstract: Munc18-interacting proteins (Mints) are multi-domain adaptors that regulate neuronal membrane trafficking, signalling and neurotransmission. Mint1 and Mint2 are highly expressed in the brain and play overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. The mechanism by which Mint1 mediates the interaction of Munc18-1 with Syntaxin1a (Sx1a) to control formation of the fusogenic SNARE complex and facilitate neurotransmission remains unclear. Here, we have studied both the specific interactions of the neuronal Mint proteins with Munc18-1 as well as their wider interactome. Using biochemical and biophysical binding approaches we identify a short acidic α -helical motif (AHM) within Mint1 and Mint2 that is necessary and sufficient for specific binding to Munc18-1. In silico modelling of this interaction with AlphaFold2 and extensive validation by structure-based mutagenesis showed that the AHM in Mint forms an α -helical structure that binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout cells we show that mutating the Mint-binding site significantly reduces the ability of Munc18-1 re-expression to rescue exocytosis. Although Munc18-1 can interact with Mint and Sx1a proteins simultaneously we find they have mutually reduced affinities, suggesting an allosteric coupling between the two proteins. Using AlphaFold2 we then examined the entire cellular network of putative interactions governed by the Mint scaffolds. Although this suggests many proposed binders do not associate directly with Mints, our analyses provide a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ARF3/ARF4 small GTPases, and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 provides experimental support that AlphaFold2 can correctly predict interactions across large-scale datasets. Overall, our data provides insights into the ersity of interactions mediated by the Mint family and shows that Mints may help facilitate a key trigger point in SNARE complex assembly and vesicle fusion.
Publisher: Cold Spring Harbor Laboratory
Date: 22-01-2022
DOI: 10.1101/2022.01.20.477115
Abstract: Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed ‘cargoes’) from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans -Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify novel retrograde cargo proteins of the Endosomal SNX-BAR Sorting Complex Promoting Exit-1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterised host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalised with the NRP1-interacting peptide of the SARS-CoV-2 Spike protein. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.
Publisher: American Chemical Society (ACS)
Date: 20-05-2011
DOI: 10.1021/BI2004119
Publisher: Rockefeller University Press
Date: 11-02-2021
Abstract: Ca2+-dependent neurotransmitter release requires synaptotagmins as Ca2+ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains—C2A and C2B—to Ca2+. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca2+ sensor in Caenorhabditis elegans. Here, we report a new Ca2+ sensor, SNT-3, which triggers delayed Ca2+-dependent neurotransmitter release. snt-1 snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca2+ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca2+ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca2+ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked litude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini–mediated membrane tethering. Our findings therefore reveal a novel dual Ca2+ sensor system in C. elegans and provide significant insights into Ca2+-regulated exocytosis.
Publisher: Elsevier BV
Date: 07-2020
Publisher: Springer Science and Business Media LLC
Date: 14-08-2012
Abstract: Two-dimensional data needs to be processed and analysed in almost any experimental laboratory. Some tasks in this context may be performed with generic software such as spreadsheet programs which are available ubiquitously, others may require more specialised software that requires paid licences. Additionally, more complex software packages typically require more time by the in idual user to understand and operate. Practical and convenient graphical data analysis software in Java with a user-friendly interface are rare. We have developed SDAR, a Java application to analyse two-dimensional data with an intuitive graphical user interface. A smart ASCII parser allows import of data into SDAR without particular format requirements. The centre piece of SDAR is the Java class GraphPanel which provides methods for generic tasks of data visualisation. Data can be manipulated and analysed with respect to the most common operations experienced in an experimental biochemical laboratory. Images of the data plots can be generated in SVG-, TIFF- or PNG-format. Data exported by SDAR is annotated with commands compatible with the Grace software. Since SDAR is implemented in Java, it is truly cross-platform compatible. The software is easy to install, and very convenient to use judging by experience in our own laboratories. It is freely available to academic users at csb/ . To download SDAR, users will be asked for their name, institution and email address. A manual, as well as the source code of the GraphPanel class can also be downloaded from this site.
Publisher: Oxford University Press (OUP)
Date: 12-02-2012
DOI: 10.1093/BIOINFORMATICS/BTS035
Abstract: Summary: Both alignment generation and visualization 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 visualization 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. Availability and implementation: SBAL has been implemented in Java to provide cross-platform compatibility. SBAL is freely available to academic users at csb/. Users will be asked for their name, institution and email address. A manual can also be downloaded from this site. The software, manual and test sets are also available as supplementary material. Contact: conan.wang@griffith.edu.au Supplementary information: Supplementary data are available at Bioinformatics online.
Publisher: Springer Science and Business Media LLC
Date: 10-2019
Publisher: Oxford University Press (OUP)
Date: 30-11-2011
DOI: 10.1093/BIOINFORMATICS/BTR664
Abstract: Summary: 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. Availability and implementation: DMAN is implemented in Java to provide a cross-platform compatibility. It is freely available to academic users at csb/. To download DMAN, users will be asked for their name, institution and email address. A manual can also be downloaded from this site. Contact: conan.wang@griffith.edu.au a.hofmann@griffith.edu.au
No related grants have been discovered for Saroja Weeratunga.