ORCID Profile
0000-0002-9770-4594
Current Organisation
University of Western Australia
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Publisher: Elsevier BV
Date: 07-2018
Publisher: Portland Press Ltd.
Date: 13-08-2021
DOI: 10.1042/BCJ20210455
Abstract: Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation–π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation–π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.
Publisher: MDPI AG
Date: 10-12-2018
DOI: 10.3390/CRYST8120460
Abstract: Chymotrypsin is a protease that is commonly used as a standard for protein crystallization and as a model system for studying serine proteases. Unliganded bovine α-chymotrypsin was crystallized at neutral pH using ammonium sulphate as the precipitant, resulting in crystals that conform to P65 symmetry with unit cell parameters that have not been reported previously. Inspection of crystallographic interfaces revealed that the major interface between any two molecules in the crystal lattice represents the interface of the biological dimer, as previously observed for crystals of unliganded α-chymotrypsin grown at low pH in space group P21.
Publisher: Elsevier BV
Date: 11-2022
Publisher: International Union of Crystallography (IUCr)
Date: 27-09-2022
DOI: 10.1107/S2059798322008579
Abstract: The recombination directionality factors from Mesorhizobium spp. (RdfS) are involved in regulating the excision and transfer of integrative and conjugative elements. Here, solution small-angle X-ray scattering, and crystallization and preliminary structure solution of RdfS from Mesorhizobium japonicum R7A are presented. RdfS crystallizes in space group P 2 1 2 1 2 1 , with evidence of eightfold rotational crystallographic/noncrystallographic symmetry. Initial structure determination by molecular replacement using ab initio models yielded a partial model (three molecules), which was completed after manual inspection revealed unmodelled electron density. The finalized crystal structure of RdfS reveals a head-to-tail polymer forming left-handed superhelices with large solvent channels. Additionally, RdfS has significant disorder in the C-terminal region of the protein, which is supported by the solution scattering data and the crystal structure. The steps taken to finalize structure determination, as well as the scattering and crystallographic characteristics of RdfS, are discussed.
Publisher: International Union of Crystallography (IUCr)
Date: 15-06-2023
DOI: 10.1107/S160057752300406X
Abstract: A new high-pressure single-crystal diffraction setup has been designed and implemented at the Australian Synchrotron for collecting molecular and protein crystal structures. The setup incorporates a modified micro-Merrill–Bassett cell and holder designed specifically to fit onto the horizontal air-bearing goniometer, allowing high-pressure diffraction measurements to be collected with little to no modification of the beamline setup compared with ambient data collections. Compression data for the amino acid, L-threonine, and the protein, hen egg-white lysozyme, were collected, showcasing the capabilities of the setup.
Publisher: Elsevier BV
Date: 02-2019
DOI: 10.1016/J.BMCL.2018.12.032
Abstract: A series of dipeptide aldehydes containing different N-terminal heterocycles was prepared and assayed in vitro against α-chymotrypsin to ascertain the importance of the heterocycle in maintaining a β-strand geometry while also providing a hydrogen bond donor equivalent to the backbone amide nitrogen of the surrogate amino acid. The dipeptide containing a pyrrole constraint (10) was the most potent inhibitor, with >30-fold improved activity over dipeptides which lacked a nitrogen hydrogen bond donor (namely thiophene 11, furan 12 and pyridine 13). Molecular docking studies of 10 bound to α-chymotrypsin demonstrates a hydrogen bond between the pyrrole nitrogen donor and the backbone carbonyl of Gly
Publisher: EMBO
Date: 23-11-2022
Abstract: High-risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA-binding protein, NONO, in neuroblastoma is associated with poor patient outcome however, there is little understanding of the mechanism of NONO-dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biochemical and genome-wide molecular analysis to reveal complex NONO-dependent regulation of gene expression. NONO forms RNA- and DNA-tethered condensates throughout the nucleus and undergoes phase separation in vitro, modulated by nucleic acid binding. CLIP analyses show that NONO mainly binds to the 5' end of pre-mRNAs and modulates pre-mRNA processing, dependent on its RNA-binding activity. NONO regulates super-enhancer-associated genes, including HAND2 and GATA2. Abrogating NONO RNA binding, or phase separation activity, results in decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA-binding activity of NONO may have therapeutic potential in this cancer context.
Publisher: Oxford University Press (OUP)
Date: 14-12-2021
DOI: 10.1093/NAR/GKAB1216
Abstract: The Drosophila behaviour/human splicing (DBHS) proteins are a family of RNA/DNA binding cofactors liable for a range of cellular processes. DBHS proteins include the non-POU domain-containing octamer-binding protein (NONO) and paraspeckle protein component 1 (PSPC1), proteins capable of forming combinatorial dimers. Here, we describe the crystal structures of the human NONO and PSPC1 homodimers, representing uncharacterized DBHS dimerization states. The structures reveal a set of conserved contacts and structural plasticity within the dimerization interface that provide a rationale for dimer selectivity between DBHS paralogues. In addition, solution X-ray scattering and accompanying biochemical experiments describe a mechanism of cooperative RNA recognition by the NONO homodimer. Nucleic acid binding is reliant on RRM1, and appears to be affected by the orientation of RRM1, influenced by a newly identified ‘β-clasp’ structure. Our structures shed light on the molecular determinants for DBHS homo- and heterodimerization and provide a basis for understanding how DBHS proteins cooperatively recognize a broad spectrum of RNA targets.
Publisher: American Society for Microbiology
Date: 03-2019
DOI: 10.1128/AAC.02281-18
Abstract: Aspergillus fumigatus infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals.
Publisher: Wiley
Date: 27-02-2017
DOI: 10.1111/FEBS.14035
Abstract: The fungal pathogen Aspergillus fumigatus has been implicated in a drastic increase in life-threatening infections over the past decade. However, compared to other microbial pathogens, little is known about the essential molecular processes of this organism. One such fundamental process is DNA replication. The protein responsible for ensuring processive DNA replication is PCNA (proliferating cell nuclear antigen, also known as the sliding cl ), which cl s the replicative polymerase to DNA. Here we present the first crystal structure of a sliding cl from a pathogenic fungus (A. fumigatus), at 2.6Å. Surprisingly, the structure bears more similarity to the human sliding cl than other available fungal sliding cl s. Reflecting this, fluorescence polarization experiments demonstrated that AfumPCNA interacts with the PCNA-interacting protein (PIP-box) motif of human p21 with an affinity (K The atomic coordinates and structure factors for the Aspergillus fumigatus sliding cl can be found in the RCSB Protein Data Bank (www.rcsb.org) under the accession code 5TUP.
Publisher: American Chemical Society (ACS)
Date: 13-08-2019
DOI: 10.1021/ACSCHEMBIO.9B00463
Abstract: Here, we report the design, synthesis, and evaluation of a series of inhibitors of
Publisher: Cold Spring Harbor Laboratory
Date: 30-11-2022
DOI: 10.1101/2022.11.30.518278
Abstract: Demixing of proteins and nucleic acids into condensed liquid phases is rapidly emerging as a ubiquitous mechanism governing the organisation of molecules within the cell. Long disordered low complexity regions (LCRs) are a common feature of proteins that form biomolecular condensates. RNA-binding proteins with prion-like composition have been highlighted as drivers of liquid demixing to form condensates such as nucleoli, paraspeckles and stress granules. Splicing factor proline- and glutamine-rich (SFPQ) is an RNA- and DNA-binding protein essential for DNA repair and paraspeckle formation. Here, we show that the shorter C-terminal LCR of SFPQ is the main region responsible for the condensation of SFPQ in vitro and in the cell. In contrast, we find that, unexpectedly, the longer N-terminal prion-like LCR of SFPQ attenuates condensation, suggesting a more regulatory role in preventing aberrant condensate formation in the cell. Our data add nuance to the emerging understanding of biomolecular condensate formation, by providing the first ex le of a common multifunctional nucleic acid-binding protein with an extensive prion-like region that serves to regulate rather than drive condensate formation.
Publisher: American Chemical Society (ACS)
Date: 03-02-2018
No related grants have been discovered for Andrew Marshall.