Browsing by Author "Sutherland-Smith AJ"

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A Single-Plasmid Inducible-Replication System for High-Yield Production of Short Ff (f1, M13 or fd)-Phage-Derived Nanorods
(John Wiley and Sons Ltd., 2025-04-01) León-Quezada RI; Miró MG; Khanum S; Sutherland-Smith AJ; Gold VAM; Rakonjac J
Ff (f1, M13 or fd) filamentous phages have been used for myriad applications including phage display, assembly of nanostructures and as carriers of agents used for diagnostic and therapeutic purposes. Recently, short Ff phage-derived functionalised nanorods have emerged as a superior alternative to full-length filamentous phages for applications from lateral flow assays to cell- and tissue-targeting. Their advantages, such as shorter length and the lack of antibiotic resistance genes, make them particularly promising for expanding the current scope of Ff bionanotechnology and biomedical applications. Limitations to the widespread use of Ff-derived nanorods include a requirement for two plasmids and the relatively low production efficiency. This is due to the presence of only the positive Ff origin of replication, allowing replication of only the positive strand. Here we describe a single-plasmid negative origin-containing inducible-replication system for nanorod production. These improvements simplify and increase nanorod production by two orders of magnitude compared with the constitutive positive origin-only production system. The high concentration of nanorods allows formation of higher-order structures, such as stacks and rafts, as imaged by transmission electron microscopy. In summary, our system will facilitate production and expand the applications of Ff-derived biological nanorods.
Beyond BLASTing: How 3D structural prediction can support or refute BLAST results
(2015-09) Daly TK; Sutherland-Smith AJ; Penny ED
Biologists know that not all BLAST results are homologs and that trees lose information at deep times. We have therefore developed a pipeline to bring increased confidence to the BLAST result. We use structural prediction to support or refute the inclusion of protein sequences into multiple sequence alignments. Ancestral sequence reconstruction (ASR) together with structural prediction can be used to create seed sequences to help find remote homologs. The process can be used to help with annotation.
In silico resurrection of the major vault protein suggests it is ancestral in modern eukaryotes
(Oxford University Press, 25/07/2013) Daly TK; Sutherland-Smith AJ; Penny ED
Vaults are very large oligomeric ribonucleoproteins conserved among a variety of species. The rat vault 3D structure shows an ovoid oligomeric particle, consisting of 78 major vault protein monomers, each of approximately 861 amino acids. Vaults are probably the largest ribonucleoprotein structures in eukaryote cells, being approximately 70 nm in length with a diameter of 40 nm--the size of three ribosomes and with a lumen capacity of 50 million Å(3). We use both protein sequences and inferred ancestral sequences for in silico virtual resurrection of tertiary and quaternary structures to search for vaults in a wide variety of eukaryotes. We find that the vault's phylogenetic distribution is widespread in eukaryotes, but is apparently absent in some notable model organisms. Our conclusion from the distribution of vaults is that they were present in the last eukaryote common ancestor but they have apparently been lost from a number of groups including fungi, insects, and probably plants. Our approach of inferring ancestral 3D and quaternary structures is expected to be useful generally.
N-terminal oligomerization drives HDAC4 nuclear condensation and neurodevelopmental dysfunction in Drosophila
(The Royal Society, 2025-10) Hawley HR; Sutherland-Smith AJ; Savoian MS; Fitzsimons HL
Histone deacetylase four (HDAC4) undergoes dynamic nucleocytoplasmic shuttling, a process critical for regulating its activity. However, aberrant nuclear accumulation of HDAC4 is associated with both neurodevelopmental and neurodegenerative disease, and in our Drosophila model, impairs normal neuronal development. Upon nuclear accumulation, HDAC4 forms biomolecular condensates, previously termed aggregates, that correlate with the severity of defects in development of the Drosophila mushroom body and adult eye. Here we determined that nuclear condensation of HDAC4 is dependent on self-oligomerization, and that impairing oligomerization reduces condensation and the severity of neurodevelopmental phenotypes in Drosophila. HDAC4 condensates are highly dynamic and are stabilized by the presence of MEF2, which promotes their formation, ultimately exacerbating phenotypic severity. These data provide insight into the role of HDAC4 condensates in normal neuronal function and suggest that their dysregulation may contribute to neurodevelopmental disorders. Consequently, targeting oligomerization of HDAC4 and its interaction with MEF2 present potential therapeutic strategies for diseases associated with HDAC4 nuclear accumulation.
Structural characterisation of methanogen pseudomurein cell wall peptide ligases homologous to bacterial MurE/F murein peptide ligases.
(2022-09) Subedi BP; Schofield LR; Carbone V; Wolf M; Martin WF; Ronimus RS; Sutherland-Smith AJ
Archaea have diverse cell wall types, yet none are identical to bacterial peptidoglycan (murein). Methanogens Methanobacteria and Methanopyrus possess cell walls of pseudomurein, a structural analogue of murein. Pseudomurein differs from murein in containing the unique archaeal sugar N-acetyltalosaminuronic acid instead of N-acetylmuramic acid, β-1,3 glycosidic bonds in place of β-1,4 bonds and only l-amino acids in the peptide cross-links. We have determined crystal structures of methanogen pseudomurein peptide ligases (termed pMurE) from Methanothermus fervidus (Mfer762) and Methanothermobacter thermautotrophicus (Mth734) that are structurally most closely related to bacterial MurE peptide ligases. The homology of the archaeal pMurE and bacterial MurE enzymes is clear both in the overall structure and at the level of each of the three domains. In addition, we identified two UDP-binding sites in Mfer762 pMurE, one at the exterior surface of the interface of the N-terminal and middle domains, and a second site at an inner surface continuous with the highly conserved interface of the three domains. Residues involved in ATP binding in MurE are conserved in pMurE, suggesting that a similar ATP-binding pocket is present at the interface of the middle and the C-terminal domains of pMurE. The presence of pMurE ligases in members of the Methanobacteriales and Methanopyrales, that are structurally related to bacterial MurE ligases, supports the idea that the biosynthetic origins of archaeal pseudomurein and bacterial peptidoglycan cell walls are evolutionarily related.
Structural characterisation of nucleotide sugar short-chain dehydrogenases/reductases from the thermophilic pseudomurein-containing methanogen Methanothermobacter thermautotrophicus ΔH
(John Wiley and Sons Ltd on behalf of Federation of European Biochemical Societies, 2025-09-03) Carbone V; Schofield LR; Edwards PJB; Sutherland-Smith AJ; Ronimus RS
Epimerases and dehydratases are widely studied members of the extendedshort-chain dehydrogenase/reductase (SDR) enzyme superfamily and areimportant in nucleotide sugar conversion and diversiﬁcation, for example,the interconversion of uridine diphosphate (UDP)-linked glucose andgalactose. Methanothermobacter thermautotrophicus contains a cluster ofgenes, the annotations of which indicate involvement in glycan biosynthesissuch as that of cell walls or capsular polysaccharides. In particular, genesencoding UDP-glucose 4-epimerase related protein (Mth375), UDP-glucose4-epimerase homologue (Mth380) and dTDP-glucose 4,6-dehydrataserelated protein (Mth373) may be involved in the biosynthesis of an unusualaminosugar in pseudomurein. In this paper, we present the structures ofMth375, an archaeal sugar epimerase/dehydratase protein (WbmF) deter-mined to a resolution of 2.0 A. The structure contains an N-terminalRossmann-fold domain with bound nicotinamide adenine dinucleotidehydride (NADH) and a C-terminal catalytic domain with bound UDP. Wealso present the structure for Mth373 co-crystallised with uridine-50-diphosphate-xylopyranose to a resolution of 1.96 A as a NAD+-dependentoxidative decarboxylase (UDP-xylose synthase; EC4.1.1.35). Molecularmodelling has also allowed for the identiﬁcation of Mth380 as aUDP-N-acetylglucosamine 4-epimerase (WbpP; EC5.1.3.7), Mth631 as aUDP-glucose 4-epimerase (GalE; EC5.1.3.2) and Mth1789 as a classicaldTDP-D-glucose 4,6-dehydratase (EC4.2.1.46). The UDP–sugar speciﬁcityof each archaeal nucleotide sugar short-chain dehydrogenase/reductase.
The Structural and Functional Characterization of Mammalian ADP-dependent Glucokinase.
(19/02/2016) Richter JP; Goroncy AK; Ronimus RS; Sutherland-Smith AJ
The enzyme-catalyzed phosphorylation of glucose to glucose-6-phosphate is a reaction central to the metabolism of all life. ADP-dependent glucokinase (ADPGK) catalyzes glucose-6-phosphate production, utilizing ADP as a phosphoryl donor in contrast to the more well characterized ATP-requiring hexokinases. ADPGK is found in Archaea and metazoa; in Archaea, ADPGK participates in a glycolytic role, but a function in most eukaryotic cell types remains unknown. We have determined structures of the eukaryotic ADPGK revealing a ribokinase-like tertiary fold similar to archaeal orthologues but with significant differences in some secondary structural elements. Both the unliganded and the AMP-bound ADPGK structures are in the "open" conformation. The structures reveal the presence of a disulfide bond between conserved cysteines that is positioned at the nucleotide-binding loop of eukaryotic ADPGK. The AMP-bound ADPGK structure defines the nucleotide-binding site with one of the disulfide bond cysteines coordinating the AMP with its main chain atoms, a nucleotide-binding motif that appears unique to eukaryotic ADPGKs. Key amino acids at the active site are structurally conserved between mammalian and archaeal ADPGK, and site-directed mutagenesis has confirmed residues essential for enzymatic activity. ADPGK is substrate inhibited by high glucose concentration and shows high specificity for glucose, with no activity for other sugars, as determined by NMR spectroscopy, including 2-deoxyglucose, the glucose analogue used for tumor detection by positron emission tomography.