Journal Articles
Permanent URI for this collectionhttps://mro.massey.ac.nz/handle/10179/7915
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Item Structural characterization of a PCP-R didomain from an archaeal nonribosomal peptide synthetase reveals novel interdomain interactions(Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology, 2021-02-17) Deshpande S; Altermann E; Sarojini V; Lott JS; Lee TV; Jez JNonribosomal peptide synthetases (NRPSs) are multimodular enzymes that produce a wide range of bioactive peptides, such as siderophores, toxins, and antibacterial and insecticidal agents. NRPSs are dynamic proteins characterized by extensive interdomain communications as a consequence of their assembly-line mode of synthesis. Hence, crystal structures of multidomain fragments of NRPSs have aided in elucidating crucial interdomain interactions that occur during different steps of the NRPS catalytic cycle. One crucial yet unexplored interaction is that between the reductase (R) domain and the peptide carrier protein (PCP) domain. R domains are members of the short-chain dehydrogenase/reductase family and function as termination domains that catalyze the reductive release of the final peptide product from the terminal PCP domain of the NRPS. Here, we report the crystal structure of an archaeal NRPS PCP-R didomain construct. This is the first NRPS R domain structure to be determined together with the upstream PCP domain and is also the first structure of an archaeal NRPS to be reported. The structure reveals that a novel helix-turn-helix motif, found in NRPS R domains but not in other short-chain dehydrogenase/reductase family members, plays a major role in the interface between the PCP and R domains. The information derived from the described PCP-R interface will aid in gaining further mechanistic insights into the peptide termination reaction catalyzed by the R domain and may have implications in engineering NRPSs to synthesize novel peptide products.Item Deciphering the roles of subcellular distribution and interactions involving the MEF2 binding region, the ankyrin repeat binding motif and the catalytic site of HDAC4 in Drosophila neuronal morphogenesis(BioMed Central Ltd, 2024-12) Tan WJ; Hawley HR; Wilson SJ; Fitzsimons HLBACKGROUND: Dysregulation of nucleocytoplasmic shuttling of histone deacetylase 4 (HDAC4) is associated with several neurodevelopmental and neurodegenerative disorders. Consequently, understanding the roles of nuclear and cytoplasmic HDAC4 along with the mechanisms that regulate nuclear entry and exit is an area of concerted effort. Efficient nuclear entry is dependent on binding of the transcription factor MEF2, as mutations in the MEF2 binding region result in cytoplasmic accumulation of HDAC4. It is well established that nuclear exit and cytoplasmic retention are dependent on 14-3-3-binding, and mutations that affect binding are widely used to induce nuclear accumulation of HDAC4. While regulation of HDAC4 shuttling is clearly important, there is a gap in understanding of how the nuclear and cytoplasmic distribution of HDAC4 impacts its function. Furthermore, it is unclear whether other features of the protein including the catalytic site, the MEF2-binding region and/or the ankyrin repeat binding motif influence the distribution and/or activity of HDAC4 in neurons. Since HDAC4 functions are conserved in Drosophila, and increased nuclear accumulation of HDAC4 also results in impaired neurodevelopment, we used Drosophila as a genetic model for investigation of HDAC4 function. RESULTS: Here we have generated a series of mutants for functional dissection of HDAC4 via in-depth examination of the resulting subcellular distribution and nuclear aggregation, and correlate these with developmental phenotypes resulting from their expression in well-established models of neuronal morphogenesis of the Drosophila mushroom body and eye. We found that in the mushroom body, forced sequestration of HDAC4 in the nucleus or the cytoplasm resulted in defects in axon morphogenesis. The actions of HDAC4 that resulted in impaired development were dependent on the MEF2 binding region, modulated by the ankyrin repeat binding motif, and largely independent of an intact catalytic site. In contrast, disruption to eye development was largely independent of MEF2 binding but mutation of the catalytic site significantly reduced the phenotype, indicating that HDAC4 acts in a neuronal-subtype-specific manner. CONCLUSIONS: We found that the impairments to mushroom body and eye development resulting from nuclear accumulation of HDAC4 were exacerbated by mutation of the ankyrin repeat binding motif, whereas there was a differing requirement for the MEF2 binding site and an intact catalytic site. It will be of importance to determine the binding partners of HDAC4 in nuclear aggregates and in the cytoplasm of these tissues to further understand its mechanisms of action.Item Small-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor(MDPI (Basel, Switzerland), 2021-02-12) Barzak FM; Ryan TM; Kvach MV; Kurup HM; Aihara H; Harris RS; Filichev VV; Harjes E; Jameson GB; Chelico LIn normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.