Browsing by Author "Ryan TM"

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    Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity
    (MDPI (Basel, Switzerland), 2022-09-06) Barzak FM; Ryan TM; Mohammadzadeh N; Harjes S; Kvach MV; Kurup HM; Krause KL; Chelico L; Filichev VV; Harjes E; Jameson GB; De la Torre JC; Andrei G
    APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2'-deoxycytidine embedded in 40-mer ssDNA was replaced by 2'-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G-ssDNA complex that gives insight into the observed "jumping" behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3-ssDNA complexes.
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    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 L
    In 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.
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    Structural and functional characterisation of the entry point to pyocyanin biosynthesis in Pseudomonas aeruginosa defines a new 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase subclass
    (Portland Press on behalf of the Biochemical Society, 2018-10) Sterritt OW; Lang EJM; Kessans SA; Ryan TM; Demeler B; Jameson GB; Parker EJ
    In Pseudomonas aeruginosa (Pae), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α2a and α2b, relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type IIA and type IIB based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism.
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    The influence of water, lanolin, urea, proline, paraffin and fatliquor on collagen D-spacing in leather
    (The Royal Society of Chemistry, 21/08/2017) Sizeland KH; Wells HC; Kelly S; Edmonds RL; Kirby NM; Hawley A; Mudie ST; Ryan TM; Haverkamp RG
    Water interacts with collagen to alter the structure at the fibrillar scale and therefore the mechanical properties of collagen. Humectants or moisturizers also alter the mechanical properties and fibril structure. The nature of these interactions and relationship between the different additives is not well understood. Changes in collagen D-spacing in leather were measured by synchrotron based small angle X-ray scattering in samples stored at various relative humidities and treated with lanolin, fatliquor, urea, proline or paraffin. The D-spacing increased with rising humidity and with increasing lanolin or fatliquor content, but not with treatment with urea, proline or paraffin. Strength increased with the addition of lanolin. Lanolin and fatliquor were shown to act as humectants whereas the other components did not act in this way. The Hofmeister effect is shown not to be a factor in the change in D-spacing, since samples treated with either proline or urea exhibited the same behavior. Different agents used in leather treatment and skin care function by different mechanisms, with collagen water retention being important for some additives but not others.