I03-Macromolecular Crystallography
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Andry Mercedes
Mavila
,
Jhon Antoni
Vargas
,
Eloy
Condori
,
Erick Giancarlo
Suclupe Farro
,
Adriano
Alves Furtado
,
Josué Manuel
López
,
Silvia Lucila
Gonzalez
,
Humberto D’muniz
Pereira
,
Jorge Luis
Marapara
,
Roger Ruiz
Paredes
,
Marianela
Cobos
,
Juan C.
Castro
,
Richard Charles
Garratt
,
Diego Antonio
Leonardo
Diamond Proposal Number(s):
[31229]
Abstract: Acetyl-CoA carboxylase (ACC) is an essential enzyme in fatty acid biosynthesis that catalyzes the formation of malonyl-CoA from acetyl-CoA. While structural studies on ACC components have largely focused on prokaryotes and higher plants, the assembly and molecular adaptations of ACC in microalgae remain underexplored. This study aimed to fill this gap by providing the first structural and evolutionary characterization of both biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) from a microalga (Ankistrodesmus sp.). Phylogenetic analysis revealed distinct evolutionary trajectories for BC and BCCP, with BC forming a chlorophyte-specific clade closely related to other oleaginous species, while BCCP displayed two distinct isoforms within green algae, resulting from gene duplication. The crystallographic structure of BC was solved in its apo (1.75 Å) and ADP-Mg2+-bound (1.90 Å) states, revealing conserved conformational changes associated with cofactor binding. BCCP from Ankistrodesmus sp. displayed a unique QLGTF/H motif instead of the canonical AMKXM biotinylation motif, suggesting loss of biotinylation capacity. However, the presence of three additional lysines in the protruding thumb loop, with Lys95 as a candidate for biotin attachment, indicates potential compensatory adaptations. SEC-MALS and pull-down assays confirmed the formation of a stable 1:1 BC-BCCP complex, and circular dichroism showed increased thermal stability of the complex, supporting its structural stability. This study highlights unique structural adaptations in Ankistrodesmus sp. ACC, emphasizing the evolutionary plasticity of BC and BCCP. These insights provide a foundation for future investigations into ACC regulation in photosynthetic organisms and offer potential biotechnological applications for optimizing lipid production in microalgae.
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Mar 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31229]
Abstract: L-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler (SW) pathway is the primary route for AsA biosynthesis and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focusses on three of them from the model system Myrciaria dubia (camu-camu): GDP-D-mannose 3',5'-epimerase (GME), L-galactose dehydrogenase (L-GalDH), and L-galactono-1,4-lactone dehydrogenase (L-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. MdL-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue from spinach. Finally, the structure of MdL-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with the FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants and the information provided should prove useful for biotechnological applications.
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Jan 2024
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31229]
Abstract: Septins are membrane-associated, GTP-binding proteins that are present in most eukaryotes. They polymerize to play important roles as scaffolds and/or diffusion barriers as part of the cytoskeleton. α-Helical coiled-coil domains are believed to contribute to septin assembly, and those observed in both human SEPT6 and SEPT8 form antiparallel homodimers. These are not compatible with their parallel heterodimeric organization expected from the current model for protofilament assembly, but they could explain the interfilament cross-bridges observed by microscopy. Here, the first structure of a heterodimeric septin coiled coil is presented, that between SEPT14 and SEPT7; the former is a SEPT6/SEPT8 homolog. This new structure is parallel, with two long helices that are axially shifted by a full helical turn with reference to their sequence alignment. The structure also has unusual knobs-into-holes packing of side chains. Both standard seven-residue (heptad) and the less common 11-residue (hendecad) repeats are present, creating two distinct regions with opposite supercoiling, which gives rise to an overall straight coiled coil. Part of the hendecad region is required for heterodimerization and therefore may be crucial for selective septin recognition. These unconventional sequences and structural features produce a metastable heterocomplex that nonetheless has enough specificity to promote correct protofilament assembly. For instance, the lack of supercoiling may facilitate unzipping and transitioning to the antiparallel homodimeric state.
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Oct 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31229]
Abstract: Septins possess a conserved guanine nucleotide-binding (G) domain that participates in the stabilization of organized hetero-oligomeric complexes which assemble into filaments, rings and network-like structures. The fruit fly, Drosophila melanogaster, has five such septin genes encoding Sep1, Sep2, Sep4, Sep5 and Pnut. Here, we report the crystal structure of the heterodimer formed between the G-domains of Sep1 and Sep2, the first from an insect to be described to date. A G-interface stabilizes the dimer (in agreement with the expected arrangement for the Drosophila hexameric particle) and this bears significant resemblance to its human counterparts, even down to the level of individual amino acid interactions. On the other hand, a model for the G-interface formed between the two copies of Pnut which occupy the center of the hexamer, shows important structural differences, including the loss of a highly favourable bifurcated salt-bridge network. Whereas wild-type Pnut purifies as a monomer, the reintroduction of the salt-bridge network results in stabilizing the dimeric interface in solution as shown by size exclusion chromatography and thermal stability measurements. Adaptive steered molecular dynamics (ASMD) reveals an unzipping mechanism for dimer dissociation which initiates at a point of electrostatic repulsion within the switch II region. Overall, the data contribute to a better understanding of the molecular interactions involved in septin assembly/disassembly
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Dec 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[29507]
Open Access
Abstract: In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learnt concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase (SoGDH from spinach), from the D-mannose/L-galactose (Smirnoff Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homologue from camu-camu, a super-accumulator of vitamin C found in the Peruvian amazon. Both enzymes are monomers in solution, have a pH optimum of 7 and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (β/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favours the oxidation reaction which ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes towards a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.
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Jun 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14493]
Open Access
Abstract: SUGARWINs are PR-4 proteins associated with sugarcane defense against phytopathogens. Their expression is induced in response to damage by Diatraea saccharalis larvae. These proteins play an important role in plant defense, in particular against fungal pathogens, such as Colletothricum falcatum (Went) and Fusarium verticillioides. The pathogenesis-related protein-4 (PR-4) family is a group of proteins equipped with a BARWIN domain, which may be associated with a chitin-binding domain also known as the hevein-like domain. Several PR-4 proteins exhibit both chitinase and RNase activity, with the latter being associated with the presence of two histidine residues H11 and H113 (BARWIN) [H44 and H146, SUGARWINs] in the BARWIN-like domain. In sugarcane, similar to other PR-4 proteins, SUGARWIN1 exhibits ribonuclease, chitosanase and chitinase activities, whereas SUGARWIN2 only exhibits chitosanase activity. In order to decipher the structural determinants involved in this diverse range of enzyme specificities, we determined the 3-D structure of SUGARWIN2, at 1.55Å by X-ray diffraction. This is the first structure of a PR-4 protein where the first histidine has been replaced by asparagine and was subsequently used to build a homology model for SUGARWIN1. Molecular dynamics simulations of both proteins revealed the presence of a flexible loop only in SUGARWIN1 and we postulate that this, together with the presence of the catalytic histidine at position 42, renders it competent as a ribonuclease. The more electropositive surface potential of SUGARWIN1 would also be expected to favor complex formation with RNA. A phylogenetic analysis of PR-4 proteins obtained from 106 Embryophyta genomes showed that both catalytic histidines are widespread among them with few replacements in these amino acid positions during the gene family evolutionary history. We observe that the H11 replacement by N11 is also present in two other sugarcane PR-4 proteins: SUGARWIN3 and SUGARWIN4. We propose that RNase activity was present in the first Embryophyta PR-4 proteins but was recently lost in members of this family during the course of evolution.
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Sep 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diego A.
Leonardo
,
Italo A.
Cavini
,
Fernanda A.
Sala
,
Deborah C.
Mendonça
,
Higor
V. D. Rosa
,
Patricia S.
Kumagai
,
Edson
Crusca Jr
,
Napoleao F.
Valadares
,
Ivo A.
Marques
,
Jose
Brandao-Neto
,
Claudia E.
Munte
,
Hans R.
Kalbitzer
,
Nicolas
Soler
,
Isabel
Uson
,
Ingemar
André
,
Ana
P. U. Araujo
,
Humberto
D'Muniz Pereira
,
Richard C.
Garratt
Diamond Proposal Number(s):
[6397, 23570, 25296]
Abstract: Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross-bridges necessary for higher order structures and thereby septin function.
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Feb 2021
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Abstract: The assembly of a septin filament requires that homologous monomers must distinguish between one another in establishing appropriate interfaces with their neighbors. To understand this phenomenon at the molecular level, we present the first four crystal structures of heterodimeric septin complexes. We describe in detail the two distinct types of G-interface present within the octameric particles which must polymerize to form filaments. These are formed between SEPT2 and SEPT6 and between SEPT7 and SEPT3, and their description permits an understanding of the structural basis for the selectivity necessary for correct filament assembly. By replacing SEPT6 by SEPT8 or SEPT11, it is possible to rationalize Kinoshita's postulate which predicts the exchangeability of septins from within a subgroup. Switches I and II, which in classical small GTPases provide a mechanism for nucleotide-dependent conformational change, have been repurposed in septins to play a fundamental role in molecular recognition. Specifically, it is switch I which holds the key to discriminating between the two different G-interfaces. Moreover, residues which are characteristic for a given subgroup play subtle, but pivotal, roles in guaranteeing that the correct interfaces are formed.
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Sep 2020
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14493, 5810]
Open Access
Abstract: Human septins 3, 9 and 12 are the only members of a specific subgroup of septins that display several unusual features, including the absence of a C-terminal coiled coil. This particular subgroup (the SEPT3 septins) are present in rod-like octameric protofilaments but are lacking in similar hexameric assemblies, which only contain representatives of the three remaining subgroups. Both hexamers and octamers can self-assemble into mixed filaments by end-to-end association, implying that the SEPT3 septins may facilitate polymerization but not necessarily function. These filaments frequently associate into higher order complexes which associate with biological membranes, triggering a wide range of cellular events. In the present work, a complete compendium of crystal structures for the GTP-binding domains of all of the SEPT3 subgroup members when bound to either GDP or to a GTP analogue is provided. The structures reveal a unique degree of plasticity at one of the filamentous interfaces (dubbed NC). Specifically, structures of the GDP and GTPγS complexes of SEPT9 reveal a squeezing mechanism at the NC interface which would expel a polybasic region from its binding site and render it free to interact with negatively charged membranes. On the other hand, a polyacidic region associated with helix α5′, the orientation of which is particular to this subgroup, provides a safe haven for the polybasic region when retracted within the interface. Together, these results suggest a mechanism which couples GTP binding and hydrolysis to membrane association and implies a unique role for the SEPT3 subgroup in this process. These observations can be accounted for by constellations of specific amino-acid residues that are found only in this subgroup and by the absence of the C-terminal coiled coil. Such conclusions can only be reached owing to the completeness of the structural studies presented here.
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May 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15991]
Open Access
Abstract: Copper-containing nitrite reductases (CuNiRs) are found in all three kingdoms of life and play a major role in the denitrification branch of the global nitrogen cycle where nitrate is used in place of dioxygen as an electron acceptor in respiratory energy metabolism. Several C- and N-terminal redox domain tethered CuNiRs have been identified and structurally characterized during the last decade. Our understanding of the role of tethered domains in these new classes of three-domain CuNiRs, where an extra cytochrome or cupredoxin domain is tethered to the catalytic two-domain CuNiRs, has remained limited. This is further compounded by a complete lack of substrate-bound structures for these tethered CuNiRs. There is still no substrate-bound structure for any of the as-isolated wild-type tethered enzymes. Here, structures of nitrite and product-bound states from a nitrite-soaked crystal of the N-terminal cupredoxin-tethered enzyme from the Hyphomicrobium denitrificans strain 1NES1 (Hd1NES1NiR) are provided. These, together with the as-isolated structure of the same species, provide clear evidence for the role of the N-terminal peptide bearing the conserved His27 in water-mediated anchoring of the substrate at the catalytic T2Cu site. Our data indicate a more complex role of tethering.
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May 2020
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