B21-High Throughput SAXS
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Cristian Andres
Carmona-Carmona
,
Giovanni
Bisello
,
Rossella
Franchini
,
Gianluigi
Lunardi
,
Roberta
Galavotti
,
Massimiliano
Perduca
,
Rui P.
Ribeiro
,
Benny Danilo
Belviso
,
Alejandro
Giorgetti
,
Rocco
Caliandro
,
Patricia M.-J.
Lievens
,
Mariarita
Bertoldi
Diamond Proposal Number(s):
[21741]
Open Access
Abstract: Aromatic amino acid decarboxylase (AADC) deficiency is a severe inherited recessive neurotransmitter disorder caused by an impairment in dopamine synthesis due to the lack/modification of AADC, the enzyme converting l-dopa to dopamine. Patients exhibit severe movement disorders and neurodevelopmental delay, with a high risk of premature mortality. Given the lack of a reliable model for the disease, we developed a dopa decarboxylase knockout model using CRISPR/Cas9 technology in the SH-SY5Y neuroblastoma cell line. This model showed a deficiency in AADC protein and activity, with an altered dopamine metabolites profile (low homovanillic acid and high 3-O-methyldopa) and a modified expression of key enzymes, such as dopamine beta-hydroxylase and monoamine oxidases, which are involved in the catecholamine pathway. We then transfected the DDC-KO cells with two AADC catalytic variants, R347Q and L353P, which resulted in loss-of-function and an altered profile of dopamine metabolites. By combining several structural approaches (X-ray crystallography, molecular dynamics, small angle X-ray scattering, dynamic light scattering, and spectroscopy), we determined that both variants alter the flexibility of the structural element to which they belong, whose integrity is essential for catalysis. This change causes a mispositioning of essential residues at the active site, leading, in turn, to an unproductive external aldimine, identifying the molecular basis for the loss-of-function. Overall, the DDC-KO model recapitulates some key features of AADC deficiency, is useful to study the molecular basis of the disease, and represents an ideal system for small molecule screening regarding specific enzyme defects, paving the way for a precision therapeutic approach.
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May 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21625]
Open Access
Abstract: NrdR is a bacterial transcriptional repressor consisting of a zinc (Zn)-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two “NrdR boxes” upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, in the last of which we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus deoxyadenosine triphosphate (dATP) or equivalent diphosphate combinations. No other combination of adenine nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR–ATP–dATP and EcoNrdR–ADP–dATP, which are the first high-resolution crystal structures of an NrdR. We have also determined cryo-electron microscopy structures of DNA-bound EcoNrdR–ATP–dATP and novel filaments of EcoNrdR–ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR–ATP–dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATP-cones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that, when loaded with the correct nucleotides, adapt to an optimal promoter-binding conformation.
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Mar 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Actin is an intrinsically dynamic protein, the function and state of which are modulated by actin-binding proteins. Actin-depolymerizing factors (ADF)/cofilins are ubiquitous actin-binding proteins that accelerate actin turnover. Malaria is an infectious disease caused by parasites of the genus Plasmodium, which belong to the phylum Apicomplexa. The parasites require two hosts to complete their life cycle: the definitive host, or the vector, an Anopheles spp. mosquito, and a vertebrate intermediate host, such as humans. Here, the malaria vector Anopheles gambiae ADF (AgADF) crystal structure is reported. AgADF has a conserved ADF/cofilin fold with six central β-strands surrounded by five α-helices with a long β-hairpin loop protruding out of the structure. The G- and F-actin-binding sites of AgADF are conserved, and the structure shows features of potential importance for regulation by membrane binding and redox state. AgADF binds monomeric ATP- and ADP-actin with a high affinity, having a nanomolar Kd, and binds effectively also to actin filaments.
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Feb 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17212, 23269]
Open Access
Abstract: Angiotensin-1-converting enzyme (ACE) is a zinc-dependent carboxypeptidase of therapeutic interest for the treatment of hypertension, inflammation and fibrosis. It consists of two homologous N and C catalytic domains, nACE and cACE, respectively. Unfortunately, the current clinically available ACE inhibitors produce undesirable side effects due to the nonselective inhibition of these domains. Through structure-based drug design, we previously identified a series of diprolyl-derived inhibitors (SG3, SG15, SG16, SG17 and SG18) in an attempt to specifically target nACE. Only one compound, SG16, possessed significant nACEselectivity. The previously determined 16-nACE crystal structure (nACE:SG16) suggested interactions with Tyr369 (Phe381 in cACE) are responsible for this selectivity. To better understand the molecular basis for the lack of selectivity in the remaining compounds, we have cocrystallised nACE in complex with SG3, SG15, SG17 and SG18 and cACE in complex with SG3, SG15, SG16 and SG18 and determined their structures at high resolution. Apart from the catalytic residues, these structures further highlight the importance of residues distal to the active site that may play an important role in the design of domain-selective inhibitors of ACE.
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Jan 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Yuqi
Yu
,
Laura N.
Jeffreys
,
Harshwardhan
Poddar
,
Adam
Hill
,
Linus
Johannissen
,
Fanzhuo
Dai
,
Michiyo
Sakuma
,
David
Leys
,
Derren J.
Heyes
,
Shaowei
Zhang
,
Nigel S.
Scrutton
Diamond Proposal Number(s):
[24447]
Open Access
Abstract: Photoreceptors control cellular processes in response to light. Most photoreceptors sense blue or red light, but the recent discovery of the cobalamin-dependent photoreceptor, CarH, has expanded the wavelength range of photoreception to other regions of the electromagnetic spectrum to include the green light region. Further identification of cobalamin-dependent green light-sensitive photoreceptors has been hampered owing to poor annotation of the light responsiveness of cobalamin-binding domains (CBDs) in public databases. Here we report a computational workflow, SignatureFinder, that uses a combination of sequence and structural analyses to identify new light-responsive CBD-containing proteins. The light response of exemplar proteins containing the proposed signature were confirmed experimentally. A structural analysis of these new photoreceptors, including the crystal structure of a new CBD domain, highlights how the signature elements interact with the cobalamin chromophore to sense light. Database mining of 128 000 CBD-containing sequences using the identified signature revealed more diverse CBD-containing photoreceptors, thereby expanding the family of green-light photoreceptors. A SignatureFinder web server is available (https://enzymeevolver.com) for wider applications, including the identification of signature sequences of other biological ligands of interest.
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Dec 2024
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I04-Macromolecular Crystallography
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Abstract: The chemosensory pathway HtChe2 from the marine bacterium Halomonas titanicae KHS3 controls the activity of a diguanylate cyclase. Constitutive activation of this pathway results in colony morphology alterations and an increased ability to form biofilm. Such characteristics resemble the behavior of the Wsp pathway of Pseudomonas. In this work, we investigate the specificity of Htc10, the only chemoreceptor coded within the HtChe2 gene cluster. The purine derivatives guanine and hypoxanthine were identified as ligands of the recombinantly produced Htc10 ligand-binding domain, with dissociation constants in the micromolar range, and its structure was solved by X-ray protein crystallography. The sensor domain of Htc10 adopts a double Cache folding, with ligands bound to the membrane-distal pocket. A high-resolution structure of the occupied guanine-binding pocket allowed the identification of residues involved in ligand recognition. Such residues were validated by site-directed mutagenesis and isothermal titration calorimetry analyses of the protein variants. Moreover, heterologous expression of Htc10 in a Pseudomonas putida mutant lacking the native Wsp chemoreceptor promoted biofilm formation, a phenotype that was further enhanced by Htc10-specific ligands. To our knowledge, this is the first description of binding specificity of a chemoreceptor that controls the activity of an associated diguanylate cyclase, opening the way for dynamic studies of the signaling behavior of this kind of sensory complex.
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Nov 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25413]
Open Access
Abstract: Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site.
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Aug 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[30305]
Open Access
Abstract: Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.
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Jul 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21625]
Open Access
Abstract: Isoprene pyrophosphates play a crucial role in the synthesis of a diverse array of essential nonsterol and sterol biomolecules and serve as substrates for posttranslational isoprenylation of proteins, enabling specific anchoring to cellular membranes. Hydrolysis of isoprene pyrophosphates would be a means to modulate their levels, downstream products, and protein isoprenylation. While NUDIX hydrolases from plants have been described to catalyze the hydrolysis of isoprene pyrophosphates, homologous enzymes with this function in animals have not yet been reported. In this study, we screened an extensive panel of human NUDIX hydrolases for activity in hydrolyzing isoprene pyrophosphates. We found that human nucleotide triphosphate diphosphatase NUDT15 and 8-oxo-dGDP phosphatase NUDT18 efficiently catalyze the hydrolysis of several physiologically relevant isoprene pyrophosphates. Notably, we demonstrate that geranyl pyrophosphate is an excellent substrate for NUDT18, with a catalytic efficiency of 2.1 × 105 m−1·s−1, thus making it the best substrate identified for NUDT18 to date. Similarly, geranyl pyrophosphate proved to be the best isoprene pyrophosphate substrate for NUDT15, with a catalytic efficiency of 4.0 × 104 M−1·s−1. LC–MS analysis of NUDT15 and NUDT18 catalyzed isoprene pyrophosphate hydrolysis revealed the generation of the corresponding monophosphates and inorganic phosphate. Furthermore, we solved the crystal structure of NUDT15 in complex with the hydrolysis product geranyl phosphate at a resolution of 1.70 Å. This structure revealed that the active site nicely accommodates the hydrophobic isoprenoid moiety and helped identify key binding residues. Our findings imply that isoprene pyrophosphates are endogenous substrates of NUDT15 and NUDT18, suggesting they are involved in animal isoprene pyrophosphate metabolism.
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Jun 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23853]
Open Access
Abstract: Mutants of alpha-1-antitrypsin cause the protein to self-associate and form ordered aggregates (‘polymers’) that are retained within hepatocytes, resulting in a predisposition to the development of liver disease. The associated reduction in secretion, and for some mutants, impairment of function, leads to a failure to protect lung tissue against proteases released during the inflammatory response and an increased risk of emphysema. We report here a novel deficiency mutation (Gly192Cys), that we name the Sydney variant, identified in a patient in heterozygosity with the Z allele (Glu342Lys). Cellular analysis revealed that the novel variant was mostly retained as insoluble polymers within the endoplasmic reticulum. The basis for this behaviour was investigated using biophysical and structural techniques. The variant showed a 40% reduction in inhibitory activity and a reduced stability as assessed by thermal unfolding experiments. Polymerisation involves adoption of an aggregation-prone intermediate and paradoxically the energy barrier for transition to this state was increased by 16% for the Gly192Cys variant with respect to the wild-type protein. However, with activation to the intermediate state, polymerisation occurred at a 3.8-fold faster rate overall. X-ray crystallography provided two crystal structures of the Gly192Cys variant, revealing perturbation within the ‘breach’ region with Cys192 in two different orientations: in one structure it faces towards the hydrophobic core while in the second it is solvent-exposed. This orientational heterogeneity was confirmed by PEGylation. These data show the critical role of the torsional freedom imparted by Gly192 in inhibitory activity and stability against polymerisation.
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Mar 2024
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