I03-Macromolecular Crystallography
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Abstract: Several archaea harbour genes that code for fructosyltransferase (FTF) enzymes. These enzymes have not been characterized yet at structure‐function level, but are of extreme interest in view of their potential role in the synthesis of novel compounds for food, nutrition and pharmaceutical applications. In this study, 3D structure of an inulin‐type fructan producing enzyme, inulosucrase (InuHj), from the archaeon Halalkalicoccus jeotgali was resolved in its apo form as well as with bound substrate (sucrose) molecule and first transglycosylation product (1‐kestose). This is the first crystal structure of an FTF from halophilic archaea. Its overall five‐bladed β‐propeller fold is conserved with previously reported FTFs, but also shows some unique features. The InuHj structure is closer to those of Gram‐negative bacteria, with exceptions such as residue E266, which is conserved in FTFs of Gram‐positive bacteria and has possible role in fructan polymer synthesis in these bacteria as compared to fructooligosaccharide (FOS) production by FTFs of Gram‐negative bacteria. Highly negative electrostatic surface potential of InuHj, due to a large amount of acidic residues, likely contributes to its halophilicity. The complex of InuHj with 1‐kestose indicates that the residues D287 in the 4B‐4C loop, Y330 in 4D‐5A and D361 in the unique α2 helix may interact with longer FOSs and facilitate the binding of longer FOS chains during synthesis. The outcome of this work will provide targets for future structure‐function studies of FTF enzymes, particularly those from archaea.
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Mar 2021
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[17057]
Open Access
Abstract: Nonhomologous end joining (NHEJ) is a DNA repair mechanism that religates double‐strand DNA breaks to maintain genomic integrity during the entire cell cycle. The Ku70/80 complex recognizes DNA breaks and serves as an essential hub for recruitment of NHEJ components. Here, we describe intramolecular interactions of the Ku70 C‐terminal domain, known as the SAP domain. Using single‐particle cryo‐electron microscopy, mass spectrometric analysis of intermolecular cross‐linking and molecular modelling simulations, we captured variable positions of the SAP domain depending on DNA binding. The first position was localized at the DNA aperture in the Ku70/80 apo form but was not observed in the DNA‐bound state. The second position, which was observed in both apo and DNA‐bound states, was found below the DNA aperture, close to the helical arm of Ku70. The localization of the SAP domain in the DNA aperture suggests a function as a flexible entry gate for broken DNA.
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Feb 2021
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B21-High Throughput SAXS
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Open Access
Abstract: N‐myc downstream‐regulated gene 1 (NDRG1) is a tumour suppressor involved in vesicular trafficking and stress response. NDRG1 participates in peripheral nerve myelination, and mutations in the NDRG1 gene lead to Charcot‐Marie‐Tooth neuropathy. The 43‐kDa NDRG1 is considered as an inactive member of the α/β hydrolase superfamily. In addition to a central α/β hydrolase fold domain, NDRG1 consists of a short N terminus and a C‐terminal region with three 10‐residue repeats. We determined the crystal structure of the α/β hydrolase domain of human NDRG1 and characterised the structure and dynamics of full‐length NDRG1. The structure of the α/β hydrolase domain resembles the canonical α/β hydrolase fold with a central β sheet surrounded by α helices. Small‐angle X‐ray scattering and CD spectroscopy indicated a variable conformation for the N‐ and C‐terminal regions. NDRG1 binds to various types of lipid vesicles, and the conformation of the C‐terminal region is modulated upon lipid interaction. Intriguingly, NDRG1 interacts with metal ions, such as nickel, but is prone to aggregation in their presence. Our results uncover the structural and dynamic features of NDRG1, as well as elucidate its interactions with metals and lipids, and encourage studies to identify a putative hydrolase activity of NDRG1.
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Jan 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Dye‐decolorizing peroxidases (DyPs) constitute a superfamily of heme‐containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A‐C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D‐type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D‐type DyP ancestor, AncDyPD‐b1. Expression of AncDyPD‐b1 in Escherichia coli results in large amounts of a heme‐containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV‐Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady‐state kinetics and the crystal structure, reveal fine pH‐dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long‐range electron transfer process.
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Jan 2021
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B21-High Throughput SAXS
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Maria S.
Eriksen
,
Oleksii
Nikolaienko
,
Erik I.
Hallin
,
Sverre
Grødem
,
Helene J.
Bustad
,
Marte I.
Flydal
,
Ian
Merski
,
Tomohisa
Hosokawa
,
Daniela
Lascu
,
Shreeram
Akerkar
,
Jorge
Cuellar
,
James J.
Chambers
,
Rory
O’connell
,
Gopinath
Muruganandam
,
Remy
Loris
,
Christine
Touma
,
Tambudzai
Kanhema
,
Yasunori
Hayashi
,
Margaret M.
Stratton
,
José M.
Valpuesta
,
Petri
Kursula
,
Aurora
Martinez
,
Clive R.
Bramham
Abstract: Activity‐regulated cytoskeleton‐associated protein (Arc) is a protein interaction hub with diverse roles in intracellular neuronal signaling, and important functions in neuronal synaptic plasticity, memory, and postnatal cortical development. Arc has homology to retroviral Gag protein and is capable of self‐assembly into virus‐like capsids implicated in the intercellular transfer of RNA. However, the molecular basis of Arc self‐association and capsid formation is largely unknown. Here, we identified a 28‐amino‐acid stretch in the mammalian Arc N‐terminal (NT) domain that is necessary and sufficient for self‐association. Within this region, we identified a 7‐residue oligomerization motif, critical for the formation of virus‐like capsids. Purified wild‐type Arc formed capsids as shown by transmission and cryo‐electron microscopy, whereas mutant Arc with disruption of the oligomerization motif formed homogenous dimers. An atomic‐resolution crystal structure of the oligomerization region peptide demonstrated an antiparallel coiled‐coil interface, strongly supporting NT‐NT domain interactions in Arc oligomerization. The NT coil–coil interaction was also validated in live neurons using fluorescence lifetime FRET imaging, and mutation of the oligomerization motif disrupted Arc‐facilitated endocytosis. Furthermore, using single‐molecule photobleaching, we show that Arc mRNA greatly enhances higher‐order oligomerization in a manner dependent on the oligomerization motif. In conclusion, a helical coil in the Arc NT domain supports self‐association above the dimer stage, mRNA‐induced oligomerization, and formation of virus‐like capsids.
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Nov 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17212]
Abstract: Angiotensin‐1 converting enzyme (ACE) is a key enzyme in the renin‐angiotensin‐aldosterone and kinin systems where it cleaves angiotensin I and bradykinin peptides, respectively. However, ACE also participates in numerous other physiological functions, can hydrolyse many peptide substrates, and has various exo‐ and endopeptidase activities. ACE achieves this complexity by containing two homologous catalytic domains (N‐ and C‐domains), which exhibit different substrate specificities.
Here we present the first open conformation structures of ACE N‐domain, and a unique closed C‐domain structure (2.0 Å) where the C‐terminus of a symmetry‐related molecule is observed inserted into the active site cavity and binding to the zinc ion. The open native N‐domain structure (1.85 Å) enables comparison with ACE2, a homologue previously observed in open and closed states. An open S2_S′‐mutant N‐domain structure (2.80 Å) includes mutated residues in the S2‐ and S′‐ subsites that effect ligand binding, but are distal to the binding site. Analysis of these structures provides important insights into how structural features of the ACE domains are able to accommodate the wide variety of substrates and allow different peptidase activities.
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Oct 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Abstract: Steroidogenesis in adrenals and gonads starts from cholesterol transport to mitochondria. This is mediated by the steroidogenic acute regulatory protein (STARD1), containing a mitochondrial import sequence followed by a cholesterol‐binding START domain. Although mutations in this protein have been linked to lipoid congenital adrenal hyperplasia (LCAH), the mechanism of steroidogenesis regulation by STARD1 remains debatable. It has been hypothesized to involve a molten‐globule structural transition and interaction with 14‐3‐3 proteins. In this study, we aimed to address the structural basis for the 14‐3‐3‐STARD1 interaction. We show that, while the isolated START domain does not interact with 14‐3‐3, this interaction is enabled by STARD1 phosphorylation at Ser57, close to the mitochondrial peptide cleavage site. Biochemical analysis of the STARD1 affinity toward 14‐3‐3 and crystal structures of 14‐3‐3 complexes with Ser57 and Ser195 phosphopeptides suggest distinct roles of site‐specific phosphorylations in recruiting 14‐3‐3, to modulate STARD1 activity, processing and import to the mitochondria. Phosphorylation at Ser195 creates a unique conditional site that could only bind to 14‐3‐3 upon partial unfolding of the START domain. Overall, our findings on the interaction between 14‐3‐3 and STARD1 may have potential clinical implications for patients with LCAH.
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Sep 2020
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[22724]
Open Access
Abstract: Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light‐dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR–Pchlide–NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a ‘lid’ to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR ‘octamer’ has been isolated and its structure investigated by cryo‐electron microscopy at 7.7 Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation – a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development.
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Aug 2020
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[20229]
Open Access
Abstract: The PII‐like protein CutA is annotated as being involved in Cu2+ tolerance, based on analysis of Escherichia coli mutants. However, the precise cellular function of CutA remains unclear. Our bioinformatic analysis reveals that CutA proteins are universally distributed across all domains of life. Based on sequence‐based clustering, we chose representative cyanobacterial CutA proteins for physiological, biochemical, and structural characterization and examined their involvement in heavy metal tolerance, by generating CutA mutants in filamentous Nostoc sp. and in unicellular Synechococcus elongatus . However, we were unable to find any involvement of cyanobacterial CutA in metal tolerance under various conditions. This prompted us to re‐examine experimentally the role of CutA in protecting E. coli from Cu2+. Since we found no effect on copper tolerance, we conclude that CutA plays a different role that is not involved in metal protection. We resolved high‐resolution CutA structures from Nostoc and S. elongatus . Similarly to their counterpart from E. coli and to canonical PII proteins, cyanobacterial CutA proteins are trimeric in solution and in crystal structure; however, no binding affinity for small signaling molecules or for Cu2+ could be detected. The clefts between the CutA subunits, corresponding to the binding pockets of PII proteins, are formed by conserved aromatic and charged residues, suggesting a conserved binding/signaling function for CutA. In fact, we find binding of organic Bis‐Tris/MES molecules in CutA crystal structures, revealing a strong tendency of these pockets to accommodate cargo. This highlights the need to search for the potential physiological ligands and for their signaling functions upon binding to CutA.
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Jul 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15991]
Open Access
Abstract: Cu‐containing nitrite reductases that convert NO2‐ to NO are critical enzymes in nitrogen‐based energy metabolism. Among organisms in the order Rhizobiales, we have identified two copies of nirK, one encoding a new class of 4‐domain CuNiR that has both cytochrome and cupredoxin domains fused at the N‐terminus and the other, a classical 2‐domain CuNiR (Br2DNiR). We report the first enzymatic studies of a novel 4‐domain CuNiR from Bradyrhizobium sp. ORS 375 (BrNiR), its genetically engineered 3‐ and 2‐domain variants, and Br2DNiR revealing up to ~500‐fold difference in catalytic efficiency in comparison with classical 2‐domain CuNiRs. Contrary to the expectation that tethering would enhance electron delivery by restricting the conformational search by having a self‐contained donor‐acceptor system, we demonstrate that 4‐domain BrNiR utilises N‐terminal tethering for down‐regulating enzymatic activity instead. Both Br2DNiR, as well as engineered 2‐domain variant of BrNiR (Δ(Cytc‐Cup) BrNiR), have 3 to 5 % NiR activity compared to the well‐characterized 2‐domain CuNiRs from Alcaligenes xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR). Structural comparison of Δ(Cytc‐Cup) BrNiR and Br2DNiR with classical 2‐domain AxNiR and AcNiR reveals structural differences of the proton transfer pathway that could be responsible for the lowering of activity. Our study provides insights into unique structural and functional characteristics of naturally‐occurring 4‐domain CuNiR and its engineered 3‐ and 2‐domain variants.The reverse protein engineering approach utilized here provides has shed light onto the broader question of the evolution of transient encounter complexes and tethered electron transfer complexes.
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Apr 2020
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