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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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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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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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19190]
Abstract: β‐propeller proteins are common in nature, where they are observed to adopt 4‐ to 10‐fold internal rotational pseudosymmetry. This size diversity can be explained by the evolutionary process of gene duplication and fusion. In this study we investigated a distorted β‐propeller protein, an apparent intermediate between two symmetries. From this template, we created a perfectly symmetric 9‐bladed β‐propeller named Cake, using computational design and ancestral sequence reconstruction. The designed repeat sequence was found to be capable of generating both 8‐fold and 9‐fold propellers which are highly stable. Cake variants with 2 to 10 identical copies of the repeat sequence were characterized by X‐ray crystallography and in solution. They were found to be highly stable, and to self‐assemble into 8‐ or 9‐ fold symmetrical propellers. These findings show that the β‐propeller fold allows sufficient structural plasticity to permit a given blade to assemble different forms, a transition from even to odd changes in blade number, and provide a potential explanation for the wide diversity of repeat numbers observed in natural propeller proteins.
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Apr 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Vibrio spp. play a vital role in the recycling of chitin in oceans, but several Vibrio strains are highly infectious to aquatic animals and humans. These bacteria require chitin for growth; thus, potent inhibitors of chitin‐degrading enzymes could serve as candidate drugs against Vibrio infections. This study examined NAG‐thiazoline (NGT)‐mediated inhibition of a recombinantly expressed GH20 β‐N‐acetylglucosaminidase, namely VhGlcNAcase from Vibrio campbellii (formerly V. harveyi) ATCC BAA‐1116. NGT strongly inhibited VhGlcNAcase with an IC50 of 11.9 ± 1.0 μm and Ki 62 ± 3 µm, respectively. NGT was also found to completely inhibit the growth of V. campbellii strain 650 with an minimal inhibitory concentration value of 0.5 µm. ITC data analysis showed direct binding of NGT to VhGlcNAcase with a Kd of 32 ± 1.2 μm. The observed ΔG°binding of −7.56 kcal·mol−1 is the result of a large negative enthalpy change and a small positive entropic compensation, suggesting that NGT binding is enthalpy‐driven. The structural complex shows that NGT fully occupies the substrate‐binding pocket of VhGlcNAcase and makes an exclusive hydrogen bond network, as well as hydrophobic interactions with the conserved residues around the −1 subsite. Our results strongly suggest that NGT could serve as an excellent scaffold for further development of antimicrobial agents against Vibrio infections.
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Mar 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[1225]
Open Access
Abstract: Trypanosomatids possess glycosome organelles that contain much of the glycolytic machinery, including phosphofructokinase (PFK). We present kinetic and structural data for PFK from three human pathogenic trypanosomatids, illustrating intriguing differences that may reflect evolutionary adaptations to differing ecological niches. The activity of Leishmania PFK – to a much larger extent than Trypanosoma PFK – is reliant on AMP for activity regulation, with 1 mm AMP increasing the L. infantum PFK (LiPFK) kcat/K0.5F6P value by 10‐fold, compared to only a 1.3‐ and 1.4‐fold increase for T. cruzi and T. brucei PFK, respectively. We also show that Leishmania PFK melts at a significantly lower (> 15 °C) temperature than Trypanosoma PFKs and that addition of either AMP or ATP results in a marked stabilization of the protein. Sequence comparisons of Trypanosoma spp. and Leishmania spp. show that divergence of the two genera involved amino acid substitutions that occur in the enzyme’s ‘reaching arms’ and ‘embracing arms’ that determine tetramer stability. The dramatic effects of AMP on Leishmania activity compared with the Trypanosoma PFKs may be explained by differences between the T‐to‐R equilibria for the two families, with the low‐melting Leishmania PFK favouring the flexible inactive T‐state in the absence of AMP. Sequence comparisons along with the enzymatic and structural data presented here also suggest there was a loss of AMP‐dependent regulation in Trypanosoma species rather than gain of this characteristic in Leishmania species and that AMP acts as a key regulator in Leishmania governing the balance between glycolysis and gluconeogenesis.
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Jan 2020
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[16289]
Abstract: The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix that includes the Golgi reassembly and stacking protein (GRASP), which was shown to be involved in cisternae stacking and lateral linkage in metazoan. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different binding partners. The conserved N terminus of the GRASP family includes two PSD‐95, DLG, and ZO‐1 (PDZ) domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content. However, PDZ1 alone mediates nearly all interactions between GRASPs and their partners. In this work, NMR, synchrotron radiation CD, and molecular dynamics (MD) were used to examine the structure, flexibility, and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual β3α1β4β5α2β6β1β2 secondary structural arrangement and NMR data indicate that the PDZ1 binding pocket is formed by a stable β2‐strand and a more flexible and unstable α2‐helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using MD simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner‐dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.
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Jan 2020
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I03-Macromolecular Crystallography
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Abstract: Coproporphyrin ferrochelatases (CpfCs, EC 4.99.1.9) insert ferrous iron into coproporphyrin III yielding coproheme. CpfCs are utilized by prokaryotic, mainly monoderm (Gram‐positive) bacteria within the recently detected coproporphyrin‐dependent heme biosynthesis pathway. Here we present a comprehensive study on CpfC from Listeria monocytogenes (LmCpfC) including the first crystal structure of a coproheme‐bound CpfC. Comparison of crystal structures of apo‐LmCpfC and coproheme‐LmCpfC allowed identification of structural rearrangements and of amino acids involved in tetrapyrrole macrocycle and Fe2+ binding. Differential scanning calorimetry of apo‐, coproporphyrin III‐ and coproheme‐LmCpfC underline the pronounced non‐covalent interaction of both coproporphyrin and coproheme with the protein (ΔTm = 11 °C compared to apo‐LmCpfC) which includes the propionates (p2, p4, p6, p7) and the amino acids Arg29, Arg45, Tyr46, Ser53 and Tyr124. Furthermore, the thermodynamics and kinetics of coproporphyrin III and coproheme binding to apo‐LmCpfC is presented as well as the kinetics of insertion of ferrous iron into coproporphyrin III‐LmCpfC that immediately leads to formation of ferric coproheme‐LmCpfC (kcat/KM = 4.7 × 105 M‐1 s‐1). We compare the crystal structure of coproheme‐LmCpfC with available structures of CpfCs with artificial tetrapyrrole macrocycles and discuss our data on substrate binding, iron insertion and substrate release in the context of the coproporphyrin‐dependent heme biosynthesis pathway.
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Dec 2019
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