I03-Macromolecular Crystallography
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Maria Giulia
Nizi
,
Mirko M.
Maksimainen
,
Sudarshan
Murthy
,
Serena
Massari
,
Juho
Alaviuhkola
,
Barbara E.
Lippok
,
Sven T.
Sowa
,
Albert
Galera-Prat
,
Renata
Prunskaite-Hyyryläinen
,
Bernhard
Lüscher
,
Patricia
Korn
,
Lari
Lehtio
,
Oriana
Tabarrini
Diamond Proposal Number(s):
[19951]
Abstract: While human poly-ADP-ribose chain generating poly-ARTs, PARP1 and 2 and TNKS1 and 2, have been widely characterized, less is known on the pathophysiological roles of the mono-ADP-ribosylating mono-ARTs, partly due to the lack of selective inhibitors. In this context, we have focused on the development of inhibitors for the mono-ART PARP10, whose overexpression is known to induce cell death. Starting from OUL35 (1) and its 4-(benzyloxy)benzamidic derivative (2) we herein report the design and synthesis of new analogues from which the cyclobutyl derivative 3c rescued cells most efficiently from PARP10 induced apoptosis. Most importantly, we also identified 2,3-dihydrophthalazine-1,4-dione as a new suitable nicotinamide mimicking PARP10 inhibitor scaffold. When it was functionalized with cycloalkyl (8a-c), o-fluorophenyl (8h), and thiophene (8l) rings, IC50 values in the 130–160 nM range were obtained, making them the most potent PARP10 inhibitors reported to date. These compounds also inhibited PARP15 with low micromolar IC50s, but none of the other tested poly- and mono-ARTs, thus emerging as dual mono-ART inhibitors. Compounds 8a, 8h and 8l were also able to enter cells and rescue cells from apoptosis. Our work sheds more light on inhibitor development against mono-ARTs and identifies chemical probes to study the cellular roles of PARP10 and PARP15.
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Jul 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19884]
Open Access
Abstract: Homomers are prevalent in bacterial proteomes, particularly among core metabolic enzymes. Homomerization is often key to function and regulation, and interfaces that facilitate the formation of homomeric enzymes are subject to intense evolutionary change. However, our understanding of the molecular mechanisms that drive evolutionary variation in homomeric complexes is still lacking. How is the diversification of protein interfaces linked to variation in functional regulation and structural integrity of homomeric complexes? To address this question, we studied quaternary structure evolution of bacterial methionine S-adenosyltransferases (MATs)—dihedral homotetramers formed along a large and conserved dimeric interface harboring two active sites, and a small, recently evolved, interdimeric interface. Here, we show that diversity in the physicochemical properties of small interfaces is directly linked to variability in the kinetic stability of MAT quaternary complexes and in modes of their functional regulation. Specifically, hydrophobic interactions within the small interface of Escherichia coli MAT render the functional homotetramer kinetically stable yet impose severe aggregation constraints on complex assembly. These constraints are alleviated by electrostatic interactions that accelerate dimer-dimer assembly. In contrast, Neisseria gonorrhoeae MAT adopts a nonfunctional dimeric state due to the low hydrophobicity of its small interface and the high flexibility of its active site loops, which perturbs small interface integrity. Remarkably, in the presence of methionine and ATP, N. gonorrhoeae MAT undergoes substrate-induced assembly into a functional tetrameric state. We suggest that evolution acts on the interdimeric interfaces of MATs to tailor the regulation of their activity and stability to unique organismal needs.
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Jul 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692]
Open Access
Abstract: Marine cyanobacteria are critical players in global nutrient cycles that crucially depend on trace metals in metalloenzymes, including zinc for CO2 fixation and phosphorus acquisition. How strains proliferating in the vast oligotrophic ocean gyres thrive at ultra-low zinc concentrations is currently unknown. Using Synechococcus sp. WH8102 as a model we show that its zinc-sensor protein Zur differs from all other known bacterial Zur proteins in overall structure and the location of its sensory zinc site. Uniquely, Synechococcus Zur activates metallothionein gene expression, which supports cellular zinc quotas spanning two orders of magnitude. Thus, a single zinc sensor facilitates growth across pico- to micromolar zinc concentrations with the bonus of banking this precious resource. The resultant ability to grow well at both ultra-low and excess zinc, together with overall lower zinc requirements, likely contribute to the broad ecological distribution of Synechococcus across the global oceans.
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Jun 2022
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I03-Macromolecular Crystallography
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Samuel L.
Freeman
,
Vera
Skafar
,
Hanna
Kwon
,
Alistair J.
Fielding
,
Peter C. E.
Moody
,
Alejandra
Martínez
,
Federico
Issoglio
,
Lucas
Inchausti
,
Pablo
Smircich
,
Ari
Zeida
,
Lucía
Piacenza
,
Rafael
Radi
,
Emma L.
Raven
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defence systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance (EPR) spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] Compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c, but is still competent for oxididation of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.
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Jun 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9475, 13467]
Open Access
Abstract: AppA, the Escherichia coli periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D-myo-Ins(2,3,4,5)P4 and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid.
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Jun 2022
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I04-Macromolecular Crystallography
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Open Access
Abstract: Gram-negative pathogens like Burkholderia pseudomallei use trimeric autotransporter adhesins such as BpaC as key molecules in their pathogenicity. Our 1.4 Å crystal structure of the membrane proximal part of the BpaC head domain shows that the domain is exclusively made of left-handed parallel β-roll repeats. This, the largest such structure solved, has two unique features. First, the core, rather than being composed of the canonical hydrophobic Ile and Val, is made up primarily of the hydrophilic Thr and Asn, with two different solvent channels. Second, comparing BpaC to all other left-handed parallel β-roll structures showed that the position of the head domain in the protein correlates with the number and type of charged residues. In BpaC, only negatively charged residues face the solvent – in stark contrast to the primarily positive surface charge of the left-handed parallel β-roll “type” protein, YadA. We propose extending the definitions of these head domains to include the BpaC-like head domain as a separate subtype, based on its unusual sequence, position and charge. We speculate that the function of left-handed parallel β-roll structures may differ depending on their position in the structure.
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Jun 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Abstract: The α-helix is pre-eminent in structural biology1 and widely exploited in protein folding2, design3 and engineering4. Although other helical peptide conformations do exist near to the α-helical region of conformational space—namely, 310-helices and π-helices5—these occur much less frequently in protein structures. Less favourable internal energies and reduced tendencies to pack into higher-order structures mean that 310-helices rarely exceed six residues in length in natural proteins, and that they tend not to form normal supersecondary, tertiary or quaternary interactions. Here we show that despite their absence in nature, synthetic peptide assemblies can be built from 310-helices. We report the rational design, solution-phase characterization and an X-ray crystal structure for water-soluble bundles of 310-helices with consolidated hydrophobic cores. The design uses six-residue repeats informed by analysing 310-helical conformations in known protein structures, and incorporates α-aminoisobutyric acid residues. Design iterations reveal a tipping point between α-helical and 310-helical folding, and identify features required for stabilizing assemblies of 310-helices. This work provides principles and rules to open opportunities for designing into this hitherto unexplored region of protein-structure space.
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Jun 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multitudinous and varied resistance mechanisms. Bacteriophage Exclusion (BREX) systems protect from phage infection by generating methylation patterns at non-palindromic 6 bp sites in host bacterial DNA, to distinguish and block replication of non-self DNA. Type 1 BREX systems are comprised of six conserved core genes. Here, we present the first reported structure of a BREX core protein, BrxA from the phage defence island of Escherichia fergusonii ATCC 35469 plasmid pEFER, solved to 2.09 Å. BrxA is a monomeric protein in solution, with an all α-helical globular fold. Conservation of surface charges and structural homology modelling against known phage defence systems highlighted that BrxA contains two helix-turn-helix motifs, juxtaposed by 180°, positioned to bind opposite sides of a DNA major groove. BrxA was subsequently shown to bind dsDNA. This new understanding of BrxA structure, and first indication of BrxA biological activity, suggests a conserved mode of DNA-recognition has become widespread and implemented by diverse phage defence systems.
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Jun 2022
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I24-Microfocus Macromolecular Crystallography
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Richard J.
Gildea
,
James
Beilsten-Edmands
,
Danny
Axford
,
Sam
Horrell
,
Pierre
Aller
,
James
Sandy
,
Juan
Sanchez-Weatherby
,
C. David
Owen
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Robin L.
Owen
,
Martin A.
Walsh
,
Graeme
Winter
Diamond Proposal Number(s):
[26986, 27088]
Open Access
Abstract: In macromolecular crystallography, radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals; for example, small-wedge data collections from micro-crystals, in situ room-temperature data collections and data collection from membrane proteins in lipidic mesophases. Whilst the indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. The identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help to optimize the scaling and merging of multiple data sets, a new program, xia2.multiplex, has been developed which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multi-crystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After the description of a number of use cases, the benefit of xia2.multiplex is demonstrated within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment-screening experiments on the SARS-CoV-2 main protease.
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Jun 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Bacterial cell division is a complex process requiring the coordination of multiple components to allow the appropriate spatial and temporal control of septum formation and cell scission. Peptidoglycan (PG) is the major structural component of the septum, and our recent studies in the human pathogen Staphylococcus aureus have revealed a complex, multistage PG architecture that develops during septation. Penicillin-binding proteins (PBPs) are essential for the final steps of PG biosynthesis; their transpeptidase activity links the peptide side chains of nascent glycan strands. PBP1 is required for cell division in S. aureus, and here, we demonstrate that it has multiple essential functions associated with its enzymatic activity and as a regulator of division. Loss of PBP1, or just its C-terminal PASTA domains, results in cessation of division at the point of septal plate formation. The PASTA domains can bind PG and thereby potentially coordinate the cell division process. The transpeptidase activity of PBP1 is also essential, but its loss leads to a strikingly different phenotype of thickened and aberrant septa, which is phenocopied by the morphological effects of adding the PBP1-specific β-lactam, meropenem. Together, these results lead to a model for septal PG synthesis where PBP1 enzyme activity is required for the characteristic architecture of the septum and PBP1 protein molecules enable the formation of the septal plate.
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Jun 2022
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