I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[21970]
Open Access
Abstract: Oral and gut microbiomes are important for the maintenance of homeostasis in the human body. Altered or disturbed mutualism between their members results in dysbiosis with local injury and subsequent systemic diseases. The high bacterial density causes intense competition among microbiome residents to acquire nutrients, including iron and heme, the latter of high importance for heme auxotrophic members of the Bacteroidetes phylum. Our main hypothesis is that the heme acquisition mechanism, with the leading role played by a novel HmuY family of hemophore-like proteins, can be used to fulfill nutritional requirements and increase virulence. We characterized HmuY homologs expressed by Bacteroides fragilis and compared their properties with the first representative of this family, the HmuY protein of Porphyromonas gingivalis. In contrast to other Bacteroidetes members, B. fragilis produces three HmuY homologs (Bfr proteins). All bfr transcripts were produced at higher levels in bacteria starved of iron and heme (fold change increase ~60, ~90, and ~70 for bfrA, bfrB, and bfrC, respectively). X-ray protein crystallography showed that B. fragilis Bfr proteins are structurally similar to P. gingivalis HmuY and to other homologs, except for differences in the potential heme-binding pockets. BfrA binds heme, mesoheme, and deuteroheme, but preferentially under reducing conditions, using Met175 and Met146 to coordinate heme iron. BfrB binds iron-free protoporphyrin IX and coproporphyrin III, whereas BfrC does not bind porphyrins. HmuY is capable of heme sequestration from BfrA, which might increase the ability of P. gingivalis to cause dysbiosis also in the gut microbiome.
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Jul 2023
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Hannah R.
Adams
,
Dimitri A.
Svistunenko
,
Michael T.
Wilson
,
Sotaro
Fujii
,
Richard W.
Strange
,
Zoe A.
Hardy
,
Priscilla A.
Vazquez
,
Tyler
Dabritz
,
Gabriel J.
Streblow
,
Colin R.
Andrew
,
Michael A.
Hough
Diamond Proposal Number(s):
[13467, 18565, 25108]
Open Access
Abstract: The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2, is fundamental to enzymology, biotechnology and human health. Cytochromes c´ (cyts c´) are a group of putative NO-binding heme proteins that fall into two families: the well characterised four alpha helix bundle fold (cyts c´-α) and an unrelated family with a largely beta sheet fold (cyts c´-β) resembling that of cytochromes P460. A recent structure of cyt c´-β from Methylococcus capsulatus Bath (McCP-β) revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas binding site. This feature, dubbed the “Phe cap”, is highly conserved within the sequences of other cyts c´-β, but is absent in their close homologues, the hydroxylamine oxidizing cytochromes P460, although some do contain a single Phe residue. Here we report an integrated structural, spectroscopic, and kinetic characterization of McCP-β complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron rich aromatic ring face of Phe 32 towards distally-bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO-sensor, soluble guanylate cyclase (sGC). Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c’−β, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.
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Apr 2023
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I24-Microfocus Macromolecular Crystallography
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Tadeo
Moreno-Chicano
,
Leiah M.
Carey
,
Danny
Axford
,
John H.
Beale
,
R. Bruce
Doak
,
Helen M. E.
Duyvesteyn
,
Ali
Ebrahim
,
Robert W.
Henning
,
Diana C. F.
Monteiro
,
Dean A.
Myles
,
Shigeki
Owada
,
Darren A.
Sherrell
,
Megan L.
Straw
,
Vukica
Šrajer
,
Hiroshi
Sugimoto
,
Kensuke
Tono
,
Takehiko
Tosha
,
Ivo
Tews
,
Martin
Trebbin
,
Richard W.
Strange
,
Kevin L.
Weiss
,
Jonathan A. R.
Worrall
,
Flora
Meilleur
,
Robin L.
Owen
,
Reza A.
Ghiladi
,
Michael A.
Hough
Diamond Proposal Number(s):
[14493]
Open Access
Abstract: Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.
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Sep 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Marcin
Bielecki
,
Svetlana
Antonyuk
,
Richard W.
Strange
,
Klaudia
Siemińska
,
John W.
Smalley
,
Paweł
Mackiewicz
,
Michał
Śmiga
,
Megan
Cowan
,
Michael J.
Capper
,
Paulina
Ślęzak
,
Mariusz
Olczak
,
Teresa
Olczak
Diamond Proposal Number(s):
[15991]
Abstract: As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensable for life and enables the microorganism to survive and multiply at the infection site. This oral pathogenic bacterium uses a newly discovered novel hmu heme uptake system with a leading role played by the HmuY hemophore-like protein, responsible for acquiring heme and increasing virulence of this periodontopathogen. We demonstrated that Prevotella intermedia produces two HmuY homologs, termed PinO and PinA. Both proteins were produced at higher mRNA and protein levels when the bacterium grew under low-iron/heme conditions. PinO and PinA bound heme, but preferentially under reducing conditions, and in a manner different to that of the P. gingivalis HmuY. The analysis of the three-dimensional structures confirmed differences between apo-PinO and apo-HmuY, mainly in the fold forming the heme-binding pocket. Instead of two histidine residues coordinating heme iron in P. gingivalis HmuY, PinO and PinA could use one methionine residue to fulfil this function, with potential support of additional methionine residue/s. The P. intermedia proteins sequestered heme only from the host albumin-heme complex under reducing conditions. Our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme coordination mode. The newer data presented in this manuscript on HmuY homologs produced by P. intermedia sheds more light on the novel mechanism of heme uptake, could be helpful in discovering their biological function, and in developing novel therapeutic approaches.
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Jan 2020
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NONE-No attached Diamond beamline
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Tadeo
Moreno Chicano
,
Ali
Ebrahim
,
Danny
Axford
,
Martin V.
Appleby
,
John H.
Beale
,
Amanda K.
Chaplin
,
Helen M. E.
Duyvesteyn
,
Reza A.
Ghiladi
,
Shigeki
Owada
,
Darren A.
Sherrell
,
Richard
Strange
,
Hiroshi
Sugimoto
,
Kensuke
Tono
,
Jonathan A. R.
Worrall
,
Robin L.
Owen
,
Michael A.
Hough
Open Access
Abstract: High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX.
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Nov 2019
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I24-Microfocus Macromolecular Crystallography
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Ali
Ebrahim
,
Tadeo
Moreno-Chicano
,
Martin V.
Appleby
,
Amanda K.
Chaplin
,
John
Beale
,
Darren A.
Sherrell
,
Helen M. E.
Duyvesteyn
,
Shigeki
Owada
,
Kensuke
Tono
,
Hiroshi
Sugimoto
,
Richard W.
Strange
,
Jonathan
Worrall
,
Danny
Axford
,
Robin L.
Owen
,
Michael A.
Hough
Diamond Proposal Number(s):
[14493]
Open Access
Abstract: An approach is demonstrated to obtain, in a sample- and time-efficient manner, multiple dose-resolved crystal structures from room-temperature protein microcrystals using identical fixed-target supports at both synchrotrons and X-ray free-electron lasers (XFELs). This approach allows direct comparison of dose-resolved serial synchrotron and damage-free XFEL serial femtosecond crystallography structures of radiation-sensitive proteins. Specifically, serial synchrotron structures of a heme peroxidase enzyme reveal that X-ray induced changes occur at far lower doses than those at which diffraction quality is compromised (the Garman limit), consistent with previous studies on the reduction of heme proteins by low X-ray doses. In these structures, a functionally relevant bond length is shown to vary rapidly as a function of absorbed dose, with all room-temperature synchrotron structures exhibiting linear deformation of the active site compared with the XFEL structure. It is demonstrated that extrapolation of dose-dependent synchrotron structures to zero dose can closely approximate the damage-free XFEL structure. This approach is widely applicable to any protein where the crystal structure is altered by the synchrotron X-ray beam and provides a solution to the urgent requirement to determine intact structures of such proteins in a high-throughput and accessible manner.
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Jul 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14493]
Open Access
Abstract: The ability to determine high-quality, artefact-free structures is a challenge in micro-crystallography, and the rapid onset of radiation damage and requirement for a high-brilliance X-ray beam mean that a multi-crystal approach is essential. However, the combination of crystal-to-crystal variation and X-ray-induced changes can make the formation of a final complete data set challenging; this is particularly true in the case of metalloproteins, where X-ray-induced changes occur rapidly and at the active site. An approach is described that allows the resolution, separation and structure determination of crystal polymorphs, and the tracking of radiation damage in microcrystals. Within the microcrystal population of copper nitrite reductase, two polymorphs with different unit-cell sizes were successfully separated to determine two independent structures, and an X-ray-driven change between these polymorphs was followed. This was achieved through the determination of multiple serial structures from microcrystals using a high-throughput high-speed fixed-target approach coupled with robust data processing.
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Nov 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[11740]
Open Access
Abstract: Porphyromonas gingivalis is considered the principal etiologic agent and keystone pathogen of chronic periodontitis. As an auxotrophic bacterium, it must acquire heme to survive and multiply at the infection site. P. gingivalis HmuY is the first member of a novel family of hemophore-like proteins. Bacterial heme-binding proteins usually use histidine-methionine or histidine-tyrosine residues to ligate heme-iron, whereas P. gingivalis HmuY uses two histidine residues. We hypothesized that other 'red complex' members, i.e ., Tannerella forsythia and Treponema denticola might utilize similar heme uptake mechanism to the P. gingivalis HmuY. Comparative and phylogenetic analyses suggested differentiation of HmuY homologs and low conservation of heme-coordinating histidine residues present in HmuY. The homologs were subjected to duplication before divergence of Bacteroidetes lineages, which could facilitate evolution of functional diversification. We found that T. denticola does not code a HmuY homolog. T. forsythia protein, termed Tfo, binds heme, but preferentially in the ferrous form, and sequesters heme from the albumin-heme complex under reducing conditions. In agreement with that, the three-dimensional structure of Tfo differs from that of HmuY in the folding of heme-binding pocket, containing two methionine residues instead of two histidine residues coordinating heme in HmuY. Heme binding to apo-HmuY is accompanied by a movement of the loop carrying the His166 residue, closing the heme-binding pocket. Molecular dynamics simulations demonstrated that this conformational change also occurs in Tfo. In conclusion, our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme-binding properties.
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Sep 2018
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I24-Microfocus Macromolecular Crystallography
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Sam
Horrell
,
Demet
Kekilli
,
Kakali
Sen
,
Robin L.
Owen
,
Florian S. N.
Dworkowski
,
Svetlana V.
Antonyuk
,
Thomas W.
Keal
,
Chin W.
Yong
,
Robert R.
Eady
,
S. Samar
Hasnain
,
Richard W.
Strange
,
Mike
Hough
Diamond Proposal Number(s):
[11175]
Open Access
Abstract: High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate `structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a `top-hat' geometry, which was rapidly transformed to a `side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.
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May 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Nitrite coordination to heme cofactors is a key step in the anaerobic production of the signaling molecule nitric oxide (NO). An ambidentate ligand, nitrite has the potential to coordinate via the N- (nitro) or O- (nitrito) atoms in a manner that can direct its reactivity. Distinguishing nitro vs nitrito coordination, along with the influence of the surrounding protein, is therefore of particular interest. In this study, we probed Fe(III) heme-nitrite coordination in Alcaligenes xylosoxidans cytochrome c′ (AXCP), an NO carrier that excludes anions in its native state but that readily binds nitrite (Kd ∼ 0.5 mM) following a distal Leu16 → Gly mutation to remove distal steric constraints. Room-temperature resonance Raman spectra (407 nm excitation) identify ν(Fe–NO2), δ(ONO), and νs(NO2) nitrite ligand vibrations in solution. Illumination with 351 nm UV light results in photoconversion to {FeNO}6 and {FeNO}7 states, enabling FTIR measurements to distinguish νs(NO2) and νas(NO2) vibrations from differential spectra. Density functional theory calculations highlight the connections between heme environment, nitrite coordination mode, and vibrational properties and confirm that nitrite binds to L16G AXCP exclusively through the N atom. Efforts to obtain the nitrite complex crystal structure were hampered by photochemistry in the X-ray beam. Although low dose crystal structures could be modeled with a mixed nitrite (nitro)/H2O distal population, their photosensitivity and partial occupancy underscores the value of the vibrational approach. Overall, this study sheds light on steric determinants of heme-nitrite binding and provides vibrational benchmarks for future studies of heme protein nitrite reactions.
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Oct 2017
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