Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model ...for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle
. The model comprises four (meta)stable intermediates (S
, S
, S
and S
) and one transient S
state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn
CaO
) cluster in the oxygen-evolving complex
. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone Q
at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok's cycle as high-resolution structures (2.04-2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S
→S
state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S
state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O
release. Thus, our results exclude peroxo-bond formation in the S
state, and the nucleophilic attack of W3 onto W2 is unlikely.
A recent paper in BMC Biology presents a general method for mix-and-inject serial crystallography, to facilitate the visualization of enzyme intermediates via time-resolved serial femtosecond ...crystallography (tr-SFX). They apply their method to resolve in near atomic detail the cleavage and inactivation of the antibiotic ceftriaxone by a β-lactamase enzyme from Mycobacterium tuberculosis. Their work demonstrates the general applicability of time-resolved crystallography, from which dynamic structures, at atomic resolution, can be obtained.See research article: https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-018-0524-5 .
In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the ...roomtemperature structures of PS II in the four (semi)stable S-states, S₁, S₂, S₃, and S₀, showing that a water molecule is inserted during the S₂ → S₃ transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O₂ formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S₂ → S₃ transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 μs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a “water wheel”-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 μs) during the S₂ → S₃ transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the ...room-temperature structures of PS II in the four (semi)stable S-states, S
, S
, S
, and S
, showing that a water molecule is inserted during the S
→ S
transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O
formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S
→ S
transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, Q
and Q
, are observed. At the donor site, tyrosine Y
and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of O
(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S
→ S
transition mirrors the appearance of O
electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
Cyclic guanosine 3′,5′‐monophosphate (cGMP) is an intracellular signalling molecule involved in many sensory and developmental processes. Synthesis of cGMP from GTP is catalysed by guanylate cyclase ...(GC) in a reaction analogous to cAMP formation by adenylate cyclase (AC). Although detailed structural information is available on the catalytic region of nucleotidyl cyclases (NCs) in various states, these atomic models do not provide a sufficient explanation for the substrate selectivity between GC and AC family members. Detailed structural information on the GC domain in its active conformation is largely missing, and no crystal structure of a GTP‐bound wild‐type GC domain has been published to date. Here, we describe the crystal structure of the catalytic domain of rhodopsin–GC (RhGC) from Catenaria anguillulae in complex with GTP at 1.7 Å resolution. Our study reveals the organization of a eukaryotic GC domain in its active conformation. We observe that the binding mode of the substrate GTP is similar to that of AC–ATP interaction, although surprisingly not all of the interactions predicted to be responsible for base recognition are present. The structure provides insights into potential mechanisms of substrate discrimination and activity regulation that may be common to all class III purine NCs.
Database
Structural data are available in Protein Data Bank database under the accession number 6SIR.
Enzymes
EC4.6.1.2.
Synthesis of cGMP, an important intracellular signalling molecule, is catalysed by guanylate cyclases. Although detailed structural information is available, current data do not provide a sufficient explanation for the substrate selectivity between guanylate and adenylate cyclases. We present the crystal structure of the GTP‐bound catalytic domain of rhodopsin–GC from Catenaria anguillulae at 1.7 Å resolution. Our study provides insights into potential mechanisms of substrate discrimination and activity regulation.
Where is crystallography going? Grimes, Jonathan M.; Hall, David R.; Ashton, Alun W. ...
Acta crystallographica. Section D, Structural biology,
February 2018, Volume:
74, Issue:
2
Journal Article
Peer reviewed
Open access
Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and ...cryo‐crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X‐ray free‐electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X‐ray crystallography in rude health, or will imaging methods, especially single‐particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide.
Macromolecular crystallography has provided results that underpin much biological discovery and there is still scope for further development; however, a revolution in electron imaging now means that it can also routinely provide detailed atomic‐level descriptions. This article attempts to tease out where crystallography is going and consider what its place might be in the new landscape.
Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding ...to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+.
Megahertz serial crystallography Wiedorn, Max O; Oberthür, Dominik; Bean, Richard ...
Nature communications,
10/2018, Volume:
9, Issue:
1
Journal Article
Peer reviewed
Open access
The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher ...than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.
Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment ...protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn
CaO
cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S
to S
), in which S
is the dark-stable state and S
is the last semi-stable state before O-O bond formation and O
evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S
), two-flash illuminated (2F; S
-enriched), and ammonia-bound two-flash illuminated (2F-NH
; S
-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S
state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn
CaO
cluster in the S
and S
states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.
Herein, the structure resulting from in situ turnover in a chemically challenging quaternary ammonium oxidative demethylation reaction was captured via crystallographic analysis and analyzed via ...single-crystal spectroscopy. Crystal structures were determined for the Rieske-type monooxygenase, stachydrine demethylase, in the unliganded state (at 1.6 Å resolution) and in the product complex (at 2.2 Å resolution). The ligand complex was obtained from enzyme aerobically cocrystallized with the substrate stachydrine (N,N-dimethylproline). The ligand electron density in the complex was interpreted as proline, generated within the active site at 100 K by the absorption of X-ray photon energy and two consecutive demethylation cycles. The oxidation state of the Rieske iron–sulfur cluster was characterized by UV–visible spectroscopy throughout X-ray data collection in conjunction with resonance Raman spectra collected before and after diffraction data. Shifts in the absorption band wavelength and intensity as a function of absorbed X-ray dose demonstrated that the Rieske center was reduced by solvated electrons generated by X-ray photons; the kinetics of the reduction process differed dramatically for the liganded complex compared to unliganded demethylase, which may correspond to the observed turnover in the crystal.
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