I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Copper nitrite reductases (CuNiRs) exhibit a strong pH dependence of their catalytic activity. Structural movies can be obtained by serially recording multiple structures (frames) from the same spot of a crystal using the MSOX serial crystallography approach. This method has been combined with on-line single crystal optical spectroscopy to capture the pH-dependent structural changes that accompany during turnover of CuNiRs from two Rhizobia species. The structural movies, initiated by the redox activation of a type-1 copper site (T1Cu) via X-ray generated photoelectrons, have been obtained for the substrate-free and substrate-bound states at low (high enzymatic activity) and high (low enzymatic activity) pH. At low pH, formation of the product nitric oxide (NO) is complete at the catalytic type-2 copper site (T2Cu) after a dose of 3 MGy (frame 5) with full bleaching of the T1Cu ligand-to-metal charge transfer (LMCT) 455 nm band (S(σ)Cys → T1Cu2+) which in itself indicates the electronic route of proton-coupled electron transfer (PCET) from T1Cu to T2Cu. In contrast at high pH, the changes in optical spectra are relatively small and the formation of NO is only observed in later frames (frame 15 in Br2DNiR, 10 MGy), consistent with the loss of PCET required for catalysis. This is accompanied by decarboxylation of the catalytic AspCAT residue, with CO2 trapped in the catalytic pocket.
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Jul 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[30305]
Open Access
Abstract: Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.
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Jul 2024
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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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I04-Macromolecular Crystallography
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Ohm
Prakash
,
Nitika
Gupta
,
Amy
Milburn
,
Liam
Mccormick
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Vishvangi
Deugi
,
Pauline
Fisch
,
Jacob
Wyles
,
N. Lowri
Thomas
,
Svetlana
Antonyuk
,
Caroline
Dart
,
Nordine
Helassa
Diamond Proposal Number(s):
[21970]
Open Access
Abstract: Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca2+-sensing protein calmodulin (CaM) are associated with LQTS, but the molecular mechanism by which these mutations lead to irregular heartbeats is not fully understood. Here, we use a multidisciplinary approach including protein biophysics, structural biology, confocal imaging and patch-clamp electrophysiology to determine the effect of the disease-associated CaM mutation E140G on CaM structure and function. We present novel data showing that mutant-regulated CaMKIIδ kinase activity is impaired with a significant reduction in enzyme autophosphorylation rate. We report the first high-resolution crystal structure of a LQTS-associated CaM variant in complex with the CaMKIIδ peptide, which shows significant structural differences, compared to the wild-type complex. Furthermore, we demonstrate that the E140G mutation significantly disrupted Cav1.2 Ca2+/CaM-dependent inactivation, while cardiac ryanodine receptor (RyR2) activity remained unaffected. In addition, we show that the LQTS-associated mutation alters CaM’s Ca2+ binding characteristics, secondary structure content and interaction with key partners involved in excitation-contraction coupling (CaMKIIδ, Cav1.2, RyR2). In conclusion, LQTS-associated CaM mutation E140G severely impacts the structure-function relationship of CaM and its regulation of CaMKIIδ and Cav1.2. This provides a crucial insight into the molecular factors contributing to CaM-mediated arrhythmias with a central role for CaMKIIδ.
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Dec 2022
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I24-Microfocus Macromolecular Crystallography
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Samuel L.
Rose
,
Seiki
Baba
,
Hideo
Okumura
,
Svetlana V.
Antonyuk
,
Daisuke
Sasaki
,
Tobias M.
Hedison
,
Muralidharan
Shanmugam
,
Derren J.
Heyes
,
Nigel S.
Scrutton
,
Takashi
Kumasaka
,
Takehiko
Tosha
,
Robert R.
Eady
,
Masaki
Yamamoto
,
S. Samar
Hasnain
Open Access
Abstract: Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.
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Jul 2022
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Abstract: Cu-containing nitrite reductases (CuNiRs) catalyze the reduction of nitrite to nitric oxide and water. CuNiRs are part of the denitrification and nitrification pathways that are not only important from bioenergetics perspective but are also the major contributors to terrestrial and oceanic nitrogen cycling. These pathways make a significantly increasing contribution to global warming by release of N2O, an ozone-depleting and greenhouse gas some 300-fold more potent than CO2. Advanced crystallographic techniques based on synchrotrons and X-ray free electron lasers have been applied to this important class of enzymes. Here we provide a detailed review of recent results obtained from a global effort.
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Mar 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15991]
Open Access
Abstract: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited condition that can cause fatal cardiac arrhythmia. Human mutations in the Ca2+ sensor calmodulin (CaM) have been associated with CPVT susceptibility, suggesting that CaM dysfunction is a key driver of the disease. However, the detailed molecular mechanism remains unclear. Focusing on the interaction with the cardiac ryanodine receptor (RyR2), we determined the effect of CPVT-associated variants N53I and A102V on the structural characteristics of CaM and on Ca2+ fluxes in live cells. We provide novel data showing that binding of both Ca2+/CaM-N53I and Ca2+/CaM-A102V to RyR23583-3603 is decreased. Ca2+/CaM:RyR23583-3603 high-resolution crystal structures highlight subtle conformational changes for the N53I variant, with A102V being similar to wild-type. We show that co-expression of CaM-N53I or CaM-A102V with RyR2 in HEK293 cells significantly increased the duration of Ca2+ events, CaM-A102V exhibited a lower frequency of Ca2+ oscillations. In addition, we show that CaMKIIδ phosphorylation activity is increased for A102V, compared to CaM-WT. This paper provides novel insight into the molecular mechanisms of CPVT-associated CaM variants and will facilitate development of strategies for future therapies.
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Dec 2021
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I03-Macromolecular Crystallography
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Samuel L.
Rose
,
Svetlana V.
Antonyuk
,
Daisuke
Sasaki
,
Keitaro
Yamashita
,
Kunio
Hirata
,
Go
Ueno
,
Hideo
Ago
,
Robert R.
Eady
,
Takehiko
Tosha
,
Masaki
Yamamoto
,
S. Samar
Hasnain
Diamond Proposal Number(s):
[15991]
Open Access
Abstract: Copper-containing nitrite reductases (CuNiRs), encoded by nirK gene, are found in all kingdoms of life with only 5% of CuNiR denitrifiers having two or more copies of nirK. Recently, we have identified two copies of nirK genes in several α-proteobacteria of the order Rhizobiales including Bradyrhizobium sp. ORS 375, encoding a four-domain heme-CuNiR and the usual two-domain CuNiR (Br2DNiR). Compared with two of the best-studied two-domain CuNiRs represented by the blue (AxNiR) and green (AcNiR) subclasses, Br2DNiR, a blue CuNiR, shows a substantially lower catalytic efficiency despite a sequence identity of ~70%. Advanced synchrotron radiation and x-ray free-electron laser are used to obtain the most accurate (atomic resolution with unrestrained SHELX refinement) and damage-free (free from radiation-induced chemistry) structures, in as-isolated, substrate-bound, and product-bound states. This combination has shed light on the protonation states of essential catalytic residues, additional reaction intermediates, and how catalytic efficiency is modulated.
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Jan 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21970]
Open Access
Abstract: Methionine adenosyltransferase (MAT) deficiency, characterized by isolated persistent hypermethioninemia (IPH), is caused by mutations in the MAT1A gene encoding MATαl, one of the major hepatic enzymes. Most of the associated hypermethioninemic conditions are inherited as autosomal recessive traits; however, dominant inheritance of hypermethioninemia is caused by an Arg264His (R264H) mutation. This mutation has been confirmed in a screening programme of newborns as the most common mutation in babies with IPH. Arg264 makes an inter-subunit salt bridge located at the dimer interface where the active site assembles. Here, it is demonstrated that the R264H mutation results in greatly reduced MAT activity, while retaining its ability to dimerize, indicating that the lower activity arises from alteration at the active site. The first crystallographic structure of the apo form of the wild-type MATαl enzyme is provided, which shows a tetrameric assembly in which two compact dimers combine to form a catalytic tetramer. In contrast, the crystal structure of the MATαl R264H mutant reveals a weaker dimeric assembly, suggesting that the mutation lowers the affinity for dimer–dimer interaction. The formation of a hetero-oligomer with the regulatory MATβV1 subunit or incubation with a quinolone-based compound (SCR0911) results in the near-full recovery of the enzymatic activity of the pathogenic mutation R264H, opening a clear avenue for a therapeutic solution based on chemical interventions that help to correct the defect of the enzyme in its ability to metabolize methionine.
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Jun 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15991]
Open Access
Abstract: Copper-containing nitrite reductases (CuNiRs) are found in all three kingdoms of life and play a major role in the denitrification branch of the global nitrogen cycle where nitrate is used in place of dioxygen as an electron acceptor in respiratory energy metabolism. Several C- and N-terminal redox domain tethered CuNiRs have been identified and structurally characterized during the last decade. Our understanding of the role of tethered domains in these new classes of three-domain CuNiRs, where an extra cytochrome or cupredoxin domain is tethered to the catalytic two-domain CuNiRs, has remained limited. This is further compounded by a complete lack of substrate-bound structures for these tethered CuNiRs. There is still no substrate-bound structure for any of the as-isolated wild-type tethered enzymes. Here, structures of nitrite and product-bound states from a nitrite-soaked crystal of the N-terminal cupredoxin-tethered enzyme from the Hyphomicrobium denitrificans strain 1NES1 (Hd1NES1NiR) are provided. These, together with the as-isolated structure of the same species, provide clear evidence for the role of the N-terminal peptide bearing the conserved His27 in water-mediated anchoring of the substrate at the catalytic T2Cu site. Our data indicate a more complex role of tethering.
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May 2020
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