Overlooked Hydrogen Bond in a Blue Copper Protein Uncovered by Neutron and Sub-Ångström Resolution X-ray Crystallography.

Metalloproteins play fundamental roles in organisms and are utilized as starting points for the directed evolution of artificial enzymes. Knowing the strategies of metalloproteins, by which they exquisitely tune their activities, will not only lead to an understanding of biochemical phenomena but also contribute to various applications. The blue copper protein (BCP) has been a renowned model system to understand the biology, chemistry, and physics of metalloproteins. Pseudoazurin (Paz), a blue copper protein, mediates electron transfer in the bacterial anaerobic respiratory chain. Its redox potential is finely tuned by hydrogen (H) bond networks; however, difficulty in visualizing H atom positions in the protein hinders the detailed understanding of the protein's structure-function relationship. We here used neutron and sub-ångström resolution X-ray crystallography to directly observe H atoms in Paz. The 0.86-Å-resolution X-ray structure shows that the peptide bond between Pro80 and the His81 Cu ligand deviates from the ideal planar structure. The 1.9-Å-resolution neutron structure confirms a long-overlooked H bond formed by the amide of His81 and the S atom of another Cu ligand Cys78. Quantum mechanics/molecular mechanics calculations show that this H bond increases the redox potential of the Cu site and explains the experimental results well. Our study demonstrates the potential of neutron and sub-ångström resolution X-ray crystallography to understand the chemistry of metalloproteins at atomic and quantum levels.

New insights into the oxidation process from neutron and X-ray crystal structures of an O2-sensitive [NiFe]-hydrogenase.

[NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F is an O2-sensitive enzyme that is inactivated in the presence of O2 but the oxidized enzyme can recover its catalytic activity by reacting with H2 under anaerobic conditions. Here, we report the first neutron structure of [NiFe]-hydrogenase in its oxidized state, determined at a resolution of 2.20 Å. This resolution allowed us to reinvestigate the structure of the oxidized active site and to observe the positions of protons in several short hydrogen bonds. X-ray anomalous scattering data revealed that a part of the Ni ion is dissociated from the active site Ni-Fe complex and forms a new square-planar Ni complex, accompanied by rearrangement of the coordinated thiolate ligands. One of the thiolate Sγ atoms is oxidized to a sulfenate anion but remains attached to the Ni ion, which was evaluated by quantum chemical calculations. These results suggest that the square-planar complex can be generated by the attack of reactive oxygen species derived from O2, as distinct from one-electron oxidation leading to a conventional oxidized form of the Ni-Fe complex. Another major finding of this neutron structure analysis is that the Cys17S thiolate Sγ atom coordinating to the proximal Fe-S cluster forms an unusual hydrogen bond with the main-chain amide N atom of Gly19S with a distance of 3.25 Å, where the amide proton appears to be delocalized between the donor and acceptor atoms. This observation provides insight into the contribution of the coordinated thiolate ligands to the redox reaction of the Fe-S cluster.

Current status of neutron crystallography in structural biology.

Hydrogen atoms and hydration water molecules in proteins are essential for many biochemical processes, especially enzyme catalysis. Neutron crystallography enables direct observation of hydrogen atoms, and reveals molecular recognition through hydrogen bonding and catalytic reactions involving proton-coupled electron transfer. The use of neutron crystallography is still limited for proteins, but its popularity is increasing owing to an increase in the number of diffractometers for structural biology at neutron facilities and advances in sample preparation. According to the characteristics of the neutrons, monochromatic or quasi-Laue methods and the time-of-flight method are used in nuclear reactors and pulsed spallation sources, respectively, to collect diffraction data. Growing large crystals is an inevitable problem in neutron crystallography for structural biology, but sample deuteration, especially protein perdeuteration, is effective in reducing background levels, which shortens data collection time and decreases the crystal size required. This review also introduces our recent neutron structure analyses of copper amine oxidase and copper-containing nitrite reductase. The neutron structure of copper amine oxidase gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions. Electron transfer via a hydrogen-bond jump and a hydroxide ion ligation in copper-containing nitrite reductase are clarified, and these observations are consistent with the results from the quantum chemical calculations. This review article is an extended version of the Japanese article, Elucidation of Enzymatic Reaction Mechanism by Neutron Crystallography, published in SEIBUTSU-BUTSURI Vol. 61, p.216-222 (2021).

Re-evaluation of protein neutron crystallography with and without X-ray/neutron joint refinement.

Protein neutron crystallography is a powerful technique to determine the positions of H atoms, providing crucial biochemical information such as the protonation states of catalytic groups and the geometry of hydrogen bonds. Recently, the crystal structure of a bacterial copper amine oxidase was determined by joint refinement using X-ray and neutron diffraction data sets at resolutions of 1.14 and 1.72 Å, respectively [Murakawa et al. (2020 ▸). Proc. Natl Acad. Sci. USA, 117, 10818-10824]. While joint refinement is effective for the determination of the accurate positions of heavy atoms on the basis of the electron density, the structural information on light atoms (hydrogen and deuterium) derived from the neutron diffraction data might be affected by the X-ray data. To unravel the information included in the neutron diffraction data, the structure determination was conducted again using only the neutron diffraction data at 1.72 Šresolution and the results were compared with those obtained in the previous study. Most H and D atoms were identified at essentially the same positions in both the neutron-only and the X-ray/neutron joint refinements. Nevertheless, neutron-only refinement was found to be less effective than joint refinement in providing very accurate heavy-atom coordinates that lead to significant improvement of the neutron scattering length density map, especially for the active-site cofactor. Consequently, it was confirmed that X-ray/neutron joint refinement is crucial for determination of the real chemical structure of the catalytic site of the enzyme.

PD-1 Expression Defines Epidermal CD8+CD103+ T Cells Preferentially Producing IL-17A and Using Skewed TCR Repertoire in Psoriasis.

In psoriasis, CD8+CD103+ memory T cells residing in the epidermis represent an effector population capable of maintaining the condition and driving a recurrence of the disease. Tissue-infiltrating CD8+ T cells expressing PD-1 are regarded as antigen-primed effector cells in others chronic inflammatory diseases. However, the expression and significance of PD-1 on skin-infiltrating CD8+ T cells in human psoriasis is not known. By analyzing skin-infiltrating T cells from human psoriasis, we found that active psoriatic epidermis contained PD-1 expressing CD8+CD103+ T cells that correlated with the disease severity and histopathology. PD-1+CD8+CD103+ T cells possessed a canonical psoriasis-specific resident memory phenotype with IL-23R expression and produced IL-17A, whereas PD-1-CD8+CD103+ T cells preferentially produced IFN-γ. The diversity of skin-infiltrating T cells was dominated by CD4+ T cells, while CD8+ T cells, especially CD8+CD103+T cells, represented an oligoclonal population in active psoriasis. In addition, PD-1+CD8+CD103+T cells used different TCR Vßs from PD-1-CD8+CD103+T cells counterpart. In the early resolved lesion, the composition and functional status of PD-1+CD8+CD103+T cells were markedly altered, while PD-1-CD8+CD103+ T cells population was minimally changed. Collectively, PD-1 expression delineates a putative pathogenic subset of epidermal CD8+CD103+ T cells, which possibly play a role in psoriasis pathogenesis.

Towards cryogenic neutron crystallography on the reduced form of [NiFe]-hydrogenase.

A membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F is a metalloenzyme that contains a binuclear Ni-Fe complex in its active site and mainly catalyzes the oxidation of molecular hydrogen to generate a proton gradient in the bacterium. The active-site Ni-Fe complex of the aerobically purified enzyme shows its inactive oxidized form, which can be reactivated through reduction by hydrogen. Here, in order to understand how the oxidized form is reactivated by hydrogen and further to directly evaluate the bridging of a hydride ligand in the reduced form of the Ni-Fe complex, a neutron structure determination was undertaken on single crystals grown in a hydrogen atmosphere. Cryogenic crystallography is being introduced into the neutron diffraction research field as it enables the trapping of short-lived intermediates and the collection of diffraction data to higher resolution. To optimize the cooling of large crystals under anaerobic conditions, the effects on crystal quality were evaluated by X-rays using two typical methods, the use of a cold nitrogen-gas stream and plunge-cooling into liquid nitrogen, and the former was found to be more effective in cooling the crystals uniformly than the latter. Neutron diffraction data for the reactivated enzyme were collected at the Japan Photon Accelerator Research Complex under cryogenic conditions, where the crystal diffracted to a resolution of 2.0 Å. A neutron diffraction experiment on the reduced form was carried out at Oak Ridge National Laboratory under cryogenic conditions and showed diffraction peaks to a resolution of 2.4 Å.

Protective role of Galectin-7 for skin barrier impairment in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) patients have a barrier disorder in association with Th2 dominant skin inflammation. Galectin-7 (Gal-7), a soluble unglycosylated lectin, is highly expressed in the stratum corneum of AD patients. However, the biological significance of increased Gal-7 expression in AD skin lesions remains unclear. OBJECTIVE: We aimed to investigate the production mechanism and functional role of Gal-7 in AD patients and IL-4/IL-13-stimulated epidermal keratinocytes. METHODS: We assessed the Gal-7 expression levels in skin lesions and sera from AD patients. Gal-7 levels were also measured in monolayered normal human epidermal keratinocytes (NHEKs) and 3-dimensional (3D)-reconstructed epidermis in the presence or absence of IL-4/IL-13 with or without Stat3, Stat6 or Gal-7 gene silencing. RESULTS: Gal-7 was highly expressed in the stratum corneum or intercellular space of AD lesional epidermis as assessed by the stratum corneum proteome analysis and immunohistochemistry. A positive correlation was noted between serum Gal-7 level and transepidermal water loss in patients with AD. These clinical findings were corroborated by our in vitro data, which showed that IL-4/IL-13 facilitated the extracellular release of endogenous Gal-7 in both monolayered NHEKs and 3D-reconstructed epidermis. This machinery was caused by IL-4/IL-13-induced cell damage and inhibited by knockdown of Stat6 but not Stat3 in NHEKs. Moreover, we performed Gal-7 knockdown experiment on 3D-reconstructed epidermis and the result suggested that endogenous Gal-7 serves as a protector from IL-4/IL-13-induced disruption of cell-to-cell adhesion and/or cell-to-extracellular matrix adhesion. CONCLUSION AND CLINICAL RELEVANCE: Our study unveils the characteristic of Gal-7 and its possible role as an alarmin that reflects the IL-4/IL-13-induced skin barrier impairment in AD.

Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing.

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.

Pathophysiology of Skin Resident Memory T Cells.

Tissue resident memory T (TRM) cells reside in peripheral, non-lymphoid tissues such as the skin, where they act as alarm-sensor cells or cytotoxic cells. Physiologically, skin TRM cells persist for a long term and can be reactivated upon reinfection with the same antigen, thus serving as peripheral sentinels in the immune surveillance network. CD8+CD69+CD103+ TRM cells are the well-characterized subtype that develops in the epidermis. The local mediators such as interleukin (IL)-15 and transforming growth factor (TGF)-ß are required for the formation of long-lived TRM cell population in skin. Skin TRM cells engage virus-infected cells, proliferate in situ in response to local antigens and do not migrate out of the epidermis. Secondary TRM cell populations are derived from pre-existing TRM cells and newly recruited TRM precursors from the circulation. In addition to microbial pathogens, topical application of chemical allergen to skin causes delayed-type hypersensitivity and amplifies the number of antigen-specific CD8+ TRM cells at challenged site. Skin TRM cells are also involved in the pathological conditions, including vitiligo, psoriasis, fixed drug eruption and cutaneous T-cell lymphoma (CTCL). The functions of these TRM cells seem to be different, depending on each pathology. Psoriasis plaques are seen in a recurrent manner especially at the originally affected sites. Upon stimulation of the skin of psoriasis patients, the CD8+CD103+CD49a- TRM cells in the epidermis seem to be reactivated and initiate IL-17A production. Meanwhile, autoreactive CD8+CD103+CD49a+ TRM cells secreting interferon-γ are present in lesional vitiligo skin. Fixed drug eruption is another disease where skin TRM cells evoke its characteristic clinical appearance upon administration of a causative drug. Intraepidermal CD8+ TRM cells with an effector-memory phenotype resident in the skin lesions of fixed drug eruption play a major contributing role in the development of localized tissue damage. CTCL develops primarily in the skin by a clonal expansion of a transformed TRM cells. CD8+ CTCL with the pagetoid epidermotropic histology is considered to originate from epidermal CD8+ TRM cells. This review will discuss the current understanding of skin TRM biology and their contribution to skin homeostasis and diseases.

Significance of IL-17A-producing CD8+CD103+ skin resident memory T cells in psoriasis lesion and their possible relationship to clinical course.

BACKGROUND: A number of studies have shown the relationship between the pathogenesis of psoriasis and skin resident memory T (TRM) cells. OBJECTIVE: To investigate the cytokine profile of TRM cells from skin lesions of psoriasis and the relationship of skin TRM cells to the future clinical course of psoriasis. METHODS: We used stocked samples of T cells that were ex vivo expanded from skin biopsies of 10 patients with psoriasis vulgaris. A half of 4-mm punch biopsy specimens was subjected to expansion of skin-infiltrating T cells using IL-2 and anti-CD3/CD28 antibody-coated microbeads. More than 106 T cells per specimen were stocked at -80°C. Defrosted cells were subjected to flow cytometric analysis. Another half of skin biopsies were subjected to immmunofluorescence staining for CD103 and other markers. RESULTS: The biopsied skin revealed CD8+CD103+ TRM cells were present in the epidermis of psoriasis and associated with acanthosis. Sorted CD103+ T cells were mostly CD8+ memory T cells expressing CD69 with a skin-homing potential. A part of CD8+CD103+ T cells produced interferon-γ, IL-17A or IL-22. Notably, CD8+CD103+ TRM cells more frequently produced IL-17A than did CD8+CD103- T cells. We retrospectively divided the 10 cases into the non-advanced therapy group, and the advanced therapy group in which systemic biologics or others were initiated within one year. The frequency of CD8+CD103+IL-17A+ TRM cells tended to be higher in the advanced therapy group. CONCLUSION: These results suggest that IL-17A-producing CD8+CD103+ TRM cells are associated with a progressive clinical course of psoriasis.

Neutron crystallography of photoactive yellow protein reveals unusual protonation state of Arg52 in the crystal.

Because of its high pKa, arginine (Arg) is believed to be protonated even in the hydrophobic environment of the protein interior. However, our neutron crystallographic structure of photoactive yellow protein, a light sensor, demonstrated that Arg52 adopts an electrically neutral form. We also showed that the hydrogen bond between the chromophore and Glu46 is a so-called low barrier hydrogen bond (LBHB). Because both the neutral Arg and LBHB are unusual in proteins, these observations remain controversial. To validate our findings, we carried out neutron crystallographic analysis of the E46Q mutant of PYP. The resultant structure revealed that the proportion of the cationic form is higher in E46Q than in WT, although the cationic and neutral forms of Arg52 coexist in E46Q. These observations were confirmed by the occupancy of the deuterium atom bound to the N η1 atom combined with an alternative conformation of the N(η2)D2 group comprising sp2 hybridisation. Based on these results, we propose that the formation of the LBHB decreases the proton affinity of Arg52, stabilizing the neutral form in the crystal.