Moreover, the “bonding defects functionalization” approach might open up new avenues for the fabrication of additional COF-based platforms for biomedical treatment.Covalent organic frameworks (COFs), an emerging class of organic porous materials, have attracted intense attention due to their versatile applications. However, the deliberate fabrication of COF-based nanomaterials for nanomedical application remains challenging due to difficulty in their size- and structure-controlled synthesis and poor aqueous dispersibility. Herein, we report two boron-dipyrromethene (BODIPY)-decorated nanoscale COFs (NCOFs), which were prepared by the Schiff-base condensation of the free end –CHO (bonding defects in COFs) on the established imine-based NCOFs with the amino-substituted organic photosensitizer BODIPY via “bonding defects functionalization” approach. Thus BODIPY has been successfully nanocrystallized via the NCOF platform, and can be used for photodynamic therapy (PDT) to treat tumors. These NCOF-based PDT agents featured nanometer size (∼110 nm), low dark toxicity, and high phototoxicity as evidenced by   •Covalent organic frameworks (COFs) are first used in tumor photodynamic therapy•BODIPY-decorated COFs are synthesized via bonding defects functionalization•in vitro and in vivoBODIPY-decorated COFs have excellent anti-tumor efficacy •COFs show great promise as nanoplatforms for biomedical applications Supramolecular Chemistry; Scaffolds in Supramolecular Chemistry; Cancer; Biomedical Materials Compared with chemotherapy and radiotherapy, PDT generates less collateral damage to normal tissues, because 1O2 is produced only in the illuminated area where the PS accumulates. In this context, some organic dyes, such as boron-dipyrromethene (BODIPY)- and porphyrin-based species   covalent decoration of the molecular PSs on the established COF framework by the existing PSM approach might also be limited by the stability and activity of the pre-embedded active precursors under the harsh COF synthetic conditions, and moreover, the incoming bulky PS molecules probably lead to crystallinity decrease, structural transformation, and even structure collapse of the pristine COFs.As a promising alternative to MOFs, s (COFs)  should bs (COFs) . Some exs (COFs) , drug des (COFs) , antibacs (COFs) , enzyme s (COFs) , biochems (COFs) , and so neration , photothneration , and aponeration , have alBased on recent reports, NCOF synthesis can be realized by the polymer-assisted solvothermal method  or with 1) (BODIPY-2I (2) and BODIPY-2H (4), by the BDF approach under the given conditions (3) and LZU-1-BODIPY-2H (5) are highly crystalline and isostructural to pristine 1, and they both feature low cytotoxicity, good biocompatibility, high cancer cell uptake, and highly efficient 1O2 generation. Therefore, they can be used as high-performing PDT agents for cancer treatment under the given in vitro and in vivo conditions.In this contribution, we report, the first of its kind, two BODIPY-decorated NCOFs, which were generated from the NCOF LZU-1 (1)  and two nditions A. Marked1) was prepared by the combination of benzene-1,3,5-tricarbaldehyde and tert-butyl (4-aminophenyl)carbamate (NBPDA) under solvothermal conditions   and BODIPY-2H (4), afforded nanoscale LZU-1-BODIPY-2I (3) and LZU-1-BODIPY-2H (5) under solvothermal conditions  (1), Synthesis of BODIPY-2I (2), Synthesis of LZU-1-BODIPY-2I (3), Synthesis of BODIPY-2H (4), and Synthesis of LZU-1-BODIPY (5) in 3 and 5 were significantly different from those of their precursors after the reaction  measurement . Owing to their structural similarity, we took 3 as an example to discuss its structural characterization in detail herein, and the corresponding detailed characterization data for 5 are provided in With the aid of nonionic surfactant polyvinyl pyrrolidone, the NCOF of LZU-1  . Furtherreaction B. The st1 and 3 are isostructural and possess good crystallinity, as revealed by their experimental powder X-ray diffraction (PXRD). As indicated in θ = 4.7° was attributed to the (100) crystal facet, and other diffraction peaks at 2θ = 8.2, 9.5, and 12.4° were assigned to the (110), (200), and (210) facets, respectively. The broad peak at 2θ = 25.7° could be indexed to the π–π stacked planes (001) of 1  of 1 . This su1 and 3 was assessed by Fourier transform infrared spectroscopy. As shown in 1 appeared at 1,621 cm−1, clearly indicating the formation of the imine linkage . As shown in 3 displayed the characteristic absorption bands of both 1 and 2. A broadband absorption of 1 and 3 throughout the whole visible light region was observed, indicating their stacked layer structures with enhanced light-harvesting capability in a wide range of visible light region due to the delocalized π electrons in the COFs. Compared with 2, the broadened characteristic absorption peak of BODIPY at 530 nm in 3 was also observed but without band-shift, indicating that the involved BODIPY species in 3 were well dispersed, as we know that aggregated BODIPY would cause the adsorption band to be significantly red-shifted  absorption spectra of -shifted . More im2 adsorption properties of 1 and 3 were measured at 77 K. As shown in 1 and 3 were 822 m3/g and 805 m3/g, respectively. The slightly decreased surface area in 3 should be caused by the introduced BODIPY species because the involved BODIPY species certainly enhanced the material weight, thus resulting in a corresponding decrease of dV/dw. Density functional theory fitting of the adsorption branches  and BODIPY-2H⊂LZU-1 (5′) were generated. As determined by the standard curve method, the loading amounts of BODIPY in 3′ and 5′ were up to 0.2149 ± 0.0875 mmol/g and 0.5130 ± 0.1763 mmol/g, respectively, which are significantly higher than those of 3 and 5. It is different from the case of 3 and 5; the BODIPY loading amount herein is size dependent, and smaller-sized 4 (9 Å) was more uploaded than its diiodio-substituted analog of 2 (11 Å), which is a typical phenomenon in the selective host-guest adsorption. In addition, the release kinetics of 3, 3′, 5, and 5′ were investigated in boiling ethanol. As expected, the encapsulated BODIPY in 3′ and 5′ was readily extracted with EtOH with a rapid BODIPY release. In contrast, no BODIPY leaching occurred in 3 and 5 under the same conditions because they were firmly anchored by the covalent imine bond. By BDF in this way, the nanocrystallization of BODIPY via COF platform has been successfully achieved, which laid a solid foundation for their applications in biomedical treatment.To further verify the covalent bonding of BODIPY species of 1, 3, and 5 was investigated by scanning electron microscopy and transmission electron microscope. As indicated in 1, 3, and 5 were uniformly distributed and their diameter was ca. 110 nm, which is further supported by the dynamic light scattering (DLS) measurement in PBS , which might be caused by the protonation of N atoms in COFs. NPs with electropositive zeta potential should be of great benefit to the tumor cell uptake owing to their high affinity for the negatively charged tumor cell membranes  illumination for 30 min, demonstrating that 3 and 5 were not affected by external light sources, even with the high-intensity lasers  irradiation was measured by using 1,3-diphenylisobenzofuran (DPBF) (1O2 probe (see Singlet Oxygen Generation in PBS in 3 and 5 under green LED irradiation (40 mW/cm2) remarkably decreased within 1 min, implying 1O2 generation. In contrast, negligible changes were observed in control experiments  under the same conditions, which indicated that 3 and 5 herein were highly efficient COF-based PSs. Because intersystem crossing was significantly promoted by heavy-atom effect   as a 1O2m effect , 3 exhib3 and 5 exhibited excellent ability to induce 1O2 generation in PBS. To evaluate their intracellular 1O2 production ability, Singlet Oxygen Sensor Green (SOSG), which is a specific green fluorescent probe for 1O2 in cells  for 4 min. This meant that 3 containing heavy iodine atoms was more efficient in intracellular 1O2 production.As shown above, in cells , was use2, 3, 4, and 5 were evaluated before intracellular PDT (see In Vitro PDT Experiment in H-tetrazolium bromide (MTT) assay . As shown in 2 with heavy iodine atoms exhibited very weak phototoxicity to HeLa and MCF-7 cells. The cell viability of HeLa and MCF-7 was up to 75.7% ± 5.2% and 79.6% ± 9.8% even with 4.0 μM of 2 under the light irradiation. Meanwhile, the free BODIPY molecule of 4 without iodine atoms showed even lower phototoxicity under the same conditions. The corresponding cell viability of HeLa and MCF-7 was more than 90%. This poor phototoxicity of 2 and 4 was clearly attributed to their poor water solubility and cell membrane permeability. In contrast to 2 and 4, 3 and 5 exhibited excellent phototoxicity. The HeLa cell viability was sharply down to 32.4% ± 3.5% even with concentration of 3 as low as 0.2 μM (BODIPY equiv.). For MCF-7 cells, a low cell viability of 14.3% ± 6.7% was observed with concentration of 3 at 0.5 μM (BODIPY equiv.). Compared with 3, 5 without heavy atoms exhibited a lower phototoxicity. For example, the cell viability of HeLa and MCF-7 was 32.8% ± 7.7% and 25.6% ± 6.9% with 1.0 μM 5 (BODIPY equiv.). Thus the nanocrystallization of 2 and 4 via COF 1 significantly enhanced their cell membrane permeability and intracellular phototoxicity, and consequently, made their practical PDT application available.The dark toxicities of T) assay  showed t3 and 5 was further confirmed by calcein-AM and propidium iodide (PI) double staining (3 or 5 (2.0 μM BODIPY equiv.) for 30 min, the proportion of the dead HeLa and MCF-7 cells (red ones) significantly increased with the prolongation of irradiation time. This result again demonstrated that 3 and 5 possessed excellent PDT efficacy, but with ignorable dark toxicity.The PDT efficacity of staining   was a hallmark event related to cell death by the mitochondrion-associated pathway  in ΔΨ , only red fluorescence of JC-1 was observed without light irradiation owing to the formation of JC-1 J-aggregate, indicating that the ΔΨ was normal and 3 or 5 was not dark toxic. When irradiated with green LED, the JC-1 fluorescence changed from red to green due to the JC-1 monomer formation, demonstrating that a significant ΔΨ decrease occurred and the cell death caused by 3 and 5 was through the mitochondrion-associated pathway. It was worth noting that 3-induced fluorescence intensity was significantly stronger than that of 5, further indicating the heavy-atom effect on photosensitization.Mitochondrial membrane potential  in ΔΨ . As show3 and 5 did not damage the lysosomes in dark. However, the intracellular red fluorescence was weakened or even disappeared under light irradiation, which demonstrated that AO was released from the lysosome into the cytoplasm (pH ∼7.2), and moreover, deprotonated in the weak alkaline environment to emit diffuse green fluorescence  . For thirescence . This ob3 and 5 were excellent COF-based nano-PSs. This encouraged us to continue to examine their in vivo anti-tumor ability , some slight signs of tumor inhibition effect were observed at the early treatment stages but then quickly recurred, and eventually there was no significant difference from the control group. The results of the 2 + laser group ruled out the individual role of the small molecular BODIPY and green laser irradiation in tumor inhibition. As expected, 3- and 5-induced PDT effectively inhibited tumor growth under green laser irradiation (1.0 W/cm2), and no recrudescent signs were observed during the whole experimental period. Compared with the 5 + laser group, the results from 3 + laser group strongly suggested that the heavy-atom effect can effectively enhance the in vivo PDT efficacy, which is entirely consistent with their in vitro PDT examination.All the aforementioned results proved that 3 and 5 had negligible adverse impact and favorable biocompatibility on organisms and were indeed suitable for in vivo anti-tumor application.Besides, COFs have picked up intense attention of researchers worldwide due to their promising applications in heterogeneous catalysis , energy 1 with –NH2 moiety on 2 or 4, the organic PSs of BODIPY were covalently attached to the COF NPs via Schiff-base condensation under the given conditions to generate the NCOF-based PSs. As a consequence, the organic BODIPY components have been successfully nanocrystallized without altering the pristine COF structure and the inherent property of BODIPY; moreover, the possible BODIPY leaching was also effectively avoided due to the covalent decoration. Indeed, both 3 and 5 featured a clear chemical composition and excellent chemical, aqueous, and photostability. In addition, their nanometer size (ca. 110 nm), low dark toxicity, and extremely high phototoxicity made them become attractive NCOF-based PSs for PDT, which was well evidenced by the in vitro and in vivo experiments (On the other hand, COFs could be considered as a type of porous crystalline organic “reactive end-group polymers.” In principle, these intrinsic bonding defects in COF matrix can be further modified and tailored to the desired application via BDF approach. As shown in eriments B.Figure in vitro experiments, we took the HeLa and MCF-7 cancer cell lines to examine the PDT of 3 and 5 in detail. Intracellular 1O2 imaging, cytostatic, and calcein-AM/PI double staining tests all supported that 3 and 5 are excellent nanomedical agents for PDT, especially 3 with heavy atoms of iodine. Furthermore, the spread of cancer cells was effectively inhibited by 3- and 5-involved PDT, as revealed by in vitro scratch assays. Notably, the in vitro PDT performance exhibited by 3 was better than those of reported BODIPY-based PDT systems (see 3, was further confirmed by the in vivo experiments, and they are able to effectively inhibit the MCF-7 xenograft growth without detectable systemic toxicity. More detailed mechanism study (3 and 5 were taken up by cancer cells via energy-dependent endocytosis, and that they were mainly localized to lysosomes and mitochondria, and consequently led to cell death through mitochondrion- and lysosome-associated pathways.In sm study C showed We can reasonably assume that the BDF approach herein is flexible and versatile, and that it can be used to construct new types of NCOF-based nanomedicines by covalently grafting various functional species such as bioactive, chemotherapeutic, and targeting agents, and so on via bonding defects decoration. In doing so, the functionality of the COF-based materials could be significantly enriched, and their application scope would be effectively expanded. To the best of our knowledge, this is the first example of NCOF-based materials for PDT. In the past two decades, nanotherapeutics, as an emerging medical treatment approach, has provided a promising way for clinical cancer treatment . A wide 2 of LZU-1 NP was performed. If so, other types of functional molecules such as antineoplastic drugs might be further grafted on the NCOF by BDF. In doing so, more advanced and effective NCOF-based medicines would be achieved.This study focused on the free end –CHO on LZU-1 by BDF, and no chemical modification on the free –NHAll methods can be found in the accompanying


Bacillus cereus (bcFlgL) and gram-negative Xanthomonas campestris (xcFlgL). bcFlgL contains one domain (D1), whereas xcFlgL adopts a two-domain structure that consists of the D1 and D2 domains. The constant D1 domain of FlgL adopts a rod structure that is generated by four longitudinal segments. This four-segment structure is recapitulated in filament and junction proteins but not in hook and rod proteins, allowing us to propose a junction-filament assembly mechanism based on a quasi-homotypic interaction. The D2 domain of xcFlgL resembles that of another junction protein, FlgK, suggesting the structural and functional relatedness of FlgL and FlgK.Bacteria move toward attractants and away from repellants by rotating their flagellum. The bacterial flagellum assembles through the ordered organization of more than 30 different proteins. Among the diverse flagellar proteins, FlgL forms the junction between the hook and the filament in the flagellum together with FlgK and provides a structural base where flagellin, a filament-forming protein, is inserted for the initiation of filament elongation. However, the functional and structural information available for FlgL is highly limited. To provide structural insights into the cross-linkage between the FlgL junction and the flagellin filament, we determined the crystal structures of FlgL from gram-positive  Moreover, in the host, the flagellum contributes to the tissue colonization and invasion of pathogenic bacteria and functions as a virulence factor. However, the host senses flagellar proteins as pathogen-associated molecular patterns using innate immune receptors for the activation of the immune response to pathogenic bacteria4.The flagellum enables bacteria to move toward attractants or away from detrimental chemicals and thus plays a key role in the dynamic responses of bacteria to diverse environments5. The basal body passes through the cell membrane and the cell wall and generates the flagellar rotation force. The basal body is also involved in the export of hook and filament proteins to the extracellular space through the tubular structure of the central rod , FlgE (hook protein), FlgK (junction protein), FlgL (junction protein), flagellin (filament protein), and FliD (cap protein), from the cell surface to the distal end  and Campylobacter jejuni FlgK proteins revealed that FlgK contains a four-helix bundle structure19. However, FlgL has not been structurally characterized, and it is thus unknown how FlgL assembles into the upper layer of the junction and links the hook to the filament at the top of FlgK.The FlgL and FlgK junction proteins that are located between the FlgE hook and the flagellin filament are indispensable for the formation of the flagellum because both FlgL-deficient bacteria and FlgK-deficient bacteria lack functional flagella and are immotileC. jejuni, it was proposed that the junction proteins are required to accommodate the symmetry mismatch between the 11-protofilament organization of the hook and the 7-protofilament organization of the filament21. However, this symmetry mismatch has been shown to occur only in C. jejuni to date. The hooks, rods, and filaments from other species are constituted by the same number (11) of protofilaments, and thus, the biological role of the junction proteins in flagellar assembly remains unclear23.In the bacterial flagellum, the hook is directly placed on the rod of the basal body without any junction proteins. However, the hook and the filament are cross-linked through the FlgL and FlgK junction proteins. Based on a structural study of the hook of Bacillus cereus (bcFlgL) and gram-negative Xanthomonas campestris (xcFlgL). Based on the structural similarity of FlgL to flagellin, we propose that flagellin assembles on top of FlgL in a manner similar to that reported for flagellin polymerization in the flagellar filament.To investigate the role of FlgL in hook-filament assembly, we have determined the crystal structures of FlgL from gram-positive Legionella pneumophila FlgL (lpFlgL), and Salmonella enterica serovar Typhimurium (S. Typhimurium) FlgL (stFlgL) for expression, purification, crystallization, and X-ray diffraction. Crystal structures for the truncated bcFlgL and xcFlgL proteins , which lack ~45 N-terminal residues and ~35 C-terminal residues, were solved by multiwavelength anomalous diffraction (MAD) phasing and single-wavelength anomalous diffraction (SAD) phasing, respectively  , the D1 domains of bcFlgL and xcFlgL were shown to be structurally homologous to those of stFlgL  and lpFlgL , which also exhibit a four-segment structure that contains the α1-α4 helices and the β3-β4 hairpin  and two-domain (D1-D2) structures, respectively Fig. . The D1 xcFlgL contains an additional domain, D2, compared to bcFlgL. In the xcFlgL structure, the α3 helix is split into the α3a and α3b helices, between which the D2 domain is inserted, extending from the middle of the S3 segment Fig. . The D2 25. In structure overlays, each of the S1, S2, and S4 segments is represented by α-helices that are identical in terms of direction and position. Moreover, the S3 segment is vertically arranged in a similar position although the S3 segment adopts diverse secondary structures . Among the flagellar components, the D1 domain of FlgL exhibits the highest structural similarity to that of flagellin, with a root-mean-square deviation (RMSD) value of 1.90 Å between the D1 domains of bcFlgL and S. Typhimurium flagellin for 140 Cα atoms  and the rod-forming protein (FlgG) Fig. 20,26. ThThe structural similarity between the D1 domains of FlgG and FlgE and between the D1 domains of FlgL and flagellin suggests that FlgE and flagellin assemble on top of FlgG and FlgL, respectively, through quasi-homotypic interactions that mimic the homotypic FlgE-to-FlgE and flagellin-to-flagellin interactions, respectively. The proposed quasi-homotypic interaction provides a clue about the role of FlgK and FlgL in the hook-filament connection. The D1 domain of flagellin adopts a four-segment structure that is completely different from the β-strand structure of the D1 domain of the FlgE hook protein. Therefore, the D1 domain of flagellin would not be able to form a direct quasi-homotypic interaction with the D1 domain of FlgE during flagellar assembly. Instead, flagellin is indirectly cross-linked to FlgE through FlgK and FlgL, which reconcile the structural incompatibility between FlgE and flagellin.C2 and xcFlgLH3) despite different crystal contacts, suggesting that the D1-D2 orientation would not change in different environments and would be recapitulated both in solution and in the flagellum. In the xcFlgL structure, the interdomain interface on the D1 domain is mainly polar, and the D1 domain of xcFlgL is therefore highly likely to exist as an independent stable domain without the D2 domain, as observed for bcFlgL. In contrast, because the interdomain interface on the D2 domain of xcFlgL is partially hydrophobic, the structural stability of the D2 domain would be achieved only in the presence of the D1 domain.In the xcFlgL structure, the β-sandwich fold of the D2 domain is affixed to the upper part of the helical structure of the D1 domain Fig. . The β5 19. Moreover, the D2 domains of FlgL and FlgK both form accessory structures that are appended to the primarily α-helical D1 domain.The β-sandwich structure of xcFlgL D2, which consists of a three-β-strand sheet and a five-β-strand sheet, is observed in the functionally related FlgK protein  Fig. 18,19. Mo27. To be consistent, the terminal regions of the flagellin D1 domain undergo structural rearrangements depending on their environments28. Our comparative analysis of the two xcFlgL structures (xcFlgLC2 and xcFlgLH3) indicates that the N-terminal and C-terminal regions of the FlgL D1 domain adopt diverse conformations, as observed for flagellin  due to a sharp directional change in the helix. Moreover, the N-terminal regions of the α1 helices are tilted in different directions in the two xcFlgL structures. Interestingly, despite the conformational differences, the terminal regions of the FlgL D1 domain have a tendency to form primarily helical structures regardless of crystal contacts.In addition to the common four-segment structure of the D1 domain, FlgL shares the structural features of the terminal regions with flagellin. The N-terminal and C-terminal regions of flagellin were shown to be highly flexible in solutionlin Fig. . In the 23. The D0 domain forms the inner ring of the flagellar tubule and plays a key role in protein polymerization and flagellar assembly. The C-terminal portion of the D0 domain invariably folds into a single α-helix. However, the N-terminal portion of the D0 domain is shaped into two types of structures depending on its length. FlgG and flagellin contain a relatively short N-terminal portion of the D0 domain that adopts a single α-helix. However, the N-terminal portion of the FlgE D0 domain is relatively long and forms a mixed structure of one α-helix and multiple β-strands. The FlgL N-terminal portion that is missing in the bcFlgL structure is relatively short (44 residues), and the terminal regions of FlgL are highly likely to form α-helices based on secondary structure prediction, suggesting that the D0 domain of FlgL adopts a coiled-coil structure that consists of two α-helices, as observed for flagellin. Thus, FlgL structurally mimics flagellin in the D1 domain and presumably in the D0 domain, supporting our proposal that FlgL would recruit flagellin to the junction through quasi-homotypic interactions, as reported for flagellin polymerization in the filament.The N-terminal and C-terminal regions of extracellular flagellar proteins have been structurally defined in FlgG, FlgE, and flagellin and have been shown to form an independent domain, D0Salmonella flagellin, in which the D0 domain was shown to enhance aggregate formation28. The gel-filtration chromatography results showed that the truncated bcFlgL, stFlgL and xcFlgL proteins that lack the D0 domain were monomeric in solution  and two flagellin molecules (flagellin and flagellin′) via the S1, S2, and S4 segments  sequence indicate that both bpFlgL and bpFlgK contain a D2 domain that would adopt a similar structure18. Thus, bpFlgL is expected to interact with bpFlgK in the flagellum through a quasi-homotypic interaction between the D2 domains of the two proteins. Similarly, the D2 domain of FlgK was proposed to interact with the variable D3 and D4 domains of FlgE19. To reveal the functional role of the D1 or D2 domains that we have proposed here, future structural studies of the FlgL-flagellin interaction are required.In the FlgL-flagellin assembly model, the D2 domain of FlgL is exposed to the outside and decorates the internal ring composed of the FlgL D1 domains Fig. . AlthougB. cereus and X. campestris, respectively, and cloned into a pET49b expression vector that was modified to express a protein containing an N-terminal His6 tag and a thrombin cleavage site31. The cloned product was transformed into E. coli strain DH5α, and the nucleotide sequence of the FlgL expression plasmid was verified by DNA sequencing. For protein expression, the expression plasmid was transformed into E. coli strains BL21 (DE3) and B834 (DE3).The bcFlgL gene  and the xcFlgL gene  were amplified by PCR from the genomic DNAs of E. coli BL21 (DE3) cells. The cells were grown in Luria-Bertani (LB) broth at 37 °C to an optical density of 0.6 at 600 nm. Protein overexpression was induced for 18 hours at 18 °C by supplementing the LB medium with 1 mM isopropyl β-D-1-thiogalactopyranoside. Selenomethionine (SeMet)-incorporated bcFlgL and xcFlgL proteins  were produced in E. coli B834 (DE3) cells using nutrient-supplemented M9 minimal medium (Molecular Dimensions) containing 40 μg/ml L-SeMet. The overexpression of SeMet proteins was induced in a manner similar to that used for the native protein.The recombinant proteins were overexpressed in 6 affinity tag was removed from FlgL by incubation with thrombin at 18 °C for 3 hours. The resulting FlgL protein was injected into a Mono Q 10/100 column  for anion-exchange chromatography and eluted with a linear gradient of sodium chloride (0–500 mM) in 20 mM Hepes, pH 7.4. The oligomeric states of the purified bcFlgL, stFlgL, and xcFlgL proteins were analyzed by gel-filtration chromatography using a Superdex 200 10/300 column  in a solution containing 20 mM Hepes, pH 7.4, and 150 mM sodium chloride.The cells were pelleted by centrifugation, resuspended in 50 mM Tris, pH 8.0, and 200 mM sodium chloride, and sonicated for cell lysis. The soluble lysate was incubated with Ni-NTA resin (Qiagen) in the presence of 10 mM imidazole at 4 °C for 1 hour for initial purification by Ni-NTA affinity chromatography. The FlgL-bound Ni-NTA resin was packed in an Econo-column (Bio-Rad) and washed with 50 mM Tris, pH 8.0, 200 mM sodium chloride, and 10 mM imidazole. The FlgL protein was eluted from the resin using 250 mM imidazole, 50 mM Tris, pH 8.0, and 200 mM sodium chloride and dialyzed against 20 mM Hepes, pH 7.4. The N-terminal His32.The crystallization conditions for FlgL were screened by the sitting-drop vapor-diffusion method using the JCSG Core Suites kit (Qiagen). The initial crystallization conditions were optimized to 15% w/v PEG 8000, 0.1 M Hepes, pH 7.4, and 0.2 M zinc acetate for the native bcFlgL protein and to 17% w/v PEG 3350 and 0.3 M magnesium nitrate for the native xcFlgL protein. SeMet-bcFlgL was crystallized in 10% w/v PEG 8000, 0.1 M sodium acetate, pH 5.4, and 0.3 M zinc acetate. SeMet-xcFlgL crystals were obtained in a solution containing 21% w/v PEG 3350 and 0.3 M magnesium nitrate (space group H3) or in a solution containing 19% w/v PEG 6000, 0.1 M Hepes, pH 7.0, and 1.0 M lithium chloride (space group C2). The crystals were transferred into a cryoprotectant solution containing either 25% glycerol or 25% ethylene glycol and then flash-frozen under a cryo-stream at −173 °C. X-ray diffraction was performed at beamlines 5C and 7A of the Pohang Accelerator Laboratory. The X-ray diffraction data were indexed, integrated, merged, and scaled using the HKL2000 program34. The models of SeMet-bcFlgL (space group C2) and SeMet-xcFlgL (space group H3) were built on the MAD and SAD maps, respectively, using the AutoBuild program in the Phenix package and the Coot program35. The structures of native bcFlgL (space group C2), SeMet-xcFlgL , and native xcFlgL  were determined by molecular replacement using a search model of SeMet-bcFlgL (space group C2) or SeMet-xcFlgL (space group H3) using the Phaser program in the CCP4 suite36. The final structures of FlgL were obtained through iterative cycles of rebuilding and refinement using the Coot and Refmac5 programs, respectively37. The structure refinement statistics are shown in Table 2+ ions, which are presumably derived from the crystallization solution .The atomic coordinates and the structure factors for FlgL  have been deposited in the Protein Data Bank (Supplementary Figures 1-4


Protobothrops snake venom that was collected in Japan.Snake venom metalloproteinases (SVMP) are widely distributed among the venoms of Crotalinae and Viperidae, and are organized into three classes  according to their size and domain structure. P-I SVMP are the smallest SVMP, as they only have a metalloproteinase (M) domain. P-II SVMP contain a disintegrin-like (D) domain, which is connected by a short spacer region to the carboxyl terminus of the M domain. P-III SVMP contain a cysteine-rich (C) domain, which is attached to the carboxyl terminus of the D domain. Some SVMP exhibit hemorrhagic activity, whereas others do not. In addition, SVMP display fibrinolytic/fibrinogenolytic (FL) activity, and the physiological functions of SVMP are controlled by their structures. Furthermore, these proteinases also demonstrate fibrinogenolytic and proteolytic activity against synthetic substrates for matrix metalloproteinases and ADAM . This article describes the structures and FL, hemorrhagic, and platelet aggregation-inhibiting activity of SVMP derived from  These symptoms are caused by a bioactive mixture of proteinases, phospholipase A lectins  ProteinaSVMP are organized into three classes according to their size and domain composition . PhylogeProtobothrops (habu) is classified into the Crotalinae family, and P. flavoviridis (Hon habu), P. elegans (Sakishima habu), and P. tokarensis (Tokara habu) are all found in Japan. Snake venom metalloproteinases (SVMP) account for at least 30% of the components of most Protobothrops venoms, suggesting that SVMP play significant roles in envenomation-related pathologies, such as bleeding, intravascular clotting, edema, thrombogenesis, inflammation, and necrosis [2 proteinase (PF-H2) and four hemorrhagic proteinases  were isolated from P. flavoviridis venom collected in Okinawa. SVMP have various physiological functions, e.g., they exhibit fibrinolytic/fibrinogenolytic (FL) and hemorrhagic activity, activate prothrombin, induce apoptosis in vascular endothelial cells, and affect platelet aggregation. The relationship between the structures and functions of metalloproteinase/disintegrin/cysteine-rich (MDC) domains has been reported previously, and specific functions of SVMP were suggested to be dependent on the structures of these domains [Protobothrops  venoms collected in Japan.necrosis ,6,7,8. Inecrosis . A non-h domains ,11. Stru domains . This arP. flavoviridis venom [Daboia russelli) venom (RVV-X) contains an additional C-type lectin-like domain [SVMP range in size from 20 to 100 kDa and have been classified into three groups, P-I to P-III , accordiis venom ,15, havee domain ,17.2+-binding sequence  and a Met-turn (Met173) bearing the typical structural features of the metzincin family of metalloproteinases [2 [Protobothrops venom, most differences were detected in the M domain. In particular, most of the variation was found in three regions  (The M domain of SVMP contains a conserved Znteinases . The aminases [2  has a ceurn Met17 bearing  92–117) A. As shop-APMSF , but are completely inhibited by EDTA (ethylenediaminetetraacetic acid). P-I SVMP are the smallest SVMP, as they only have an M domain. Therefore, the hemorrhagic and proteolytic activities of P-I SVMP are generally weaker than those of P-III SVMP, which have a C domain containing a hypervariable region (HVR) (positions 382–403 in P-I SVMP are single-chain polypeptides with molecular masses of about 25 kDa (200–210 amino acid residues). The proteolytic activities of these enzymes are not influenced by P-II SVMP contain a canonical D domain, which is connected by a sort spacer region to the carboxyl terminus of the M domain. In general, snake venom disintegrins are generated by the proteolytic processing of large precursor P-II SVMP ,30,31, aP-III SVMP are classified into the following subclasses: P-IIIa/b, which exhibit hemorrhagic and proteolytic activity and inhibit platelet aggregation; P-IIIc, which induce apoptosis in human umbilical vein endothelial cells (HUVEC); and P-IIId, which activate factor X or prothrombin. When the amino acid sequences of the DC domains of class P-II integrin precursors  [561-Val562 bond in plasminogen to generate two-chain plasmin, which results in fibrinolytic activity [FL enzymes and thrombin-like enzymes are widely distributed among the venoms of Crotalinae and Viperidae. The FL enzymes and thrombin-like enzymes found in snake venom act on fibrinogen and fibrin, leading to the defibrinogenation of blood and fibrinolysis, which reduces blood viscosity ,36,37. TI and II , prefereI and II . Tissue- (LV-PA) , specifiactivity ,42.Trimeresurus mucrosquamatus [Crotalus atrox [2 proteinase derived from P. tokarensis venom (PT-H2) [Gloydius blomhoffii sinaiticus venom [2 proteinase [P. flavoviridis venom, in which the Asp75 residue of the PT-H2 proteinase from P. tokarensis venom has been replaced with an Asn75 residue . It is suggested that Asp75 is an important amino acid residue for fibrinolytic activation because PT-H2 proteinase has a different substrate specificity from PF-H2 proteinase.The FL activity of SVMP involves the selective cleavage of the Aα chain of fibrinogen rather than the Bβ chain; however, these proteinases do not produce fibrin clots. On the other hand, this is not the case for some of SVMPs found in the venoms of quamatus  or Crotaus atrox , which d (PT-H2)  and FE-3us venom , can prooteinase  found in residue , is not 2 are obviously different. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of fibrin that had been dissolved by PT-H2 or urokinase are shown in 2 completely digested the α and β chains, but barely affected the γ chain or the fibrin polymer [The fibrinolytic activation mechanisms of urokinase and PT-H polymer .P. flavoviridis venom and characterized in a previous study [2 proteinase (P-I SVMP) from the same snake venom (PF-H2), which has a low molecular weight, does not exhibit hemorrhagic activity [P. flavoviridis venom displayed a high degree of homology compared with those of SVMP from other snake venoms , Lys (or Arg), His, Gln, Phe, and Gly (or Ala), respectively. Most of the replaced amino acid residues are amino acids that influence protein-protein interactions between basic or hydrophobic amino acids. It is suggested that the hemorrhagic effects of P-I SVMP are controlled by theese amino acid residues. These amino acid residues are an important target for functional analyses of P-I SVMP.Marked hemorrhaging, edema, necrosis, thrombogenesis, hypotension, and pain occur immediately after snakebites involving Viperidae and Crotalinae. Hemorrhagic SVMP have been isolated from many snake venoms and are considered to be one of the causes of the abovementioned symptoms. In vivo, hemorrhagic SVMP act on the peripheral vasculature, which causes red blood cells to leak from the affected blood vessels. Therefore, the hemorrhagic SVMP present in the snake venoms of Viperidae and Crotalinae are considered to be lethal factors. HR2a and HR2b (P-I SVMP), which have low molecular weights, and HR1A and HR1B (P-III SVMP), which have high molecular weights, were isolated from us study . The H2 activity . The hemactivity ,22,23 ise venoms . The dife venoms B. On theith HR2a A. FurtheP. flavoviridis venom, and nine elegantin [P. elegans venom.Platelet aggregation is a key event in thrombus formation and is dependent on the binding of adhesive proteins to αIIbβ3 integrin molecules on the platelet surface. Platelet adhesion proteins, such as fibrinogen and von Willebrand factor, contain the tripeptide segment Arg-Gly-Asp (RGD) as a cell recognition site. Snake venom proteins, including disintegrins and SVMP, inhibit platelet aggregation ,49. Snaklegantin ,53 isofoP. flavoviridis and P. elegans. In addition, HR1A, HR1B [Protobothrops snake venom in Japan. HR1A and HR1B derived from P. flavoviridis venom undergo autoproteolysis and are released as single major fragments of 32 and 34 kDa, respectively, containing D and C domains. It is suggested that these fragments inhibit platelet aggregation [P. elegans venom had a marked effect on adenosine diphosphate (ADP)- and collagen-induced platelet aggregation  and rapidly inhibited ADP-induced platelet aggregation. SV-PAD-2 can be subclassified as a P-IIIc SVMP based on the fact that it undergoes dimerization because its molecular mass according to SDS-PAGE was 110 kDa under non-reducing conditions and 52 kDa under reducing conditions [On the other hand, P-I and P-III SVMP, which inhibit platelet aggregation, have been purified from the venoms of 1A, HR1B , and SV-1A, HR1B , which aregation . The cDNregation ,29 were regation  and exhiregation ,25,26. Hnditions .Bothrops moojeni venom metalloproteinase (BmooMP) α-II, inhibit ADP-, collagen- and ristocetin-induced platelet aggregation. BmooMPα-II is a non-hemorrhagic P-I SVMP that inhibits platelet aggregation [50: 1.3 μM for ristocetin-induced platelet aggregation; 3.2 μM for collagen-induced platelet aggregation) [2 inhibited ADP-induced platelet aggregation (IC50: 8 μM). As PT-H2 inhibits platelet aggregation to a different extent to Bar-I, it is likely that PT-H2 inhibits platelet aggregation via a different mechanism from Bar-I. Many of the platelet aggregation inhibitors isolated from SVMP are disintegrin domain-containing P-II or P-III SVMP. In a recent investigation, I found that PT-H2 inhibited ADP-induced platelet aggregation. I suggest that PT-H2 also inhibits platelet aggregation via the cleavage of platelet integrins.Recently, it has been reported that P-I SVMP, such as barnettlysin-I (Bar-I) and regation . In addiegation) . Shancheegation) . FurtherProtobothrops species (P. flavoviridis (hon habu), P. elegans (sakishima habu), and P. tokarensis (tokara habu)) venom collected in Japan. The venoms of these snakes contain many SVMPs that exhibit a variety of physiological and biological functions. SVMPs range in size from 20 to 100 kDa and have been classified into three groups, P-I to P-III, according to their M, D, and C domain characteristics. P-III SVMP display various physiological functions associated with the DC domain. In the current study, it was suggested that the DC domain  influences the substrate specificity of SVMP, and the M domain is thought to function like a pair of scissors during substrate cleavage. However, P-I SVMP that displayed various substrate specificities were recently purified from several snake venoms.In this paper, we have described the structures and functions of SVMP derived from Protobothrops venom, most differences were detected in the M domain, so it may be considered that the functions of SVMP are influenced by these particular amino acid residues in the M domain. The SVMPs isolated from all Protobothrops venoms collected in Japan exhibited similar structures. The SVMPs examined in the present study might be useful for clarifying the relationship between the M domain architecture and function.As for the amino acid sequences of the SVMP found in


Congenital lactase deficiency (CLD) is a severe autosomal recessive genetic disorder that affects the functional capacity of the intestinal protein lactase-phlorizin hydrolase (LPH). This disorder is diagnosed already during the first few days of the newborn’s life due to the inability to digest lactose, the main carbohydrate in mammalian milk. The symptoms are similar to those in other carbohydrate malabsorption disorders, such as congenital sucrase-isomaltase deficiency, and include severe osmotic watery diarrhea. CLD is associated with mutations in the translated region of the LPH gene that elicit loss-of-function of LPH. The mutations occur in a homozygote or compound heterozygote pattern of inheritance and comprise missense mutations as well as mutations that lead to complete or partial truncations of crucial domains in LPH, such as those linked to the folding and transport-competence of LPH and to the catalytic domains. Nevertheless, the identification of the mutations in CLD is not paralleled by detailed genotype/protein phenotype analyses that would help unravel potential pathomechanisms underlying this severe disease. Here, we review the current knowledge of CLD mutations and discuss their potential impact on the structural and biosynthetic features of LPH. We also address the question of whether heterozygote carriers can be symptomatic for CLD and whether genetic testing is needed in view of the severity of the disease. Carbohydrates, also sugars and starches, constitute one main source of energy in our daily diet. Depending on their structural complexity, these compounds are categorized under monosaccharide, disaccharide, oligosaccharide or polysaccharide groups ,2. CarboThe failure to digest disaccharides to monosaccharides due to enzyme deficiency results in carbohydrate malabsorption that causes oftentimes watery acidic diarrhea . OsmoticSI is associated with several malabsorption disorders, such as congenital sucrase-isomaltase deficiency (CSID) , irritabLactose intolerance is another intestinal digestion disorder that is linked to reduced or complete loss of LPH activity. Two major types of lactose intolerance exist, primary lactase deficiency, also called adult-type hypolactasia, and congenital lactase deficiency (CLD) . In mostUnlike in several forms of genetically-determined or acquired forms of carbohydrate malabsorption disorders, congenital lactase deficiency is suspected in neonates of a few days of age with the onset of watery diarrhea after the start of breastfeeding, usually in the absence of vomiting, with adequate intake and no food refusal ,20. If tN-glycosylation site in this domain is the site for its association with the lectin chaperone calnexin, thus facilitating the folding of the entire LPH molecule [N-acylsphingosines and flavonoid glycosides [per se and shares striking similarities with bacterial ß galactosidase. Domain IV contains the lactase active site and is anchored to the membrane via one single pass domain containing the lactase active site. Unlike domain III, this domain is not transport-competent per se and requires the presence of domain III to be trafficked along the secretory pathway  [LPH is composed of four homologous structural domains, which together cooperate to ensure a competent trafficking and efficient polarized sorting of the protein to the apical membrane . Recent molecule . Domain ycosides . It is tHßfinal) .N- and O-glycosylation of LPH is involved in the regulation of the trafficking efficiency to the apical membrane and the enzymatic capacity [N- and O-glycosylated LPH is almost 4 fold more active than the N-glycosylated enzyme [LPH is exclusively expressed in the enterocytes, and its processing comprises three main steps that implicate the concerted interaction of the different domains . The fircapacity ,34. In fThe pattern of inheritance of CLD can be both homozygote and compound heterozygote. Several different mutations in the coding region of LPH have been meanwhile characterized, the majority of them in a study with 32 Finish patients. Around 80% of the patients were homozygous for a stop codon at tyrosine 1390 (Y1390X) that results in a truncated protein. The presence of this mutation, referred to as the Fin (major) mutation, was explored in the LPH gene in a cohort of more than 550 individuals in Finland and revealed frequencies varying from 1:35 to 1:133. Other mutations, S1666fsX1722 and S218fsX224, resulted in a frameshift and a premature stop codon or an amino acid substitution (Q268H and G1363S). The Fin (major) Y1390X mutation was found in two more patients of Finnish origin in a compound heterozygote pattern of inheritance with either R1587H or V565fsX567 . Finallyper se, these findings highlight the central role of this domain in the overall folding pro-LPH. Remarkably, the G1363S creates an additional N-glycosylation site and confers a temperature-sensitive phenotype on the LPH-G1363S mutant, which acquires transport-competence and can exit the ER at a temperature of 20°C. These characteristics suggest that a longer exposure of this mutant to the folding machinery in the ER, for instance to the chaperones calnexin and BiP, may help restore the correct folding of the LPH domains. Chaperone-based therapies can thus be a promising strategy in the treatment of CLD patients carrying this mutation [The sophisticated interaction between the different domains of pro-LPH, each with its specific role in the context of folding, trafficking competence, polarized sorting, and enzymatic function of pro-LPH could be destabilized or severely altered by mutations in either one of these domains. Until present, only two published studies have analyzed the genotype-phenotype relationship in CLD at the molecular and cellular levels. One of these studies focused on the impact of the G1363S mutation, which was characterized in Finnish, Turkish, and Iraqi patients and is localized in domain III and in close proximity downstream the phlorizin hydrolase active site. This mutation elicited misfolding, not only in domain III but also of entire pro-LPH resulting in a transport-incompetent LPH-G1363S that was intracellularly blocked in the ER. Given that domain III is an autonomous and transport-competent mutation .Another study focused on the biochemical features of two mutations, Y1473X and D1796fs, which were found as a compound heterozygous pattern in a Japanese infant with CLD . These mAlthough detailed analyses of the other known mutation in CLD do not exist, the pathogenic severity can be predicted if a mutation has generated a truncated protein or if a mutation is located in a region that is critically important in the context of folding and acquisition of transport competence. For instance, the mutations Y1390X and E1612X generate pro-LPH mutants that lack the critical domains IV and also the transmembrane domain, which plays a primordial role in the homodimerization of pro-LPH ,38. SincThe mutations Q268H and S688P are located in the profragment of pro-LPH that exerts a central role as an intramolecular chaperone in the initial folding of pro-LPH prior to homodimerization. The S688P was predicted to be possibly damaged by PolyPhen, and the substitution of a basic histidine for uncharged glutamine in Q268H was likely to alter the secondary structure of the profragment ,35. FinaObviously, the majority of the mutations, if not all, that have been so far characterized in CLD, whether in a homozygous or a compound heterozygous pattern of inheritance can be considered to be pathogenic, unlike mutations of SI in CSID that have been categorized into three major classes according to their trafficking behavior and grade of functional severity ,35.Until present, it is unknown whether a single normal parental allele in conjunction with the diseased one would be enough to generate and process LPH that can sufficiently digest dietary lactose and would provide knowledge on the lactose digestive capacity of heterozygote carriers of severe pathogenic mutations in the LPH gene. The question that should be addressed in this context is whether an interaction between a wild type LPH monomer with another LPH mutant monomer resulting in a heterodimer is potentially possible. This assumption stems from the fact that wild type LPH dimerizes in the ER to an active and transport-competent enzyme. One of the absolute requirements for this early event along the secretory pathway is the existence of an intact, that is, non-mutated transmembrane domain. It can be, therefore, hypothesized that an interaction between wild type LPH and LPH mutants containing transmembrane domains, such as LPH-G1363S, LPH-Q268H, LPH-S688P, and LPH-R1587H, may take place. Further, the trafficking and enzymatic function of the resulting heterodimers would be dictated by the severity of the mutation itself. It would be tempting, therefore, to address this question in future co-expression studies of wild type LPH and its transmembrane domains carrying mutants. Obviously, this type of interaction excludes the truncated mutants that lack the transmembrane domains, such as LPH-Y1390X, LPH-E1612X, LPH-D1796fs, LPH-Y1473X, LPH-S1150Pfs*19, LPH-S1666fsX1722, and LPH-S218fsX224. Nevertheless, interaction via the luminal region of these mutants rather than the transmembrane anchor cannot be excluded, provided minimal folding requirements are fulfilled, such as correct folding of domains involved in dimerization of LPH. In a recent study, this hypothesis has been examined for two truncated LPH mutants, LPH-D1796fs, LPH-Y1473X, in a Japanese infant. In co-expression studies, neither the LPH-Y1473X nor LPH-D1796fs were retained in the same experimental sample as wild type LPH clearly indicating that these mutant forms do not interact with wild type LPH . HoweverThe current progress made in the identification of several mutations in CLD is certainly essential at the clinical level, facilitates the genetic testing, and moreover shows that this disease is more frequent than previously thought. Nevertheless, this progress is not paralleled by functional and biochemical analysis of the LPH mutants. In particular, the analysis of the effect of missense mutations on the folding pattern, function, and pathogenicity of LPH mutants determine the grade of severity of the protein phenotype. These studies are also crucial for the heterozygote in light of the potential interaction of wild type and mutant LPH via the transmembrane domain, resulting in the effect on the overall functional performance and trafficking competence of a potential heterodimeric LPH. Furthermore, despite the view that a truncated protein is a priori not active or is transport-competent, correct folding or partial folding of the extracellular domain may still have the capability to form dimers with wild type LPH and should be studied. Until solid data on the effect of these mutations on the heterozygotes are generated and given the severity of this disease and life threat in the early days for the newborn, genetic testing of the LPH gene is recommended for potential heterozygote carriers.


Defective NCS-1 can be deleterious to cells and has been linked to serious neuronal disorders like autism. Here, we review recent studies describing at the single molecule level the structural and mechanistic details of the folding and misfolding processes of the non-myristoylated NCS-1. By manipulating one molecule at a time with optical tweezers, the conformational equilibria of the Ca2+-bound, Mg2+-bound and apo states of NCS-1 were investigated revealing a complex folding mechanism underlain by a rugged and multidimensional energy landscape. The molecular rearrangements that NCS-1 undergoes to transit from one conformation to another and the energetics of these reactions are tightly regulated by the binding of divalent ions (Ca2+ and Mg2+) to its EF-hands. At pathologically high Ca2+ concentrations the protein sometimes follows non-productive misfolding pathways leading to kinetically trapped and potentially harmful misfolded conformations. We discuss the significance of these misfolding events as well as the role of inter-domain interactions in shaping the energy landscape and ultimately the biological function of NCS-1. The conformational equilibria of NCS-1 are also compared to those of calmodulin (CaM) and differences and similarities in the behavior of these proteins are rationalized in terms of structural properties.The human neuronal calcium sensor-1 (NCS-1) is a multispecific two-domain EF-hand protein expressed predominantly in neurons and is a member of the NCS protein family. Structure-function relationships of NCS-1 have been extensively studied showing that conformational dynamics linked to diverse ion-binding is important to its function. NCS-1 transduces Ca Calcium signaling is mediated by calcium binding proteins that sense changes in cellular concentration of Ca2+ ions and respond by interacting with downstream regulatory targets, further cascading the signal. One example of such calcium sensor proteins is the family of neuronal calcium sensors (NCSs), which are expressed primarily in neurons and photoreceptor cells  and K+ (Kv4)-channels  and C-domain  into an on-pathway intermediate state I1; then EF4 binds a calcium ion, to induce the full folding of the C-domain (intermediate state I2). The last calcium ion finally binds to EF2 to fold the N-domain and thus the entire protein reaches its native state (N state). In this folding process, the C-domain always folds first and acts as “internal chaperone” for the correct folding of the N-domain. If the C-domain does not reach its native state, as in the knockout mutant NCS1EF4, the N-domain fails to fold correctly. This asymmetrical folding process that always starts with the folding of the C-domain probably reflects the asymmetrical structure of the protein, as further discussed below.In the presence of activating concentrations of calcium the folding process of non-myristoylated NCS-1 gets considerably more complex  are populated by NCS-1, differing in extension and occupancy probability, with M1 being significantly populated only at high [Ca2+]. Figure Once an unfolded NCS-1 molecule has collapsed into the on-pathway intermediate state I2, it can either transit into I1 to eventually reach the native state, or take alternative pathways that lead to misfolded states  with the EF hands 2 and 3 mispaired (16% probability), and the other two leading to either the on-pathway intermediate F12 (42% probability) or the on-pathway intermediate F34 (42% probability), with either the N- or the C-domain, respectively, fully folded. The folding process of CaM can thus start with equal probability from the N- or the C-domain, while that of NCS-1 always start from the C-domain and always through the same U to I2 transition. For CaM, F12 and F34 can then both transit into the native state, although with different probabilities as in 50% of the cases F12 takes a misfolding pathway leading to the off-pathway intermediate state F123, where EF3 is collapsed onto the folded N-domain in a non-native conformation. The native state of CaM can thus be reached from two distinct intermediate states through two different folding pathways, unlike the native state of NCS-1 that can be accessed only from I1. Moreover, the native unidimensional folding process of NCS-1 is characterized by a bottleneck where the protein can either proceed towards its native state by overcoming the large activation barrier separating I2 from I1 or take misfolding pathways in a calcium dependent manner. No bottleneck characterizes the folding network of CaM where the interconversions between the different molecular states are controlled by similar activation barriers. In addition, despite CaM folding rates and folding mechanism having been shown to be highly dependent on calcium concentration (Stigler and Rief, The folding and misfolding mechanisms of the other NCS family members and of other EF hand calcium binding proteins are still largely uncharacterized and the question remains: is it likely that the same principles of folding and misfolding can be inferred for these similar proteins? Other related proteins have been studied with regards to folding and stability using ensemble methods with varying degrees of details (Yamniuk et al., Where do the differences in the folding networks of CaM and NCS-1 originate from? The answer may lie in inter-domain interactions and the coupling free energies that are more developed in NCS-1. Proteins such as CaM have a flexible linker connecting their two domains, allowing a high degree of conformational variation between them and at the same time imposes separation of their folding funnels (Gifford et al., 2+-bound, Mg2+-bound and apo forms of the non-myristoylated NCS-1 have been investigated at the single molecule level using optical tweezers. We have illustrated the rugged and multistate energy landscape arising from these studies, which underlays the calcium-dependent folding and misfolding trajectories of NCS-1. We have also discussed the importance that the conformational sensitivity of NCS-1 might have for its calcium sensing actions. Finally, we have made comparison to the very similar calcium binding EF hand protein CaM and have highlighted differences and similarities between the folding networks of the two proteins, which are likely to have arisen due to their different structural and functional features across the domains.We have outlined how the Ca2+ have been associated with aging cells and linked to the development of major neurological diseases such as bipolar disorder and Alzheimer’s (Toescu and Vreugdenhil, For NCS-1, the calcium-dependent modulation of misfolding pathways is highly interesting and suggests an important link between the conformational space, calcium dysregulation, and neurodegeneration. Interestingly, sustained elevated levels of free CaThe behavior of NCS-1 outlined in this review article may also help rationalize how NCS-1 is able to have such promiscuous interactions with over 20 reported binding partners (Burgoyne, DC designed and wrote the article; carried out bibliographic research. BK assisted in structural details and biological aspects, and contributed in the final revisions of the manuscript. PH designed and wrote the article. CC designed and wrote the article; prepared the figures. All authors have provided final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The processing map can be divided into four domains according to its graphic features. Among the four domains, the optimal domain is in the temperature and strain rate range of 850–925 °C and 0.001–0.1 s−1, where peak efficiency η is 0.54 and the main microstructural evolution is DRX . When the alloy is processed in the α + β phase field, the temperature and strain rate range of 800–850 °C and 3–10 s−1 should be avoided, where instability parameter ξ is negative and the microstructural feature is flow localization. When the alloy is processed in the β phase field, DRV (dynamic recovery) and slight DRX of β phase is the main microstructural characteristics in the range of 1000–1050 °C and 0.001–0.02 s−1. However, flow localization of β phase is the main microstructural feature in the range of 1000–1050 °C and 1–10 s−1, which should be avoided.The Arrhenius-type constitutive equation is mostly used to describe flow behaviors of material. However, no processing map has been constructed directly according to it. In this study, a novel computational method was applied for establishing the processing map for Ti-6Al-4V alloy in the temperature and strain rate range of 800–1050 °C and 0.001–10 s P is clarified into two parts: G and J. The G content represents the power which is converted into temperature rise. And the J co-content represents the power dissipated through the microstructure change, such as phase transformation, recovery, recrystallization, cavity formation and so forth [The processing map proposed by Prasad et al.  is widelso forth . The conso forth . The comso forth  was deriterature ,8,9,10, terature  proposedCu alloy . Althougperature . HoweverThe two-phase α/β titanium alloy Ti-6Al-4V has been widely used in the aerospace industry due to its high strength-to-weight ratio, good corrosion resistance and excellent mechanical properties. However, there are many difficulties in controlling its microstructure and mechanical properties due to the narrow processing time and temperature window . As ment−1. The computing method of processing maps based on the traditional power law equation and the Arrhenius-type constitutive equation were compared. The results indicate that the novel processing map is more effective. According to the present processing map, the stable domains and unstable domains were clarified and were validated by the microstructural observations. Eventually, optimized processing parameters for hot deformation of Ti-6Al-4V alloy was suggested.In this study, isothermal hot compression tests were conducted in the temperature range of 800–1050 °C and the strain rate range of 0.001–10 s−1, 0.01 s−1, 0.1 s−1, 1 s−1 and 10 s−1 respectively. Specimens were heated to the deformation temperatures at a rate of 5 °C/s and held for 5 min using a Gleeble-3500 Thermal/Mechanical Simulator . Subsequently, specimens were isothermally compressed up to total true strain of 0.9 and air cooled to room temperature. A thin layer of graphite was used to minimize the friction between the specimen and the anvils. Partial compression results  are from previous study [The material used in this study was a hot-rolled and annealed Ti-6Al-4V alloy bar with a nominal chemical composition of Ti-6.2Al-4.6V. The β-transus temperature was measured at about 995 °C using the metallographic method. Cylindrical specimens with 8 mm diameter and 12 mm height were machined from the annealed bar. The deformation temperature ranged from 800 °C to 1050 °C at an interval of 50 °C. The stain rates were 0.001 sus study , in whicus study  in this 3 + 85% H2O for 10 s. Transmission electron microscopy  samples were prepared by mechanical grinding to about 40 μm followed by twin-jet electropolishing  using the solution of 6% perchloric acid, 59% methyl alcohol and 30% mutual. Electron backscatter diffraction  samples were made, first with metallographically polishing and finally by being electro-polished in the same solution with TEM. The EBSD data was analyzed by the tango program using the HKL-Channel 5.Microstructural observations of the compressed specimens were made on the sectional plane parallel to the compression axis in the central regions. For scanning electron microscopy  observations, specimen surfaces were prepared by metallographical polishing and followed by chemical etching in a solution consisting of 5% HF + 5% HNOThe flow curves in −1) or flow softening  when dynamic softening balances or exceeds the work hardening. In addition, the sudden drop of flow stress  beyond the peak stress called the discontinuous yielding behavior was observed. The discontinuous yielding behavior has been found in many titanium alloys, such as Ti55 [The flow curves show different shapes in  as Ti55 , Ti60 [2 as Ti55 , Ti-6.5A as Ti55  and Ti-1 as Ti55 . The mai as Ti55 .G content, J co-content and total power P can be expressed in Equation (1) [σ is the flow stress (MPa), −1). When workpiece is an ideal linear dissipater, the J reaches its maximum value Gη can be used to represent the power dissipated through microstructure change. The variations of η values with strain rate and temperature constitute the power dissipation map at a given strain. The domains with high η values are considered to be safe domains and microstructure mechanisms are believed to be DRX, DRV, spheroidization or superplasticity (SP) [η values [J to replace D in Equation (3), then derived Equation (4) [The efficiency of power dissipation ity (SP) . Howeverη values . Therefoη values  is applition (4) .(3)dDdεξ values with strain rate and temperature constitute the instability map which depicts instability regions. In the negative ξ values regions, microstructure mechanisms are believed to be adiabatic shear band, flow localization, dynamic strain aging or twinning. Eventually, the processing map is established by superimposing the instability map over the power dissipation map.The variations of In this study, the true strain of 0.8 is close to the true strain of final compression state. Therefore, this strain was used to construct the processing map. In this section, both methods of the traditional power law equation and Arrhenius-type constitutive equation were carried out to compute the processing maps. Then, the effectiveness of the two methods was compared.In this section, the processing map was directly constructed by using the method proposed by Prasad et al. . But theThe power law constitutive equation was shown in Equation (5).η and ξ can be easily derived by combining Equations (1)–(4) and Equation (5), as expressed in Equation (6) and Equation (7).Then, the values of η and ξ, the value of strain rate sensitivity exponent m should be acquired first. The value of m can be obtained using Equation (8) by taking natural logarithm of both sides of Equation (5).For calculating the values of For a given strain and temperature, the plot of lnσ versus ln Substituting Equation (9) into Equation (8) yields Equation (10):η and ξ can be easily derived by combining Equations (6), (7) and (10). Eventually, the processing map was established, as shown in η and the shaded regime is the unstable domains with negative ξ values. In −1. The power dissipation efficiency η is about 0.49 in the domain. Domain B approximately locates in the range of 800–900 °C and 3–10 s−1. The values of ξ is negative in the domain. Domain C is approximately in the range of 930–1020 °C and 3–10 s−1. The peak efficiency η is 0.61 in the domain. Domain D approximately spreads over the range of 930–1000 °C and 0.001–0.01 s−1. The power dissipation efficiency η is about 0.2 in the domain.Then, the values of In the previous study , the ArrQ is the activation energy (kJ·mol−1), R is the universal gas constant (8.3145 J·mol−1·K−1), T is the absolute temperature (K), A, n′, β, α and n are the material constants.The Arrhenius-type constitutive equation is shown in Equation (11).J can be represented as:From Equations (1) and (11), the dissipater power co-content η was calculated as Equation (13):The value of η can be achieved by substituting the Arrhenius-type constitutive equation into Equation (13). The integral of the Arrhenius-type constitutive equation in Equation (13) can be easily calculated with MATLAB R2016a using function int.The value of ξ is difficult to be calculated directly. The computational method proposed by Murty [ξ, as expressed in Equation (14).In Equation (4), the value of by Murty  was emplm can be calculated as Equation (15):The values of strain rate sensitivity exponent m in Equation (15), the value of dσ/d In order to obtain ξ can be calculated by substituting m and η into Equation (14). Eventually, the processing map was established by superimposing the instability map over the power dissipation map, as shown in −1. The peak efficiency η is 0.54 in the domain. The domain is considered to be safe domain. Microstructure mechanisms such as DRX and DRV may occur in this domain. Domain B locates approximately in the range of 800–850 °C and 3–10 s−1. The values of ξ are negative in the domain. Flow instability mechanisms such as the adiabatic shear band and flow localization may occur in this domain. Domain C lies approximately in the range of 1000–1050 °C and 1–10 s−1. Domain D locates approximately in the range of 1000–1050 °C and 0.001–0.02 s−1. The power dissipation efficiency η is about 0.42 in the domain. In addition, the iso-efficiency contours in Then, the value of η and ξ. The maximum power dissipation efficiency 0.54 occurs in Domain A in η is 0.42 in Domain D in η is smaller in Domain D in Although both processing maps can be divided into four domains, there are many differences in the values and scopes of It is commonly acknowledged that the processing map alone is not sufficient to determine the optimum processing parameters and it is needed to be confirmed through microstructural characterization . Thus, mThe SEM image and IPF (inverse pole figure) map of the as-received specimen are shown in The IPF map of compressed specimen corresponding to the Domain A is represented in ξ values also indicate flow instability in domain C. No micro-cracking is found in this domain. Thus, β phase flow localization is the main mechanism of softening in this domain.−1. Flow behaviors were analyzed. The processing maps based on the traditional power law equation and Arrhenius-type constitutive equation were constructed and compared. Microstructural characteristics in different domains were studied. The results of microstructure characterization agree well with the results of the novel processing map. The following conclusions are drawn from this study.In this study, the hot deformation characteristics of Ti-6Al-4V alloy were investigated using isothermal compression tests in the temperature range of 800–1050 °C and strain rate range of 0.001–10 s(1) A novel computing method of processing map was constructed according to the Arrhenius-type constitutive equation. The results of the novel processing map fit engineering reality better than the traditional processing map.−1.(2) The novel processing map was divided into four domains according to its graphic features. The peak efficiency with a maximum value 0.54 lies in the temperature and strain rate range of 850–925 °C and 0.001–0.1 s−1. This domain is considered to be the optimal hot working window. However, flow localization occurs in the temperature and strain rate range of 800–850 °C and 3–10 s−1.(3) In the α + β phase field, DRX is the main microstructural characteristics in the temperature and strain rate range of 850–925 °C and 0.001–0.1 s−1. Flow localization of β phase is the main microstructural characteristics in the temperature and strain rate range of 1000–1050 °C and 1–10 s−1.(4) In the β phase field, DRV and slight DRX of β phase are the main microstructural feature in the temperature and strain rate range of 1000–1050 °C and 0.001–0.02 s


Oxidative stress plays a vital role in the initiation and progression of age-related neurodegenerative diseases. Ameliorating oxidative damage is therefore considered as a beneficial strategy for the treatment of age-related neurodegenerative disorders. Probucol (Prob), a lipid-lowering prototype agent, was reported to treat cardiovascular diseases, chronic kidney disease and diabetes mellitus. However, whether Prob has an effect on age-related neurodegenerative diseases remains unknown. In the study, it was found that Prob ameliorated D-galactose  induced cognitive deficits and neuronal loss in the hippocampal CA1 region. Moreover, Prob alleviated ROS and MDA levels by elevating SOD, GSH-PX and HO-1 mRNA and protein expressions, and improving plasmic and cerebral SOD and GSH-PX activities in D-gal treated mice. Furthermore, Prob promoted the dissociation of Keap1/Nrf2 complex leading to the accumulation of Nrf2 in nucleus, implying that the improved anti-oxidant property of Prob is mediated by Keap1/Nrf2 pathway. The study firstly demonstrates the favorable effects of Prob against cognitive impairments in a senescent mouse model, rendering this compound a promising agent for the treatment or prevention of age-related neurodegenerative disease. Aging is a complicated multifactorial process causing a gradual decline in physiological function and the capability of an organism to survive. Oxidative damage has been increasingly considered as a contributing factor in the reduction of physiologic function occurring during the aging process . IncreasOxidative damage in CNS may be ascribed to the failure of antioxidant defenses or the increased production of reactive oxygen species (ROS) . The accProbucol (Prob) is a phenolic lipid-lowering prototype agent clinically used to treat cardiovascular diseases , 16. In D-galactose -induced aging model in rodents has been extensively considered as an animal model for brain aging in a variety of anti-aging studies since D-gal, which induces oxidative injury in CNS, resulting in memory and synaptic dysfunction , mimics As shown in The spatial learning and memory was detected using MWM test. After four-day training, escape latency and percentage of time in the target quadrant on day 5 were recorded. As illustrated in The retention memory was evaluated using the 24h retention trial of the passive avoidance test. The results showed that higher memory errors and poorer retention latency in D-gal group than that in the control group was observed  and 2B, It has been documented that neurons in hippocampal CA1 region are highly susceptible to oxidative stress, which may cause neuronal loss and morphological change. Thus, in the present study, the hippocampal neurons in the CA1 region were chosen to be stained by H&E and morphology of neurons in the CA1 region was observed using 200×light microscopy. The results showed that the hippocampal neurons in the control group were arranged in an organized pattern with clear boundaries, whereas the hippocampal neurons of D-gal group exhibited loose and irregular arrangements and condensed nucleus was observed . ImportaNeuronal numbers in the CA1 region were also evaluated using Nissl staining. The results revealed that the number of Nissl positive neurons in D-gal group was remarkably decreased compared with the control group, while Prob treatment inhibited the reduction of neuronal numbers induced by D-gal , indicatCollectively, these results suggest that Prob treatment has an ability to alleviate hippocampal neuronal loss in D-gal-induced aging mice.As shown in The amelioration of oxidative stress by Prob in D-gal-induced aging mice suggests that Prob may influence the activities of antioxidant enzymes. The results showed that the activities of SOD and GSH-PX in the plasma and brains of D-gal group was significantly reduced compared to control group  and 4G. As illustrated in SOD, GSH-PX and Ho-1 gene is regulated by Keap1/Nrf2 pathway, we speculated that Prob might have impacts on Keap1/Nrf2 signaling expression. The results of Western blot assay showed that the levels of Keap1 and Nrf2 in D-gal group did not exhibit significant difference compared to control group . SOD, GSH-PX activity kits, MDA and ROS detection kits were purchased from Nanjing Jiancheng Bioengineering Institute . Keap1, Nrf2, SOD and GSH-PX antibodies were from Santa Cruz Biotechnology, Inc. . Ho-1 antibody was from Merck Millipore . Other reagents were all provided from Sigma-Aldrich unless otherwise noted.Six- to eight-week-old adult male Kunming strain mice obtained from Xuzhou Medical University were randomly divided into five groups: Control group, Control+ Prob high-dosage group, D-gal group, D-gal+ Prob high-dosage group and D-gal+ Prob low-dosage group. The low- and high-dosage Prob-treated groups were intraperitoneally injected with 10 and 20mg/kg Prob daily, respectively; D-gal-treated groups were given subcutaneously with D-gal (150mg/kg) for six weeks. The treatment regimens of Prob and D-gal were according to previous studies , 35. ConCognitive function was detected by Morris Water Maze (MWM) test as previously described , 37. ThePassive avoidance test was performed for measuring memory retention in mice according to a previous study . BrieflyThe H&E staining assay was performed according to previous studies , 40. BriNissl staining was performed according to the manufacturer’s instructions . In brief, ethanol-dehydrated samples were prepared and stained with Nissl staining solution for 10min at 37 °C. The total number of cells at CA1 region in each section was counted.TUNEL staining assay was used to determine the degree of cell apoptosis in hippocampal CA1 region according to the manufacturer’s instructions . Sections were also stained for neuronal marker microtubule-associated protein (MAP2) , with nuclei stained with DAPI, to identify neurons in hippocampal CA1 region. The percentages of neurons with apoptotic nuclei (both MAP2 and TUNEL positive) were then quantified.RT-PCR assay was performed as previously described , 42. AftWestern blot assay was conducted as previously described , 43. TheTotal cholesterol level was measured in plasma using an enzymatic method according to the oxidase/peroxidase system using commercial kit reagents .SOD, GSH-PX activity and MDA level were detected according to SOD, GSH activity and MDA detection assay kit instructions, respectively. In brief, action buffers were added to the supernatants of tissue samples. The mixtures were incubated at 37 °C and the optical densities were measured at 550nm, 412nm, and 532nm using visible spectrophotometer for SOD, GSH-PX activities and MDA, respectively. The activities of SOD and GSH-PX were expressed as the μmoL per g of protein sample, and the level of MDA was expressed as nmoL/mL.ROS level was detected according to ROS detection assay kit instruction. Briefly, 100nM DCFH-DA was added to single-cell suspension for 30min. Then cells were collected, washed and detected at 485nm excitation wavelength and 525nm emission wavelength. The fluorescence intensity of ROS was expressed as fluorescence intensity per μg protein.Co-IP assay was performed according to a previous study . BrieflyFor H&E, Nissl and TUNEL/MAP double staining assays, cells were analyzed according to a previous study . BrieflyThe data of each group were presented as mean±SEM. One-way analysis of variance (ANOVA) was used to analyze the variance using SPSS software for Windows 20.0. P<0.05 was considered to be statistically significant.


Parkinson’s disease (PD) is a chronic, progressive movement disorder that affects nearly 7 million people globally and 1 million people in the United States. Although there are pharmacological and surgical interventions to treat the symptoms of PD, no targeted therapeutics that reverse the effects of the disease are currently available. Since identification of the first LRRK2 mutations in PD patients in 2004, more than 40 mutations have been found in both familial and sporadic forms of PD. Because of the association of LRRK2 to PD, the structure of the WD40 domain dimer of human LRRK2 presented here will help elucidate the pathogenesis of certain WD40 mutations and provide structure-based template for potential therapeutic interventions. Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein with both a Ras of complex (ROC) domain and a kinase domain (KD) and, therefore, exhibits both GTPase and kinase activities. Human genetics studies have linked LRRK2 as a major genetic contributor to familial and sporadic Parkinson’s disease (PD), a neurodegenerative movement disorder that inflicts millions worldwide. The C-terminal region of LRRK2 is a Trp-Asp-40 (WD40) domain with poorly defined biological functions but has been implicated in microtubule interaction. Here, we present the crystal structure of the WD40 domain of human LRRK2 at 2.6-Å resolution, which reveals a seven-bladed WD40 fold. The structure displays a dimeric assembly in the crystal, which we further confirm by measurements in solution. We find that structure-based and PD-associated disease mutations in the WD40 domain including the common G2385R polymorphism mainly compromise dimer formation. Assessment of full-length LRRK2 kinase activity by measuring phosphorylation of Rab10, a member of the family of Rab GTPases known to be important kinase substrates of LRRK2, shows enhancement of kinase activity by several dimerization-defective mutants including G2385R, although dimerization impairment does not always result in kinase activation. Furthermore, mapping of phylogenetically conserved residues onto the WD40 domain structure reveals surface patches that may be important for additional functions of LRRK2. Collectively, our analyses provide insights for understanding the structures and functions of LRRK2 and suggest the potential utility of LRRK2 kinase inhibitors in treating PD patients with WD40 domain mutations. Parkinson’s disease (PD) is a severe neurodegenerative disorder that often inflicts older people, for which there is neither an objective test for diagnosis nor an effective cure. About 1–2% of the population above age 65 in the world live with PD 2. The exa2+ showed a domain-swapped dimeric architecture  and consists of armadillo repeats (ARM), ankyrin repeats (ANK), leucine-rich repeats (LRR), Ras of complex (ROC), C-terminal of ROC (COR), kinase domain (KD), and the Trp-Asp-40 (WD40) domain A. LRRK2 6 kDa anditecture . Howeveritecture . Consistitecture . A recenitecture , 14. In itecture , and fulitecture . TherefoThe C-terminal WD40 domain of LRRK2 is required for LRRK2-induced neurotoxicity , 18 and 26Here, we report the crystal structure of human LRRK2 WD40 domain determined at 2.6-Å resolution. The structure reveals a canonical seven-bladed WD40 fold and a surprising dimeric state in the crystal. We confirmed that isolated WD40 domain forms concentration-dependent dimers in solution and that structure-based interface mutations mainly disrupt the dimerization. Notably, the WD40 domain mutation G2385R is located at the dimerization interface and compromised dimerization. In contrast to previously identified reduction in kinase activity, our data on Rab10 phosphorylation indicated an enhanced kinase activity, supporting the overarching hypothesis that pathogenesis of PD is mediated through hyperactivation of LRRK2. Similar to G2385R, two structure-based mutants H2391D and R2394E at the WD40 dimerization interface also increased LRRK2 kinase activity. We further mapped sequence conservation onto the WD40 domain structure and found surface patches that may be important for additional functions of LRRK2.SI Appendix, Table S1), diffraction data collected on a crystal soaked with transplatinum (II) diammine dichloride gave sufficient anomalous signals at the wavelength of 0.98 Å, which is a high energy remote wavelength of the platinum L-III edge. A total of three platinum sites were identified . We determined the WD40 domain crystal structure using the single wavelength anomalous diffraction (SAD) method and refined it to 2.6-Å resolution . The ordered part of the structure extends from residue 2,142 to residue 2,498 and has a few gaps, and the last 30 residues in the construct are disordered.We expressed the WD40 domain of human LRRK2 (residue 2142–2527) in insect Sf9 cells. The protein was purified, and diffracting crystals were obtained. We initially attempted molecular replacement structure determination of the LRRK2 WD40 domain using multiple known WD40 domain structures in the Protein Data Bank (PDB). No viable solution was obtained, likely because of the weak homology between the LRRK2 WD40 domain and WD40 domain structures in the PDB. We therefore searched for heavy atom derivatives using soaking of preformed native crystals. After screening 13 heavy atom compounds . The electrostatic potential plot reveals a highly charged surface with positively and negatively charged patches . All blades comprise four anti-parallel β-strands. The first six blades run from β2–β5, β6–β9, β10–β13, β14–β17, β18–β21, and β22–β25 continuously, and the last and seventh blade contains β26–β28 and β1 from the N-terminal end to complete the fold, forming a stabilized ring-like structure .The structure shows a canonical seven-bladed WD40 domain architecture B, with a patches C. A stru patches  revealed patches , the apo patches , the ana patches , and the patches  . Previous structural and biochemical studies have indicated that one surface (top) of the WD40 domain is more frequently used for protein–protein interactions than the opposing surface . The dimerization interface is large and buries approximately ∼1,200 Å2 surface area per monomer. Residues that bury more than 40 Å surface areas are marked . In addition to surface area burial, a number of potential hydrogen bonding and salt bridge interactions exist, among D2388 of one subunit and H2391 and S2345 of the symmetric subunit, among R2394 of one subunit and main chain atoms of M2408 and Y2410 of the symmetric subunit, and between E2395 of one subunit and Y2346 of the symmetric subunit  A. The in subunit B.Given the dimeric association of the WD40 domain in the crystal, we determined whether it also forms dimers in solution. We used multiangle light scattering (MALS) to measure the molecular mass of the MBP-tagged LRRK2 WD40 domain as it eluted from an in-line size exclusion column. The calculated molecular mass of an MBP-tagged LRRK2 WD40 domain monomer is roughly ∼83 kDa. The MALS measurement showed that the LRRK2 WD40 domain peak when injected at 2 mg/mL corresponded to 164.0 kDa (2% error) in molecular mass, corresponding to a dimer C. TherefSI Appendix, Fig. S2) and investigated whether mutations on these residues affect the dimerization of the LRRK2 WD40 domain in solution . Using gel filtration chromatography and matched with concentrations, WD40 domain mutants L2343D, F2344A, S2345D, R2394E, E2395R, and S2409A were shown to interfere with dimerization, while M2408E had little influence on dimerization. The D2388K and H2391D mutants did not express well. Consistent with the above analysis, the gel filtration elution positions of the R2394E mutant showed delays relative to the wild-type (WT) at all four tested concentrations .We next identified residues that may be important for dimerization based on the structure (www.molgen.ua.ac.be/PDmutDB/) . Similarly, D2175H, T2356I, and L2439I also compromised dimerization likely because they altered the protein structure, while Y2189C did not affect the dimer interaction . The R2143H variant did not express well. A recent study reported three additional WD40 variants N2308D, N2313S, and S2350I in the mainland Chinese population  H. Y2189CDmutDB/) , T2356I DmutDB/) , and L24DmutDB/)  are buriDmutDB/)  and R214DmutDB/)  are mutaDmutDB/)  and V239DmutDB/)  mutants pulation H.SI Appendix, Table S3). LRRK2 autophosphorylation on S1292 . In contrast, in the G2019S background, the LRRK2 WD40 domain mutations exerted similar effects as in the WT background, with H2391D, R2394E, and the disease-related G2385R moderately increasing Rab10 phosphorylation . It has been shown previously that G2019S mutation alone does not change Rab10 phosphorylation . Of note, previous studies reported that G2385R did not lead to an obvious change in autophosphorylation . Consistent with previous work, the R1441G mutation considerably suppressed Ser935 phosphorylation . We also investigated the remaining WD40 domain disease-associated variants on LRRK2 kinase activity using Rab10 phosphorylation. In contrast to G2385R, these variants did not cause a significant difference in Rab10 phosphorylation .Because R1441G and G2019S are two recurrent PD variants, we also generated the same sets of LRRK2 WD40 domain mutations in these backgrounds. In the R1441G background, addition of the LRRK2 WD40 domain mutations did not further alter Rab10 phosphorylation . The blade V surface partially overlaps with the WD40 dimerization interface, and it is likely that this surface, as well as the conserved top surface, is used for additional intramolecular or intermolecular interactions to regulate LRRK2 function.A completely different scenario is that the surface on blade V, identified by mutants H2391D, R2394E, and the disease-related G2385R, or the conserved top surface on blade III and IV D, may beWhile further studies will be required to dissect these scenarios to elucidate the exact role of the WD40 domain and its dimerization in the function of LRRK2, our data clearly showed moderately increased LRRK2 kinase activity for the PD-associated G2385R variant. Hence, patients with WD40 mutations that are relatively common in certain Asian populations might benefit from receiving LRRK2 kinase inhibitors. These data also support the general concept that like all other LRRK2 pathogenic mutations, WD40 mutations also result in activation of LRRK2 and promote Rab10 phosphorylation.Human LRRK2 WD40 domain (residues 2142–2527) was expressed in Sf9 insect cells via baculovirus infection and purified to homogeneity.www.wwpdb.org [PDB ID codes 6DLO (50) and 6DLP (51)].The LRRK2 WD40 domain was crystallized using hanging drop vapor diffusion. The structure was determined by anomalous diffraction phasing and refined to 2.6-Å resolution. The atomic coordinates and structure factors have been deposited in the Protein Data Bank, To measure the molecular mass of the WD40 domain protein in solution, we used a three-angle light scattering detector and a refractive index detector, which were coupled to a chromatography system.Proteins were electrophoretically transferred onto nitrocellulose membrane. After incubation with antibodies, protein bands were acquired via near infrared fluorescent detection using Odyssey CLx imaging.Supplementary File