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Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies. [ABSTRACT FROM AUTHOR]

CC chemokine ligand 5 (CCL5) and CCL3 are critical for immune surveillance and inflammation. Consequently, they are linked to the pathogenesis of many inflammatory conditions and are therapeutic targets. Oligomerization and glycosaminoglycan (GAG) binding of CCL5 and CCL3 are vital for the functions of these chemokines. Our structural and biophysical analyses of human CCL5 reveal that CCL5 oligomerization is a polymerization process in which CCL5 forms rod-shaped, double-helical oligomers. This CCL5 structure explains mutational data and offers a unified mechanism for CCL3, CCL4, and CCL5 assembly into high-molecular-weight, polydisperse oligomers. A conserved, positively charged BBXB motif is key for the binding of CC chemokines to GAG. However, this motif is partially buried when CCL3, CCL4, and CCL5 are oligomerized; thus, the mechanism by which GAG binds these chemokine oligomers has been elusive. Our structures of GAG-bound CCL5 and CCL3 oligomers reveal that these chemokine oligomers have distinct GAG-binding mechanisms. The CCL5 oligomer uses another positively charged and fully exposed motif, KKWVR, in GAG binding. However, residues from two partially buried BBXB motifs alongwith other residues combine to form a GAG-binding groove in the CCL3 oligomer. The N termini of CC chemokines are shown to be involved in receptor binding and oligomerization. We also report an alternative CCL3 oligomer structure that reveals how conformational changes in CCL3 N termini profoundly alter its surface properties and dimer-dimer interactions to affect GAG binding and oligomerization. Such complexity in oligomerization and GAG binding enables intricate, physiologically relevant regulation of CC chemokine functions. [ABSTRACT FROM AUTHOR]

CC chemokine ligands (CCLs) are 8- to 14-kDa signaling proteins involved in diverse immune functions. While CCLs share similar tertiary structures, oligomerization produces highly diverse quaternary structures that protect chemokines from proteolytic degradation and modulate their functions. CCL18 is closely related to CCL3 and CCL4 with respect to both protein sequence and genomic location, yet CCL18 has distinct biochemical and biophysical properties. Here, we report a crystal structure of human CCL18 and its oligomerization states in solution based on crystallographic and small-angle X-ray scattering analyses. Our data show that CCL18 adopts an α-helical conformation at its N-terminus that weakens its dimerization, explaining CCL18's preference for the monomeric state. Multiple contacts between monomers allow CCL18 to reversibly form a unique open-ended oligomer different from those of CCL3, CCL4, and CCL5. Furthermore, these differences hinge on proline 8, which is conserved in CCL3 and CCL4 but is replaced by lysine in human CCL18. Our structural analyses suggest that a mutation of proline 8 to alanine stabilizes a type 1 β-turn at the N-terminus of CCL4 to prevent dimerization but prevents dimers from making key contacts with each other in CCL3. Thus, the P8A mutation induces depolymerization of CCL3 and CCL4 by distinct mechanisms. Finally, we used structural, biochemical, and functional analyses to unravel why insulin-degrading enzyme degrades CCL3 and CCL4 but not CCL18. Our results elucidate the molecular basis for the oligomerization of three closely related CC chemokines and suggest how oligomerization shapes CCL chemokine function. [ABSTRACT FROM AUTHOR]

Human presequence protease (hPreP) is an M16 metalloprotease localized in mitochondria. There, hPreP facilitates proteostasis by utilizing an ~13,300-Å3 catalytic chamber to degrade a diverse array of potentially toxic peptides, including mitochondrial presequences and ∖-amyloid (A∖), the latter of which contributes to Alzheimer disease pathogenesis. Here, we report crystal structures for hPreP alone and in complex with A∖, which show that hPreP uses size exclusion and charge complementation for substrate recognition. These structures also reveal hPreP-specific features that permit a diverse array of peptides, with distinct distributions of charged and hydrophobic residues, to be specifically captured, cleaved, and have their amyloidogenic features destroyed. SAXS analysis demonstrates that hPreP in solution exists in dynamic equilibrium between closed and open states, with the former being preferred. Furthermore, A∖ binding induces the closed state and hPreP dimerization. Together, these data reveal the molecular basis for flexible yet specific substrate recognition and degradation by hPreP. [ABSTRACT FROM AUTHOR]

Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid β (Aβ). Thus, IDE retards the progression of Alzheimer's disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ~18-Å opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into β-strands for their polymerization into amyloid fibrils, they also use such β-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Aβ, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N- and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides. [ABSTRACT FROM AUTHOR]

Zinc metalloprotease 1 (Zmp1), a Mycobacterium tuberculosis 75 kDa secreted enzyme, mediates key stages of tuberculosis disease progression. The biological activity of Zmp1 presumably stems from its ability to degrade bacterium- and/or host-derived peptides. The crystal structures of Zmp1 and related M13 metalloproteases, such as neprilysin and endothelin-converting enzyme-1 were determined only in the closed conformation, which cannot capture substrates or release proteolytic products. Thus, the mechanisms of substrate binding and selectivity remain elusive. Here we report two open-state cryo-EM structures of Zmp1, revealed by our SAXS analysis to be the dominant states in solution. Our structural analyses reveal how ligand binding induces a conformational switch in four linker regions to drive the rigid body motion of the D1 and D2 domains, which form the sizable catalytic chamber. Furthermore, they offer insights into the catalytic cycle and mechanism of substrate recognition of M13 metalloproteases for future therapeutic innovations. [Display omitted] • Cryo-EM analysis reveals open-state structures of M. tuberculosis Zmp1 • SEC-SAXS reveals a substrate-induced transition from open to closed state • Structural analysis reveals the mechanism of the open-closed transition • Electrostatic analysis suggests a mechanism for M13 protease substrate recognition M13 family enzymes must transition between open and closed states to degrade their substrates. Liang et al. utilized cryo-EM and SEC-SAXS to determine the mechanistic basis for this transition in Zmp1, an M13 protease from Mycobacterium tuberculosis , and propose a unified model for M13 family substrate recognition. [ABSTRACT FROM AUTHOR]

Presequence protease (PreP) is a proteostatic enzyme that plays a key role in the maintenance of mitochondrial health. Defects in PreP stability are associated with neurological disorders in humans, and altered activity of this enzyme modulates the progress of Alzheimer's disease-like pathology in mice. As agonists that boost PreP proteolytic activity represent a promising therapeutic avenue, we sought to determine the structural basis for the action of benzimidazole derivatives (3c and 4c), first reported by Vangavaragu et al. (Eur. J. Med. Chem. 76 (2014) 506–516) that enhance the activity of PreP. However, we found the published procedure for the synthesis of 3c yielded aldimine A instead. We then developed an alternative synthesis and obtained 3c , termed compound C, and an alternative benzimidazole derivative, termed compound B. We tested compounds A , B and C for their ability to enhance the activities of human PreP. In contrast to the previous report, we observed that none of the compounds A , B , or C (3c) modulated the catalytic activity of human PreP. Here we report our findings on the mis-identification of the reported benzimidazoles and the lack of biological activity of such compounds on human PreP. Thus, PreP modulators for PreP-based therapies remain to be discovered. Image 1 • Fail to replicate synthesis of benzimidazole derivatives reported as PreP enhancers. • Report a new method for synthesis and structure validation of such compounds. • Reassign structures of incorrectly reported small benzimidazole derivatives. • Such small benzimidazole derivatives were not found to enhance PreP activity. [ABSTRACT FROM AUTHOR]

When a tunnel passes through weak and fragmented surrounding rock, excavation disturbance can lead to significant deformation. To mitigate this, advanced support measures like the umbrella arch are necessary. This study investigates the effectiveness of the umbrella arch in controlling deformation of the surrounding rock, focusing on the super shallow-buried spillway section of the Shenzhen East Transit Expressway in China. Both numerical modeling and field monitoring were used to assess the support effect of the umbrella arch. The mechanism of the support was analyzed, revealing that the umbrella arch primarily prevents collapse of the surrounding rock by creating a small compression area. It is recommended that a starting segment with the same length as the tunnel diameter be reserved for the construction of long umbrella arches to maximize their supporting effect. The proposed numerical model was calibrated using field monitoring data, and the mechanical responses of an 80-m-long umbrella arch during construction were thoroughly analyzed. The research results presented in this paper can serve as a reference for the design of umbrella arches in similar projects in the future. [ABSTRACT FROM AUTHOR]

The spindle-shaped virus SSV1 of the hyperthermophilic archaeon Sulfolobus shibatae encodes an integrase (SSV1 Int). Here, the crystal structure of the C-terminal catalytic domain of SSV1 Int is reported. This is the first structural study of an archaeal tyrosine recombinase. Structural comparison shows that the C-terminal domain of SSV1 Int possesses a core fold similar to those of tyrosine recombinases of both bacterial and eukaryal origin, apart from the lack of a conserved helix corresponding to αI of Cre, indicating conservation of these enzymes among all three domains of life. Five of the six catalytic residues cluster around a basic cleft on the surface of the structure and the nucleophile Tyr314 is located on a flexible loop that stretches away from the central cleft, supporting the possibility that SSV1 Int cleaves the target DNA in a trans mode. Biochemical analysis suggests that the N-terminal domain is responsible for the dimerization of SSV1 Int. The C-terminal domain is capable of DNA cleavage and ligation, but at efficiencies significantly lower than those of the full-length protein. In addition, neither the N-terminal domain alone nor the C-terminal domain alone shows a strong sequence preference in DNA binding. Therefore, recognition of the core-type sequence and efficient catalysis by SSV1 Int presumably requires covalent linkage and interdomain communication between the two domains. [ABSTRACT FROM AUTHOR]

[Display omitted] • Degradation of Aβ by E111Q-IDE via a non-chaperonin mechanism is demonstrated. • Zinc binding to Aβ was found to form aggregates that do not fit to the IDE's catalytic cavity. • Zinc binding to Aβ inactivates E111Q-IDE's catalytic function, whereas zinc removal restores its function. • E111Q-IDE presented a reduced activity on Aβ aggregation kinetics when compared with the wild-type IDE. It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimer's amyloid-β (Aβ). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on Aβ degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of Aβ(1–40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of Aβ(1–40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of Aβ fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length Aβ(1–40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to Aβ(1–40) inactivates cf-E111Q-IDE's catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on Aβ degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDE's function. [ABSTRACT FROM AUTHOR]

Insulin-degrading enzyme, IDE, is a metalloprotease implicated in the metabolism of key peptides such as insulin, glucagon, β-amyloid peptide. Recent studies have pointed out its broader role in the cell physiology. In order to identify new drug-like inhibitors of IDE with optimal pharmacokinetic properties to probe its multiple roles, we ran a high-throughput drug repurposing screening. Ebselen, cefmetazole and rabeprazole were identified as reversible inhibitors of IDE. Ebselen is the most potent inhibitor (IC 50 (insulin) = 14 nM). The molecular mode of action of ebselen was investigated by biophysical methods. We show that ebselen induces the disorder of the IDE catalytic cleft, which significantly differs from the previously reported IDE inhibitors. IDE inhibition by ebselen can explain some of its reported activities in metabolism as well as in neuroprotection. Image 1 [ABSTRACT FROM AUTHOR]

Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the “elbow” regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab–antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones. [ABSTRACT FROM AUTHOR]

Insulin degrading enzyme (IDE) is a zinc metalloprotease that degrades small amyloid peptides such as amyloid-â and insulin. So far the dearth of IDE-specific pharmacological inhibitors impacts the understanding of its role in the physiopathology of Alzheimer's disease, amyloid-â clearance, and its validation as a potential therapeutic target. Hit 1 was previously discovered by high-throughput screening. Here we describe the structure-activity study, that required the synthesis of 48 analogues. We found that while the carboxylic acid, the imidazole and the tertiary amine were critical for activity, the methyl ester was successfully optimized to an amide or a 1,2,4-oxadiazole. Along with improving their activity, compounds were optimized for solubility, lipophilicity and stability in plasma and microsomes. The docking or co-crystallization of some compounds at the exosite or the catalytic site of IDE provided the structural basis for IDE inhibition. The pharmacokinetic properties of best compounds 44 and 46 were measured in vivo . As a result, 44 (BDM43079) and its methyl ester precursor 48 (BDM43124) are useful chemical probes for the exploration of IDE's role. [ABSTRACT FROM AUTHOR]

Fucokinase/L-fucose-1-P-guanylyltransferase (FKP) is a bifunctional enzyme which converts L-fucose to Fuc-1-P and thence to GDP-L-fucose through a salvage pathway. The molecular weights of full-length FKP (F-FKP) and C-terminally truncated FKP (C-FKP, residues 300-949) are 105.7 and 71.7 kDa, respectively. In this study, both recombinant F-FKP and C-FKP were expressed and purified. Size-exclusion chromatography experiments and analytical ultracentrifugation results showed that both F-FKP and C-FKP are trimers. Native F-FKP protein was crystallized by the vapour-diffusion method and the crystals belonged to space group P212121 and diffracted synchrotron X-rays to 3.7 Å resolution. The crystal unit-cell parameters are a = 91.36, b = 172.03, c = 358.86 Å, α = β = γ = 90.00°. The three-dimensional features of the F-FKP molecule were observed by cryo-EM (cryo-electron microscopy). The preliminary cryo-EM experiments showed the F-FKP molecules as two parallel disc-shaped objects stacking together. Combining all results together, it is assumed that there are six FKP molecules in one asymmetric unit, which corresponds to a calculated Matthews coefficient of 2.19 Å3 Da−1 with 43.83% solvent content. These preliminary crystallographic and cryo-EM microscopy analyses provide basic structural information on FKP. [ABSTRACT FROM AUTHOR]

The concentration of total suspended matter (TSM) is an important parameter for evaluating lake water quality. We determined in situ hyperspectral data and TSM concentration data for Changdang Lake, China, to establish a TSM concentration inversion model. The model was applied using 60 Sentinel-2 images acquired from 2016 to 2021 to determine the temporal and spatial distribution of TSM concentration. Remote sensing images were also utilized to monitor the effect of ecological dredging in Changdang Lake. The following results were obtained: (1) Compared with four existing models, the TSM concentration inversion model established in this study exhibited higher accuracy and was suitable for Changdang Lake. (2) TSM concentrations obtained for the period 2016–2021 were higher in spring and summer, and lower in autumn and winter. (3) The dredging process influenced a small area of the surrounding water body, resulting in higher TSM concentrations. However, a subsequent reduction in TSM concentrations indicated that the ecological dredging project might improve the water quality of Changdang Lake to a considerable extent. Therefore, it was inferred that the use of Sentinel-2 images was effective for the long-term monitoring of water quality changes in small and medium-sized lakes. [ABSTRACT FROM AUTHOR]

Increasing evidences suggest that the type I interferon α (IFNα) plays a critical role in the etiopathogenesis of systemic lupus erythematosus (SLE), which makes it a promising therapeutic target for the treatment of the disease. By screening a large size non-immune human antibody library, we have developed a human single-chain antibody (ScFv) AIFNα1bScFv01 and corresponding whole antibody AIFNα1bIgG01 to human interferon α1b (IFNα1b) with high specificity and high affinity. The IgG antibody could down-regulate the expression of ISG15 and IFIT-1 induced by either recombinant IFNα1b or naïve IFNα from SLE patients' sera, and reduced total serum IgG and IgM antibodies level in a pristane-primed lupus-like mouse model. The crystal structure of AIFNα1bScFv01-IFNα1b complex solved to 2.8 Å resolution revealed that both Pro26-Gln40 region in loop AB and Glu147-Arg150 region in helix E of IFNα1b contribute to binding with AIFNα1bScFv01. Four residues of above two regions (Leu30, Asp32, Asp35 and Arg150) are critical for the formation of antigen-antibody complexes. AIFNα1bScFv01 shares partial epitopes of IFNα1b with its receptor IFNAR2 but with much higher binding affinity to IFNα1b than IFNAR2. Thus, AIFNα1bIgG01 exhibits its neutralizing activity through competition with IFNAR2 to bind with IFNα and prevents the activation of IFNα-mediated signaling pathway. Our results highlight the potential use of the human antibody for modulating the activity of IFNα in SLE. [ABSTRACT FROM AUTHOR]

A subset of tumour necrosis factor receptor (TNFR) superfamily members contain death domains in their cytoplasmic tails. Death receptor 6 (DR6) is one such member and can trigger apoptosis upon the binding of a ligand by its cysteine-rich domains (CRDs). The crystal structure of the ectodomain (amino acids 1-348) of human death receptor 6 (DR6) encompassing the CRD region was phased using the anomalous signal from S atoms. In order to explore the feasibility of S-SAD phasing at longer wavelengths (beyond 2.5 Å), a comparative study was performed on data collected at wavelengths of 2.0 and 2.7 Å. In spite of sub-optimal experimental conditions, the 2.7 Å wavelength used for data collection showed potential for S-SAD phasing. The results showed that the Rano/ Rp.i.m. ratio is a good indicator for monitoring the anomalous data quality when the anomalous signal is relatively strong, while d′′/sig( d′′) calculated by SHELXC is a more sensitive and stable indicator applicable for grading a wider range of anomalous data qualities. The use of the `parameter-space screening method' for S-SAD phasing resulted in solutions for data sets that failed during manual attempts. SAXS measurements on the ectodomain suggested that a dimer defines the minimal physical unit of an unliganded DR6 molecule in solution. [ABSTRACT FROM AUTHOR]