Amyloid-like Fibrils Research Articles

Zinc Metal Ion Affected the Structural Stability of Amyloid-Like Nanofibrils

Synthetic peptides that self-assemble into well-defined structures with a cross-β arrangement are called amyloid-like fibrils. Amyloids are associated with a list of disorders and neuro-degenerative diseases, such as Alzheimer's and Parkinson`s disease. We previously showed that amyloid-like nanofibrils with a repeating motif “IHIH” were functional fibrils. They were able to bind a metal ion through imidazole moieties and mimic the native carbonic anhydrase enzyme by hydrolysing the CO2 molecule. Thus, these synthetic amyloid fibrils were suggest-ed to be good candidates to moderate and update the modern enzymatic molecules. This study aims to shed a light on the stability of these amyloid nanofibrils over a study period of 25 days, in the presence/absence of a metal ion. The work continued for approximately 7 months in the Biochemistry department, School of Life Sciences at the University of Sussex in the United Kingdom. A set of designed peptides with a repeating motif “IHIH” were ex-plored, based on some structural studies. Short and long peptides with free ends as well as closed ends were investigated. Peptides allowed to self-assemble with and without a metal ion (zinc) were then examined using circular dichroism, fluorimetry and electron microscopy for structural biophysical analysis. Regardless of the metal ion contribution, peptides showed stable secondary structures with a -sheet conformation for the incubation time of 25 days. Their morphologies did not appear to change over time. However, the presence of a zinc ion has an effect on the secondary structure of the mature fibrils. Results indicated that fibrils grown with the zinc ion have a significantly higher propensity to form -sheets secondary structures during incubation time. The presence of a zinc ion also affected the dimensions of the amyloid-like fibrils by the end of the study course, at which point they significantly re-duced. This effect of zinc ion on synthetic amyloid fibrils has not been previously reported. The stabilities of the zinc-nanofibrils point to their potential for use in modifying or updating the enzyme-mimic analytical reactions. The effect of adding zinc on the fibrillation seems to be crucial. Although it apparently improved the -sheet assembly, it affected the width/length of the synthetic amyloids. This effect could be promising toward reducing the generation of amyloid fibrils and ultimately understanding the pathogenesis of Alzheimer disease.

Cytoplasmic TDP-43 De-mixing Independent of Stress Granules Drives Inhibition of Nuclear Import, Loss of Nuclear TDP-43, and Cell Death

While cytoplasmic aggregation of TDP-43 is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia, how aggregates form and what drives its nuclear clearance have not been determined. Here we show that TDP-43 at its endogenous level undergoes liquid-liquid phase separation (LLPS) within nuclei in multiple cell types. Increased concentration of TDP-43 in the cytoplasm or transient exposure to sonicated amyloid-like fibrils is shown to provoke long-lived liquid droplets of cytosolic TDP-43 whose assembly and maintenance are independent of conventional stress granules. Cytosolic liquid droplets of TDP-43 accumulate phosphorylated TDP-43 and rapidly convert into gels/solids in response to transient, arsenite-mediated stress. Cytoplasmic TDP-43 droplets slowly recruit importin-α and Nup62 and induce mislocalization of RanGap1, Ran, and Nup107, thereby provoking inhibition of nucleocytoplasmic transport, clearance of nuclear TDP-43, and cell death. These findings identify a neuronal cell death mechanism that can be initiated by transient-stress-induced cytosolic de-mixing of TDP-43.

Cyclized NDGA modifies dynamic \u03b1-synuclein monomers preventing aggregation and toxicity

Growing evidence implicates α-synuclein aggregation as a key driver of neurodegeneration in Parkinson’s disease (PD) and other neurodegenerative disorders. Herein, the molecular and structural mechanisms of inhibiting α-synuclein aggregation by novel analogs of nordihydroguaiaretic acid (NDGA), a phenolic dibenzenediol lignan, were explored using an array of biochemical and biophysical methodologies. NDGA analogs induced modest, progressive compaction of monomeric α-synuclein, preventing aggregation into amyloid-like fibrils. This conformational remodeling preserved the dynamic adoption of α-helical conformations, which are essential for physiological membrane interactions. Oxidation-dependent NDGA cyclization was required for the interaction with monomeric α-synuclein. NDGA analog-pretreated α-synuclein did not aggregate even without NDGA-analogs in the aggregation mixture. Strikingly, NDGA-pretreated α-synuclein suppressed aggregation of naïve untreated aggregation-competent monomeric α-synuclein. Further, cyclized NDGA reduced α-synuclein-driven neurodegeneration in Caenorhabditis elegans. The cyclized NDGA analogs may serve as a platform for the development of small molecules that stabilize aggregation-resistant α-synuclein monomers without interfering with functional conformations yielding potential therapies for PD and related disorders.

Macrophage-Mediated Phagocytosis and Dissolution of Amyloid-Like Fibrils in Mice, Monitored by Optical Imaging

Light chain-associated amyloidosis is characterized by the extracellular deposition of amyloid fibrils in abdominothoracic organs, skin, soft tissue, and peripheral nerves. Phagocytic cells of the innate immune system appear to be ineffective at clearing the material; however, human light chain amyloid extract, injected subcutaneously into mice, is rapidly cleared in a process that requires neutrophil activity. To better elucidate the phagocytosis of light chain fibrils, a potential method of cell-mediated dissolution, amyloid-like fibrils were labeled with the pH-sensitive dye pHrodo red and a near infrared fluorophore. After injecting this material subcutaneously in mice, optical imaging was used to quantitatively monitor phagocytosis and dissolution of fibrils concurrently. Histologic evaluation of the residual fibril masses revealed the presence of CD68+, F4/80+, ionized calcium binding adaptor molecule 1- macrophages containing Congo red-stained fibrils as well as neutrophil-associated proteins with no evidence of intact neutrophils. These data suggest an early infiltration of neutrophils, followed by extensive phagocytosis of the light chain fibrils by macrophages, leading to dissolution of the mass. Optical imaging of this novel murine model, coupled with histologic evaluation, can be used to study the cellular mechanisms underlying dissolution of synthetic amyloid-like fibrils and human amyloid extracts. In addition, it may serve as a test bed to evaluate investigational opsonizing agents that might serve as therapeutic agents for lightchain-associated amyloidosis.

Residue-Specific Solvation-Directed Thermodynamic and Kinetic Control over Peptide Self-Assembly with 1D/2D Structure Selection.

Understanding the self-organization and structural transformations of molecular ensembles is important to explore the complexity of biological systems. Here, we illustrate the crucial role of cosolvents and solvation effects in thermodynamic and kinetic control over peptide association into ultrathin Janus nanosheets, elongated nanobelts, and amyloid-like fibrils. We gained further insight into the solvation-directed self-assembly (SDSA) by investigating residue-specific peptide solvation using molecular dynamics modeling. We proposed the preferential solvation of the aromatic and alkyl domains on the peptide backbone and protofibril surface, which results in volume exclusion effects and restricts the peptide association between hydrophobic walls. We explored the SDSA phenomenon in a library of cosolvents (protic and aprotic), where less polar cosolvents were found to exert a stronger influence on the energetic balance at play during peptide propagation. By tailoring cosolvent polarity, we were able to achieve precise control of the peptide nanostructures with 1D/2D shape selection. We also illustrated the complexity of the SDSA system with pathway-dependent peptide aggregation, where two self-assembly states (i.e., thermodynamic equilibrium state and kinetically trapped state) from different sample preparation methods were obtained.

Allura red rapidly induces amyloid-like fibril formation in hen egg white lysozyme at physiological pH.

Allura red (AR) is an artificial azo dye mostly used in food industries and has potential health risks. We examined the role of AR in amyloidogenesis using hen egg white lysozyme (HEWL) at pH 7.0. The amyloidogenic induction properties of AR in HEWL were identified by circular dichroism (CD), turbidity, intrinsic fluorescence, light scattering, transmission electron microscopy (TEM), and molecular dynamic simulation studies. Turbidity and light scattering measurements showed that HEWL becomes aggregated in the presence of 0.03-15.0 mM of AR at pH 7.0 but not at very low AR concentrations (0.01-0.28 mM). However, AR-induced aggregation is a kinetically rapid process, with no observable lag phase and saturation within 6 s. The kinetics results suggested that the HEWL aggregation induced by AR is very rapid. The CD results demonstrated that the total β-sheet content of HEWL was increased in the AR treated samples. The TEM results are established that AR-induced aggregates had amyloid-like structures. Molecular dynamics simulations analysis showed that the bound AR-HEWL structures were highly favored compared to unbound structures. The mechanism of AR-induced amyloid fibril formation may involve electrostatic, hydrogen bonding, and hydrophobic interactions.

Hydrogel Formation by an Aromatic Analogue of a β-Amyloid Fragment, Aβ16–22: A Scaffold for 3D Cell Culture

A short 7-residue stretch from β-amyloid, Aβ16–22 (Ac-KLVFFAE-am), yields typical amyloid-like fibrils at neutral pH. The Phe–Phe cassette present in the peptide is believed to be critical for its self-assembly. We report that the aromatic analogue Aβ16–22(F20Y) forms self-supporting soft gels at concentrations ≥2 mM even though the end-capped parent peptide does not form hydrogel up to 20 mM (1.8% w/w) concentration. The hydrogel is made up of distinct amyloid-like fibers. The storage modulus of 20 mM gel is ∼3–5-fold higher than the loss modulus in the 2–3000 rad/s angular frequency range, indicating distinct elastic properties. The hydrogel supports the growth of rat pancreatic cells (RIN-5F), human embryonic kidney cells (HEK-293), baby hamster kidney cells (BHK-21), and human neuroblastoma cells (IMR-32). The cells grow in clusters as is anticipated in a three-dimensional matrix. The rat pancreatic cells produced insulin, suggesting that they are functional inside the gel.

Ubiquitin binds the amyloid β peptide and interferes with its clearance pathways.

Several lines of evidence point to a compromised proteostasis associated with a reduction of the Ubiquitin Proteasome System (UPS) activity in patients affected by Alzheimer's Disease (AD) and suggest that the amyloid β peptide (Aβ) is an important player in the game. Inspired also by many reports, underlining the presence of ubiquitin (Ub) in the amyloid plaques of AD brains, here we set out to test whether Ub may bind the Aβ peptide and have any effect on its clearance pathways. By using an integrated array of MALDI-TOF/UPLC-HRMS, fluorescence, NMR, SPR, Microscale Thermophoresis (MST) and molecular dynamics studies, we consistently demonstrated that Aβ40 binds Ub with a 1 : 1 stoichiometry and K d in the high micromolar range. In particular, we show that the N-terminal domain of the Aβ peptide (through residues D1, E3 and R5) interacts with the C-terminal tail of Ub (involving residues K63 and E64), inducing the central region of Aβ (14HQKLVFFAEDVGSNK28) to adopt a mixed α-helix/β-turn structure. ELISA assays, carried out in neuroblastoma cell lysates, suggest that Aβ competitively binds Ub also in the presence of the entire pool of cytosolic Ub binding proteins. Ub-bound Aβ has a lower tendency to aggregate into amyloid-like fibrils and is more slowly degraded by the Insulin Degrading Enzyme (IDE). Finally, we observe that the water soluble fragment Aβ1-16 significantly inhibits Ub chain growth reactions. These results evidence how the non-covalent interaction between Aβ peptides and Ub may have relevant effects on the regulation of the upstream events of the UPS and pave the way to future in vivo studies addressing the role played by Aβ peptide in the malfunction of proteome maintenance occurring in AD.

Inhibition of the Formation of Amyloid-Like Fibrils with Spices, Especially Cloves

During the study of inhibition of amyloid fibril formation, a-chymotrypsin protein was developed in 55% ethanol at pH 7.0. We investigated the inhibitory effect of different spices on amyloid fibril formation using turbidity measurements and Congo red binding assays. We found that all spices except the black pepper and caraway seed prevented fibril formation. The highest inhibition was measured with the clove, which reduced the amount of aggregates by 90%. We studied the inhibitory effect of the cloves at different concentrations on aggregation, it was found that the inhibitory activity of clove is dependent on concentration. We have measured the total phenolic content of the spice extracts too. Based on all these findings we have come to the following conclusion: Our results indicate that spices can contain other compounds too — not only phenolic compounds — which influence the formation of amyloid fibrils, and the effectiveness of various phenolic compounds are different.

Eduscho Coffee Extract Effectively Inhibits the Formation of Amyloid-like Fibrils by Trypsin in Aqueous Ethanol

In this work we used an in vitro trypsin aggregation model to show that certain commercial coffee extracts can inhibit protein aggregation. Aggregation experiments were performed using several spectroscopic methods and a dye binding assay, such as turbidity, Congo red (CR) and electronic circular dichroism (ECD), that was further supported by transmission electron microscopy (TEM). A correlation was found between the anti-aggregation properties and the total phenolic content of the coffee extracts. The results revealed that the greatest effect was exerted by the Eduscho coffee extract. It was found that the inhibitory effect of this extract was concentration dependent. Using size exclusion chromatography, we demonstrated that the inhibitory effect of the Eduscho coffee extract on the formation of amyloid-like fibrils was due to its capacity to stabilize the oligomeric form of the protein.

A C-type lectin from Bothrops leucurus snake venom forms amyloid-like aggregates in RPMI medium and are efficiently phagocytosed by peritoneal macrophages

Lectins are carbohydrate-binding proteins that play important roles in the immune system. Under specific conditions, lectins can form amyloids, proteinaceous aggregates rich in cross β-strand structures. A Ca++-dependent lectin, isolated from Bothrops leucurus snake venom (BLL) has demonstrated relevant biological activities such as antibacterial and antitumor activity. In this work, we aimed to study the interaction of BLL with macrophages. The formation of amyloid structures by BLL in a cell culture medium, the effects of the lectin on macrophage morphology and cytokine production were investigated. BLL amyloid-like fibrils in RMPI medium, pH 7.2, at 37 °C was confirmed by binding of Congo Red, Thioflavin T and electron microscopy. Neither binding of amyloid markers nor fibrillar structures were found when the lectin was incubated in RPMI plus galactose, the specific BLL-binding carbohydrate. Several phagocytic compartments containing fibrillar structures were observed in BLL-treated macrophages in RPMI medium for 24 h; these compartments showed an apple-green birefringence after Congo Red staining and were positive for thioflavin S and anti-amyloid antibody, indicating the presence of amyloid-like fibrils. No fibrillar material and no labeling were observed when the macrophages were treated with BLL plus galactose or cytochalasin B, an inhibitor of phagocytosis. BLL did not affect the viability of the cells. A significant release of proinflammatory (TNF-α, IL-6, INF-ϒ and IL-1β) and regulatory (IL-10) cytokines was observed in BLL-treated macrophages. Taken together, our results shed light on the structural organization of BLL, improving knowledge about the interaction of lectin with macrophages. The phagocytosis of amyloid-like aggregates together with the proinflammatory response induced by BLL may open new perspectives for the use of this lectin as an interesting model to study cytokines and the production of other mediators as well as understand the mechanisms occurring in human immune cells during amyloid protein deposition.

Amyloid-like Fibrils from a Diphenylalanine Capped with an Aromatic Fluorenyl.

The self-assembly behavior of a diphenylalanine amphiphile blocked at the C-terminus with a 9-fluorenylmethyl ester and stabilized at the N-terminus with a trifluoroacetate (TFA) anion, TFA·FF-OFm, has been examined. At low peptide concentration (0.5 mg/mL), long amyloid-like fibrils, which come from the fusion of two or more helical ribbons and/or thinner fibrils, organized in bundles or as individual entities are detected. Microbeam synchrotron radiation infrared spectroscopy has shown that TFA·FF-OFm molecules in amyloid-like fibrils arrange, forming antiparallel β-sheets. Alteration of the experimental conditions to prioritize the thermodynamic contribution with respect to the kinetic one in the self-assembly process inhibits the organization of amyloid-like structures in favor of the formation of conventional fibrous structures. On the basis of experimental observations, a structural model where the individual antiparallel β-sheets are oriented in parallel has been proposed for TFA·FF-OFm amyloid-like fibrils.

Changing times: Fluorescence-lifetime analysis of amyloidogenic SF-IAPP fusion protein

In a number of conformational diseases, intracellular accumulation of proteins bearing non-native conformations occurs. The search for compounds that are capable of hindering the formation and accumulation of toxic protein aggregates and fibrils is an urgent task. Present fluorescent methods of fibrils’ detection prevent simple real-time observations. We suppose to use green fluorescent protein fused with target protein and fluorescence lifetime measurement technique for this purpose.The recombinant proteins analyzed were produced in E. coli. Mass spectrometry was used for the primary structure of the recombinant proteins and post-translational modifications identification. The fluorescence lifetime of the superfolder green fluorescent protein (SF) and the SF protein fused with islet amyloid polypeptide (SF-IAPP) were studied in polyacrylamide gel using Fluorescent-Lifetime Imaging Microscopy (FLIM).It was shown that the SF average fluorescence lifetime in gel slightly differs from that of the SF-IAPP monomer under these conditions. SF-IAPP does not lose the ability to form amyloid-like fibrils. Under the same conditions (in polyacrylamide gel), SF and SF-IAPP monomers have similar fluorescence time characteristics and the average fluorescence lifetime of SF-IAPP in fibrils significantly decreases.We propose the application of FLIM to the measurement of average fluorescence lifetimes of fusion proteins (amyloidogenic protein-SF) in the context of studies using cellular models of conformational diseases.

Grapefruit Seed Extract Inhibits the Formation of Amyloid-like Fibrils by Trypsin in Aqueous Ethanol

Several natural compounds deriving from plants are known to be efficient anti-amyloid aggregation agents. In this study, anti-aggregation activity of grapefruit seed extract was investigated using trypsin as a model protein in aqueous ethanol at pH 7.0. Using turbidity measurement, Congo red (CR) binding assay, electronic circular dichroism (ECD) and transmission electron microscopy (TEM), we found that grapefruit seed extract has ability to inhibit trypsin amyloidlike fibril formation in vitro, and effectiveness increases with growing concentration of grapefruit seed extract. The total phenolic content of it was determined. The results showed that in addition to the polyphenolic compounds some other compounds are also responsible for the fibril formation inhibitory effect. We indicated it for the first time that limonin has anti-fibrillation effect.

The yeast GRASP Grh1 displays a high polypeptide backbone mobility along with an amyloidogenic behavior

GRASPs are proteins involved in cell processes that seem paradoxical: responsible for shaping the Golgi cisternae and involved in unconventional secretion mechanisms that bypass the Golgi. Despite its physiological relevance, there is still a considerable lack of studies on full-length GRASPs. Our group has previously reported an unexpected behavior of the full-length GRASP from the fungus C. neoformans: its intrinsically-disordered characteristic. Here, we generalize this finding by showing that it is also observed in the GRASP from S. cerevisae (Grh1), which strongly suggests it might be a general property within the GRASP family. Furthermore, Grh1 is also able to form amyloid-like fibrils either upon heating or when submitted to changes in the dielectric constant of its surroundings, a condition that is experienced by the protein when in close contact with membranes of cell compartments, such as the Golgi apparatus. Intrinsic disorder and fibril formation can thus be two structural properties exploited by GRASP during its functional cycle.

Methods for Structural Analysis of Amyloid Fibrils in Misfolding Diseases.

Many proteins and peptides are able to self-assemble in solution in vitro and in vivo to form amyloid-like fibrils. These fibrils share common structural characteristics. In order for a fibril to be characterized as amyloid, it is expected to fit certain criteria including the composition of cross-β. Here we describe how the formation of amyloid fibrils can be characterized in vitro using a variety of methods including circular dichroism and intrinsic tyrosine/tryptophan fluoresence to follow conformational changes; Thioflavin and/or ThS assembly to monitor nucleation and growth; transmission electron microscopy to visualize fibrillar morphology and X-ray fiber diffraction to examine cross-β structure.

Insulin adsorption onto zinc oxide nanoparticle mediates conformational rearrangement into amyloid-prone structure with enhanced cytotoxic propensity

BackgroundInjection localized amyloidosis is one of the most prevalent disorders in type II diabetes mellitus (TIIDM) patients relying on insulin injections. Previous studies have reported that nanoparticles can play a role in the amyloidogenic process of proteins. Hence, the present study deals with the effect of zinc oxide nanoparticles (ZnONP) on the amyloidogenicity and cytotoxicity of insulin. MethodsZnONP is synthesised and characterized using XRD, Zeta Sizer, UV-Visible spectroscope and TEM. The characterization is followed by ZnONP interaction with insulin, which is studied employing fluorescence spectroscopes, isothermal titration calorimetry and molecular dynamics simulations. The interaction leads insulin conformational rearrangement into amyloid-like fibril, which is studied using thioflavin T dye binding assay, circular dichroism spectroscopy and TEM, followed by cytotoxicity propensity using Alamar Blue dye reduction assay. ResultsInsulin has very weak interaction with ZnONP interface. Insulin at studied concentration forms amorphous aggregates at physiological pH, whereas in presence of ZnONP interface amyloid-like fibrils are formed. While the amyloid-like fibrils are cytotoxic to MIN6 and THP-1 cell lines, insulin and ZnONP individual solutions and their fresh mixtures enhance the cells proliferation. ConclusionsThe presence of ZnONP interface enhances insulin fibrillation at physiological pH by providing a favourable template for the nucleation and growth of insulin amyloids. General significanceThe studied protein-nanoparticle system from protein conformational dynamics point of view throws caution over nanoparticle use in biological applications, especially in vivo applications, considering the amyloidosis a very slow but non-curable degenerative disease.

X-ray Crystallographic Structure of a Teixobactin Derivative Reveals Amyloid-like Assembly.

This paper describes the X-ray crystallographic structure of a derivative of the antibiotic teixobactin and shows that its supramolecular assembly through the formation of antiparallel β-sheets creates binding sites for oxyanions. An active derivative of teixobactin containing lysine in place of allo-enduracididine assembles to form amyloid-like fibrils, which are observed through a thioflavin T fluorescence assay and by transmission electron microscopy. A homologue, bearing an N-methyl substituent, to attenuate fibril formation, and an iodine atom, to facilitate X-ray crystallographic phase determination, crystallizes as double helices of β-sheets that bind sulfate anions. β-Sheet dimers are key subunits of these assemblies, with the N-terminal methylammonium group of one monomer and the C-terminal macrocycle of the other monomer binding each anion. These observations suggest a working model for the mechanism of action of teixobactin, in which the antibiotic assembles and the assemblies bind lipid II and related bacterial cell wall precursors on the surface of Gram-positive bacteria.

Cu/Zn-superoxide dismutase forms fibrillar hydrogels in a pH-dependent manner via a water-rich extended intermediate state.

Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins.

Structural and functional properties of antimicrobial protein L5 of Lysоbacter sp. XL1.

The Gram-negative bacterium Lysobacter sp. XL1 secretes into the extracellular space five bacteriolytic enzymes that lyse the cell walls of competing microorganisms. Of special interest are homologous lytic proteases L1 and L5. This work found protein L5 to possess Gly-Gly endopeptidase and N-acetylmuramoyl-L-Ala amidase activities with respect to staphylococcal peptidoglycan. Protein L5 was found to be capable of aggregating into amyloid-like fibril structures. The crystal structure of protein L5 was determined at a 1.60-Å resolution. Protein L5 was shown to have a rather high structural identity with bacteriolytic protease L1 of Lysobacter sp. XL1 and α-lytic protease of Lysobacter enzymogenes at a rather low identity of their amino acid sequences. Still, the structure of protein L5 was revealed to have regions that differed from their equivalents in the homologs. The revealed structural distinctions in L5 are suggested to be of importance in exhibiting its unique properties.