Tandem-repeat lectins: structural and functional insights.

Multivalency in lectins plays a pivotal role in influencing glycan cross-linking, thereby affecting lectin functionality. This multivalency can be achieved through oligomerization, the presence of tandemly repeated carbohydrate recognition domains, or a combination of both. Unlike lectins that rely on multiple factors for the oligomerization of identical monomers, tandem-repeat lectins inherently possess multivalency, independent of this complex process. The repeat domains, although not identical, display slightly distinct specificities within a predetermined geometry, enhancing specificity, affinity, avidity and even oligomerization. Despite the recognition of this structural characteristic in recently discovered lectins by numerous studies, a unified criterion to define tandem-repeat lectins is still necessary. We suggest defining them multivalent lectins with intrachain tandem repeats corresponding to carbohydrate recognition domains, independent of oligomerization. This systematic review examines the folding and phyletic diversity of tandem-repeat lectins and refers to relevant literature. Our study categorizes all lectins with tandemly repeated carbohydrate recognition domains into nine distinct folding classes associated with specific biological functions. Our findings provide a comprehensive description and analysis of tandem-repeat lectins in terms of their functions and structural features. Our exploration of phyletic and functional diversity has revealed previously undocumented tandem-repeat lectins. We propose research directions aimed at enhancing our understanding of the origins of tandem-repeat lectin and fostering the development of medical and biotechnological applications, notably in the design of artificial sugars and neolectins.

A trimeric glycosylated GH45 cellulase from the red abalone (Haliotis rufescens) exhibits endo and exoactivity.

The red abalone (Haliotis rufescens) represents North America's most important aquaculture species. Its hepatopancreas is rich in cellulases and other polysaccharide-degrading enzymes, which provide it the remarkable ability to digest cellulose-rich macroalgae; nevertheless, its cellulolytic systems are poorly explored. This manuscript describes some functional and structural properties of an endogenous trimeric glycosylated endoglucanase from H. rufescens. The purified enzyme showed a molecular mass of 23.4 kDa determined by MALDI-TOF mass spectrometry, which behaved as a homotrimer in gel filtration chromatography and zymograms. According to the periodic acid-Schiff reagent staining, detecting sugar moieties in SDS-PAGE gel confirmed that abalone cellulase is a glycoprotein. Hydrolysis of cello-oligosaccharides and p-nitrophenyl-ß-D-glucopyranosides confirmed its endo/exoactivity. A maximum enzyme activity toward 0.5% (w/v) carboxymethylcellulose of 53.9 ± 1.0 U/mg was achieved at 45°C and pH 6.0. We elucidated the abalone cellulase primary structure using proteases and mass spectrometry methods. Based on these results and using a bioinformatic approach, we identified the gene encoding this enzyme and deduced its full-length amino acid sequence; the mature protein comprised 177 residues with a calculated molecular mass of 19.1 kDa and, according to sequence similarity, it was classified into the glycosyl-hydrolase family 45 subfamily B. An AlphaFold theoretical model and docking simulations with cellopentaose confirmed that abalone cellulase is a ß-sheet rich protein, as also observed by circular dichroism experiments, with conserved catalytic residues: Asp26, Asn109, and Asp134. Interestingly, the AlphaFold-Multimer analysis indicated a trimeric assembly for abalone cellulase, which supported our experimental findings. The discovery and characterization of these enzymes may contribute to developing efficient cellulose bioconversion processes for biofuels and sustainable bioproducts.

Study of peripheral domains in structure-function of isocitrate lyase (ICL) from Pseudomonas aeruginosa.

The capacity of Pseudomonas aeruginosa to assimilate nutrients is essential for niche colonization and contributes to its pathogenicity. Isocitrate lyase (ICL), the first enzyme of the glyoxylate cycle, redirects isocitrate from the tricarboxylic acid cycle to render glyoxylate and succinate. P. aeruginosa ICL (PaICL) is regarded as a virulence factor due to its role in carbon assimilation during infection. The AceA/ICL protein family shares the catalytic domain I, triosephosphate isomerase barrel (TIM-barrel). The carboxyl terminus of domain I is essential for Escherichia coli ICL (EcICL) of subfamily 1. PaICL, which belongs to subfamily 3, has domain II inserted at the periphery of domain I, which is believed to participate in enzyme oligomerization. In addition, PaICL has the α13-loop-α14 (extended motif), which protrudes from the enzyme core, being of unknown function. This study investigates the role of domain II, the extended motif, and the carboxyl-terminus (C-ICL) and amino-terminus (N-ICL) regions in the function of the PaICL enzyme, also as their involvement in the virulence of P. aeruginosa PAO1. Deletion of domain II and the extended motif results in enzyme inactivation and structural instability of the enzyme. The His6-tag fusion at the C-ICL protein produced a less efficient enzyme than fusion at the N-ICL, but without affecting the acetate assimilation or virulence. The PaICL homotetrameric structure of the enzyme was more stable in the N-His6-ICL than in the C-His6-ICL, suggesting that the C-terminus is critical for the ICL quaternary conformation. The ICL-mutant A39 complemented with the recombinant proteins N-His6-ICL or C-His6-ICL were more virulent than the WT PAO1 strain. The findings indicate that the domain II and the extended motif are essential for the ICL structure/function, and the C-terminus is involved in its quaternary structure conformation, confirming that in P. aeruginosa, the ICL is essential for acetate assimilation and virulence.

Structural and functional analysis of a tandem repeat galacturonic acid-binding lectin from the sea hare Aplysia californica.

A d-galacturonic acid-specific lectin, named AcL, was purified from the sea hare Aplysia californica by galactose-agarose affinity chromatography. AcL has a molecular mass of 27.5 kDa determined by MALDI-TOF mass spectrometry. This lectin shows a good affinity for d-galacturonic acid and a lower affinity for galactosides: raffinose, melibiose, α and ß-lactose, and d-galactose. We determined the amino acid sequence of AcL by trypsin digestion and subsequent peptide analysis by mass spectrometry, resulting in a 238 amino acid protein with a theoretical molecular mass of 26.4 kDa. The difference between the theoretical and experimental values can be attributed to post-translational modifications. Thiol-disulfide quantification discerned five disulfide bonds and three free cysteines. The structure of Acl is mainly comprised of beta sheets, determined by circular dichroism, and predicted with AlphaFold. Theoretical models depict three nearly identical tandem domains consisting of two beta sheets each. From docking analysis, we identified AcL glycan-binding sites as multiple conserved motifs in each domain. Furthermore, phylogenetic analysis based on its structure and sequence showed that AcL and its closest homologues (GalULs) form a clear monophyletic group, distinct from other glycan-binding proteins with a jelly-roll fold: lectins of types F and H. GalULs possess four conserved sequence regions that distinguish them and are either ligand-binding motifs or stabilizing network hubs. We suggest that this new family should be referred to as GalUL or D-type, following the traditional naming of lectins; D standing for depilans, the epithet for the species (Aplysia depilans) from which a lectin of this family was first isolated and described.

Structural and evolutionary insights into the multidomain galectin from the red abalone Haliotis rufescens with specificity for sulfated glycans.

Galectins are an evolutionarily ancient family of lectins characterized by their affinity for ß-galactosides and a conserved binding site in the carbohydrate recognition domain (CRD). These lectins are involved in multiple physiological functions, including the recognition of glycans on the surface of viruses and bacteria. This feature supports their role in innate immune responses in marine mollusks. Here, we identified and characterized a galectin, from the mollusk Haliotis rufescens (named HrGal), with four CRDs that belong to the tandem-repeat type. HrGal was purified by affinity chromatography in a galactose-agarose resin and exhibited a molecular mass of 64.11 kDa determined by MALDI-TOF mass spectrometry. The identity of HrGal was verified by sequencing, confirming that it is a 555 amino acid protein with a mass of 63.86 kDa. This protein corresponds to a galectin reported in GenBank with accession number AHX26603. HrGal is stable in the presence of urea, reducing agents, and ions such as Cu2+ and Zn2+. The recombinant galectin (rHrGal) was purified from inclusion bodies in the presence of these ions. A theoretical model obtained with the AlphaFold server exhibits four non-identical CRDs, with a ß sandwich folding and the representative motifs for binding ß-galactosides. This allows us to classify HrGal within the tandem repeat galectin family. On the basis of a phylogenetic analysis, we found that the mollusk sequences form a monophyletic group of tetradomain galectins unrelated to vertebrate galectins. HrGal showed specificity for galactosides and glucosides but only the sulfated sugars heparin and ι-carrageenan inhibited its hemagglutinating activity with a minimum inhibitory concentration of 4 mM and 6.25 X 10-5% respectively. The position of the sulfate groups seemed crucial for binding, both by carrageenans and heparin.

Crystal structure of a C-type lysozyme from Litopenaeus vanamei exhibiting a high binding constant to its chitotriose inhibitor.

Although information about invertebrate lysozymes is scarce, these enzymes have been described as components of the innate immune system, functioning as antibacterial proteins. Here we describe the first thermodynamic and structural study of a new C-type lysozyme from a Pacific white shrimp Litopenaeus vannamei (LvL), which has shown high activity against both Gram (+) and Gram (-) bacteria including Vibrio sp. that is one of the most severe pathogens in penaeid shrimp aquaculture. Compared with hen egg-white lysozyme, its sequence harbors a seven-residue insertion from amino acid 97 to 103, and a nine-residue extension at the C-terminus only found in penaeid crustaceans, making this enzyme one of the longest lysozyme reported to date. LvL was crystallized in the presence and absence of chitotriose. The former crystallized as a monomer in space group P61 and the latter in P212121 with two monomers in the asymmetric unit. Since the enzyme crystallized at a pH where lysozyme activity is deficient, the ligand could not be observed in the P61 structure; therefore, we performed a docking simulation with chitotriose to compare with the hen egg lysozyme crystallized in the presence of the ligand. Remarkably, additional amino acids in LvL caused an increase in the length of α-helix H4 (residues 97-103) that is directly related to ligand recognition. The Ka for chitotriose (4.1 × 105 M-1), as determined by Isothermal Titration Calorimetry, was one order of magnitude higher than those for lysozymes from hen and duck eggs. Our results revealed new interactions of chitiotriose with residues in helix H4.

A biophysical and structural study of two chitinases from Agave tequilana and their potential role as defense proteins.

Plant chitinases are enzymes that have several functions, including providing protection against pathogens. Agave tequilana is an economically important plant that is poorly studied. Here, we identified a chitinase from short reads of the A. tequilana transcriptome (AtChi1). A second chitinase, differing by only six residues from the first, was isolated from total RNA of plants infected with Fusarium oxysporum (AtChi2). Both enzymes were overexpressed in Escherichia coli and analysis of their sequences indicated that they belong to the class I glycoside hydrolase family19, whose members exhibit two domains: a carbohydrate-binding module and a catalytic domain, connected by a flexible linker. Activity assays and thermal shift experiments demonstrated that the recombinant Agave enzymes are highly thermostable acidic endochitinases with Tm values of 75 °C and 71 °C. Both exhibit a molecular mass close to 32 kDa, as determined by MALDI-TOF, and experimental pIs of 3.7 and 3.9. Coupling small-angle x-ray scattering information with homology modeling and docking simulations allowed us to structurally characterize both chitinases, which notably show different interactions in the binding groove. Even when the six different amino acids are all exposed to solvent in the loops located near the linker and opposite to the binding site, they confer distinct kinetic parameters against colloidal chitin and similar affinity for (GlnNAc)6, as shown by isothermal titration calorimetry. Interestingly, binding is more enthalpy-driven for AtChi2. Whereas the physiological role of these chitinases remains unknown, we demonstrate that they exhibit important antifungal activity against chitin-rich fungi such as Aspergillus sp. DATABASE: SAXS structural data are available in the SASBDB database with accession numbers SASDDE7 and SASDDA6. ENZYMES: Chitinases (EC3.2.1.14).

The role of conserved non-aromatic residues in the Lactobacillus amylovorus α-amylase CBM26-starch interaction.

It is generally accepted that carbohydrate binding modules (CBMs) recognize their carbohydrate ligands by hydrophobic and CH-π interactions. Point mutations of one CBM26 of the Lactobacillus amylovorus α-amylase starch-binding domain (LaCBM26) showed that conserved non-aromatic residue are essential in the starch recognition function of the domain, as the mutation of a single glutamine (Q68L) eliminates binding to starch and ß-cyclodextrin, even in the presence of aromatic amino acids necessary for ligand binding. The secondary structure of mutated proteins was verified and showed no differences from the wild-type domain. However, random mutations of five residues involved in binding (Y18, Y20, Q68, E74, and F77) did cause change in the secondary structure of the protein, which also causes loss of function. Much of the diversity introduced in the LaCBM26 was probably incompatible with the appropriate folding of these proteins, suggesting that the domain has little tolerance to change.

Data concerning secondary structure and alpha-glucans-binding capacity of the LaCBM26.

Carbohydrate-binding modules (CBMs) are auxiliary domains into glycoside-hydrolases that allow the interaction between the insoluble substrate and the solubilized enzyme, through hydrophobic, CH-π interactions and hydrogen bonds. Here, we present the data article related to the interaction of one LaCBM26 and some mutated proteins with soluble α-glucans determined by enzyme-linked carbohydrate-binding assay, isothermal titration calorimetry (ITC), and affinity gel electrophoresis (AGE). The data of the behavior of proteins in presence and absence of substrate analyzed by circular dichroism CD and thermofluor are also presented. These results are complementary to the research article "The role of conserved non-aromatic residues in the Lactobacillus amylovorus α-amylase CBM26-starch interaction" (Armenta et al., 2019).

Stabilizing an amyloidogenic λ6 light chain variable domain.

Light chain amyloidosis is a lethal disease where vital organs are damaged by the fibrillar aggregation of monoclonal light chains. λ6a is an immunoglobulin light chain encoded by the germ-line gene segment implicated in this disease. AR is a patient-derived germ-line variant with a markedly low thermodynamic stability and prone to form fibrils in vitro in less than an hour. Here, we sought to stabilize this domain by mutating some residues back to the germ-line sequence, and the most stabilizing mutations were the single-mutant AR-F21I and the double-mutant AR-F21/IV104L, both located in the hydrophobic core. While mutation Arg25Gly in 6aJL2 destabilized the domain, mutating Gly25 back to arginine in AR did not contribute to stabilization as expected. Crystallographic structures of AR and 6a-R25G were generated to explain this discrepancy. Finally, 6a-R25G crystals revealed an octameric assembly which was emulated into 6aJL2 and AR crystals by replicating their structural parameters and suggesting a common assembly pattern. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession numbers 5IR3 and 5C9K.

Molecular and functional characterization of a glycosylated Galactose-Binding lectin from Mytilus californianus.

Lectins play crucial roles for innate immune responses in invertebrates by recognizing and eliminating pathogens. In this study, a lectin from the mussel Mytilus californianus (MCL) was identified and characterized. The lectin was purified by affinity chromatography in α-lactose-agarose resin showing an experimental molecular mass of 18000 Da as determined by SDS-PAGE and MALDI-TOF mass spectrometry. It was specific for binding d-galactose and N-Acetyl-d-galactosamine that contained carbohydrate moieties that were also inhibited by melibiose and raffinose. It had the ability to agglutinate all types of human erythrocytes, as well as rabbit red blood cells. Circular dichroism analyzes have indicated that this lectin possessed an α/ß fold with a predominance of ß structures. This was consistent with the structure of the protein that was determined by the X-ray diffraction techniques. MCL was crystallized in the space group C21 and it diffracted to 1.79 Å resolution. Two monomers were found in the asymmetric unit and they formed dimers in solution. The protein has shown to be a member of the ß-trefoil family, with three sugar binding sites per monomer. In accord with fluorescence-based thermal shift assays, we observed that the MCL Tm increased about 10 °C in the presence of galactose. Furthermore, we have determined the complete amino acid sequence by cDNA sequencing. The gene had two ORF2 proteins, one resulting in a 180 residue protein with a theoretical molecular mass of 20227 Da, and another resulting in a 150 residue protein with a theoretical molecular mass of 16911 Da. The difference between the theoretical and experimental values was due to the presence of a glycosylation that was observed by the glycosylation assay. A positive microbial agglutination and a growth inhibition activity were observed against Gram-negative and Gram-positive bacteria. The M. californianus lectin is the fourth member of the recently proposed new family of lectins that have been reported to date, occurring only in mollusks belonging to the family Mytilidae. It is the first member to be glycosylated and with a strong tendency to form large oligomers.

Production of bioactive conjugated linoleic acid by the multifunctional enolase from Lactobacillus plantarum.

Lactobacillus plantarum α-enolase, a multifunctional-anchorless-surface protein belonging to the conserved family of enolases with a central role in glycolytic metabolism, was characterized to have a side role in the intricate metabolism of biohydrogenation of linoleic acid, catalyzing the formation of bioactive 9-cis-11-trans-CLA through dehydration and isomerization of 10-hydroxy-12-cis-octadecenoic acid. The identity of the enolase was confirmed through mass spectrometric analysis that showed the characteristic 442 amino acid sequence with a molecular mass of 48.03kDa. The enolase was not capable of using linoleic acid directly as a substrate but instead uses its hydroxyl derivative 10-hydroxi-12-cis-octadecenoic acid to finally form bioactive conjugated linoleic acid. Biochemical optimization studies were carried out to elucidate the conditions for maximum production of 9-cis-11-trans-CLA and maximum stability of α-enolase when catalyzing this reaction. Furthermore, through structural analysis of the protein, we propose the binding sites of substrate and product molecules that were characterized as two hydrophobic superficial pockets located at opposite ends of the enolase connected through a channel where the catalysis of dehydration and isomerization might occur. These results prove that multifunctional α-enolase also plays a role in cell detoxification from polyunsaturated fatty acids such as linoleic acid, along with the linoleate isomerase complex.

Functional characterization of a fatty acid double-bond hydratase from Lactobacillus plantarum and its interaction with biosynthetic membranes.

Hydrogenation of linoleic acid and other polyunsaturated fatty acids is a detoxification mechanism that is present in the Lactobacillus genus of lactic bacteria. The first stage in this multi-step process is hydration of the substrate with formation of 10-hydroxy-9-cis-octadecenoic acid due to fatty-acid hydratase activity that has been detected only in the membrane-associated cell fraction; however, its interaction with the cell membrane is unknown. To provide information in this respect we characterized the homotrimeric 64.7 kDa-native protein from Lactobacillus plantarum; afterwards, it was reconstituted in proteoliposomes and analyzed by confocal fluorescence microscopy. The results showed that hydratase is an extrinsic-membrane protein and hence, the enzymatic reaction occurs at the periphery of the cell. This location may be advantageous in the detoxifying process since the toxic linoleic acid molecule can be bound to hydratase and converted to non-toxic 10-hydroxy-9-cis-octadecenoic acid before it reaches cell membrane. Additionally, we propose that the interaction with membrane periphery occurs through electrostatic contacts. Finally, the structural model of L. plantarum hydratase was constructed based on the amino acid sequence and hence, the putative binding sites with linoleic acid were identified: site 1, located in an external hydrophobic pocket at the C-terminus of the protein and site 2, located at the core and in contact with a FAD molecule. Interestingly, it was found that the linoleic acid molecule arranges around a methionine residue in both sites (Met154 and Met81, respectively) that acts as a rigid pole, thus playing a key role in binding unsaturated fatty acids.

Structural analysis of the endogenous glycoallergen Hev b 2 (endo-ß-1,3-glucanase) from Hevea brasiliensis and its recognition by human basophils.

Endogenous glycosylated Hev b 2 (endo-ß-1,3-glucanase) from Hevea brasiliensis is an important latex allergen that is recognized by IgE antibodies from patients who suffer from latex allergy. The carbohydrate moieties of Hev b 2 constitute a potentially important IgE-binding epitope that could be responsible for its cross-reactivity. Here, the structure of the endogenous isoform II of Hev b 2 that exhibits three post-translational modifications, including an N-terminal pyroglutamate and two glycosylation sites at Asn27 and at Asn314, is reported from two crystal polymorphs. These modifications form a patch on the surface of the molecule that is proposed to be one of the binding sites for IgE. A structure is also proposed for the most important N-glycan present in this protein as determined by digestion with specific enzymes. To analyze the role of the carbohydrate moieties in IgE antibody binding and in human basophil activation, the glycoallergen was enzymatically deglycosylated and evaluated. Time-lapse automated video microscopy of basophils stimulated with glycosylated Hev b 2 revealed basophil activation and degranulation. Immunological studies suggested that carbohydrates on Hev b 2 represent an allergenic IgE epitope. In addition, a dimer was found in each asymmetric unit that may reflect a regulatory mechanism of this plant defence protein.

Different contribution of conserved amino acids to the global properties of triosephosphate isomerases.

It is generally assumed that the amino acids that exist in all homologous enzymes correspond to residues that participate in catalysis, or that are essential for folding and stability. Although this holds for catalytic residues, the function of conserved noncatalytic residues is not clear. It is not known if such residues are of equal importance and have the same role in different homologous enzymes. In humans, the E104D mutation in triosephosphate isomerase (TIM) is the most frequent mutation in the autosomal diseases named "TPI deficiencies." We explored if the E104D mutation has the same impact in TIMs from four different organisms (Homo sapiens, Giardia lamblia, Trypanosoma cruzi, and T. brucei). The catalytic properties were not significantly affected by the mutation, but it affected the rate and extent of formation of active dimers from unfolded monomers differently. Scanning calorimetry experiments indicated that the mutation was in all cases destabilizing, but the mutation effect on rates of irreversible denaturation and transition-state energetics were drastically dependent on the TIM background. For instance, the E104D mutation produce changes in activation energy ranging from 430 kJ mol(-1) in HsTIM to -78 kJ mol(-1) in TcTIM. Thus, in TIM the role of a conserved noncatalytic residue is drastically dependent on its molecular background. Accordingly, it would seem that because each protein has a particular sequence, and a distinctive set of amino acid interactions, it should be regarded as a unique entity that has evolved for function and stability in the organisms to which it belongs.

Effects of a buried cysteine-to-serine mutation on yeast triosephosphate isomerase structure and stability.

All the members of the triosephosphate isomerase (TIM) family possess a cystein residue (Cys126) located near the catalytically essential Glu165. The evolutionarily conserved Cys126, however, does not seem to play a significant role in the catalytic activity. On the other hand, substitution of this residue by other amino acid residues destabilizes the dimeric enzyme, especially when Cys is replaced by Ser. In trying to assess the origin of this destabilization we have determined the crystal structure of Saccharomyces cerevisiae TIM (ScTIM) at 1.86 Å resolution in the presence of PGA, which is only bound to one subunit. Comparisons of the wild type and mutant structures reveal that a change in the orientation of the Ser hydroxyl group, with respect to the Cys sulfhydryl group, leads to penetration of water molecules and apparent destabilization of residues 132-138. The latter results were confirmed by means of Molecular Dynamics, which showed that this region, in the mutated enzyme, collapses at about 70 ns.

Difficult macromolecular structures determined using X-ray diffraction techniques.

Macromolecular crystallography has been, for the last few decades, the main source of structural information of biological macromolecular systems and it is one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. In addition, it is also an extremely powerful tool for drug design. Recent technological and methodological developments in macromolecular X-ray crystallography have allowed solving structures that until recently were considered difficult or even impossible, such as structures at atomic or subatomic resolution or large macromolecular complexes and assemblies at low resolution. These developments have also helped to solve the 3D-structure of macromolecules from twin crystals. Recently, this technique complemented with cryo-electron microscopy and neutron crystallography has provided the structure of large macromolecular machines with great precision allowing understanding of the mechanisms of their function.

Crystal structure of Cu / Zn superoxide dismutase from Taenia solium reveals metal-mediated self-assembly.

Taenia solium is the cestode responsible for porcine and human cysticercosis. The ability of this parasite to establish itself in the host is related to its evasion of the immune response and its antioxidant defence system. The latter includes enzymes such as cytosolic Cu/Zn superoxide dismutase. In this article, we describe the crystal structure of a recombinant T. solium Cu/Zn superoxide dismutase, representing the first structure of a protein from this organism. This enzyme shows a different charge distribution at the entrance of the active channel when compared with human Cu/Zn superoxide dismutase, giving it interesting properties that may allow the design of specific inhibitors against this cestode. The overall topology is similar to other superoxide dismutase structures; however, there are several His and Glu residues on the surface of the protein that coordinate metal ions both intra- and intermolecularly. Interestingly, one of these ions, located on the ß2 strand, establishes a metal-mediated intermolecular ß-ß interaction, including a symmetry-related molecule. The factors responsible for the abnormal protein-protein interactions that lead to oligomerization are still unknown; however, high metal levels have been implicated in these phenomena, but exactly how they are involved remains unclear. The present results suggest that this structure could be useful as a model to explain an alternative mechanism of protein aggregation commonly observed in insoluble fibrillar deposits.

Crystal structure of human cystatin C stabilized against amyloid formation.

Human cystatin C (HCC) is a family 2 cystatin inhibitor of papain-like (C1) and legumain-related (C13) cysteine proteases. In pathophysiological processes, the nature of which is not understood, HCC is codeposited in the amyloid plaques of Alzheimer's disease or Down's syndrome. The amyloidogenic properties of HCC are greatly increased in a naturally occurring L68Q variant, resulting in fatal cerebral amyloid angiopathy in early adult life. In all crystal structures of cystatin C studied to date, the protein has been found to form 3D domain-swapped dimers, created through a conformational change of a beta-hairpin loop, L1, from the papain-binding epitope. We have created monomer-stabilized human cystatin C, with an engineered disulfide bond (L47C)-(G69C) between the structural elements that become separated upon domain swapping. The mutant has drastically reduced dimerization and fibril formation properties, but its inhibition of papain is unaltered. The structure confirms the success of the protein engineering experiment to abolish 3D domain swapping and, in consequence, amyloid fibril formation. It illustrates for the first time the fold of monomeric cystatin C and allows verification of earlier predictions based on the domain-swapped forms and on the structure of chicken cystatin. Importantly, the structure defines the so-far unknown conformation of loop L1, which is essential for the inhibition of papain-like cysteine proteases.

A single mutation at the sheet switch region results in conformational changes favoring lambda6 light-chain fibrillogenesis.

Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although lambda chains, particularly those belonging to the lambda6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the lambda6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the V(L) (variable region of the light chain)-V(L) interface. This mutant crystallized in two orthorhombic polymorphs, P2(1)2(1)2(1) and C222(1). In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222(1) lattice showed the establishment of intermolecular beta-beta interactions that involved the N-terminus and beta-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the V(L) interface in lambda6 LCs.