Unfolding and Aggregation Pathways of Variable Domains from Immunoglobulin Light Chains.

Light chain amyloidosis is the most common form of systemic amyloidosis. This disease is caused by the formation and deposition of amyloid fibers made from immunoglobulin light chains. Environmental conditions such as pH and temperature can affect protein structure and induce the development of these fibers. Several studies have shed light on the native state, stability, dynamics, and final amyloid state of these proteins; however, the initiation process and the fibril formation pathway remain poorly understood structurally and kinetically. To study this, we analyzed the unfolding and aggregation process of the 6aJL2 protein under acidic conditions, with temperature changes, and upon mutation, using biophysical and computational techniques. Our results suggest that the differences in amyloidogenicity displayed by 6aJL2 under these conditions are caused by traversing different aggregation pathways, including unfolded intermediates and the formation of oligomers.

The Common Bean Small Heat Shock Protein Nodulin 22 from Phaseolus vulgaris L. Assembles into Functional High-Molecular-Weight Oligomers.

Small heat shock proteins (sHsps) are present in all domains of life. These proteins are responsible for binding unfolded proteins to prevent their aggregation. sHsps form dynamic oligomers of different sizes and constitute transient reservoirs for folding competent proteins that are subsequently refolded by ATP-dependent chaperone systems. In plants, the sHsp family is rather diverse and has been associated with the ability of plants to survive diverse environmental stresses. Nodulin 22 (PvNod22) is an sHsp of the common bean (Phaseolus vulgaris L.) located in the endoplasmic reticulum. This protein is expressed in response to stress (heat or oxidative) or in plant roots during mycorrhizal and rhizobial symbiosis. In this work, we study its oligomeric state using a combination of in silico and experimental approaches. We found that recombinant PvNod22 was able to protect a target protein from heat unfolding in vitro. We also demonstrated that PvNod22 assembles into high-molecular-weight oligomers with diameters of ~15 nm under stress-free conditions. These oligomers can cluster together to form high-weight polydisperse agglomerates with temperature-dependent interactions; in contrast, the oligomers are stable regarding temperature.

Zinc and Copper Ions Induce Aggregation of Human ß-Crystallins.

Cataracts are defined as the clouding of the lens due to the formation of insoluble protein aggregates. Metal ions exposure has been recognized as a risk factor in the cataract formation process. The γ and ß crystallins are members of a larger family and share several structural features. Several studies have shown that copper and zinc ions induce the formation of γ-crystallins aggregates. However, the interaction of metal ions with ß-crystallins, some of the most abundant crystallins in the lens, has not been explored until now. Here, we evaluate the effect of Cu(II) and Zn(II) ions on the aggregation of HßA1, as a representative of the acidic form, and HßB2, as a representative of the basic ß-crystallins. We used several biophysical techniques and computational methods to show that Cu(II) and Zn(II) induce aggregation following different pathways. Both metal ions destabilize the proteins and impact protein folding. Copper induced a small conformational change in HßA1, leading to high-molecular-weight light-scattering aggregates, while zinc is more aggressive towards HßB2 and induces a larger conformational change. Our work provides information on the mechanisms of metal-induced aggregation of ß-crystallins.

Combining Experimental Data and Computational Methods for the Non-Computer Specialist.

Experimental methods are indispensable for the study of the function of biological macromolecules, not just as static structures, but as dynamic systems that change conformation, bind partners, perform reactions, and respond to different stimulus. However, providing a detailed structural interpretation of the results is often a very challenging task. While experimental and computational methods are often considered as two different and separate approaches, the power and utility of combining both is undeniable. The integration of the experimental data with computational techniques can assist and enrich the interpretation, providing new detailed molecular understanding of the systems. Here, we briefly describe the basic principles of how experimental data can be combined with computational methods to obtain insights into the molecular mechanism and expand the interpretation through the generation of detailed models.

DNA binding induces a cis-to-trans switch in Cre recombinase to enable intasome assembly.

Mechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution nuclear magnetic resonance (NMR) spectroscopy to discover the link between intrinsic flexibility and function in Cre recombinase. Transverse relaxation-optimized spectroscopy (TROSY) NMR spectra show the N-terminal and C-terminal catalytic domains (CreNTD and CreCat) to be structurally independent. Amide 15N relaxation measurements of the CreCat domain reveal fast-timescale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein-protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and intermolecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative autoinhibitory conformation for the αN region of free Cre, wherein it packs in cis over the protein DNA binding surface and active site. Moreover, binding to loxP DNA induces a conformational change that dislodges the C terminus, resulting in a cis-to-trans switch that is likely to enable protein-protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a reexamination of the mechanisms by which this widely utilized gene-editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.

Aggregation pathways of human γ D crystallin induced by metal ions revealed by time dependent methods.

Cataract formation is a slow accumulative process due to protein aggregates promoted by different factors over time. Zinc and copper ions have been reported to induce the formation of aggregates opaque to light in the human gamma D crystallin (HγD) in a concentration and temperature dependent manner. In order to gain insight into the mechanism of metal-induced aggregation of HγD under conditions that mimic more closely the slow, accumulative process of the disease, we have studied the non-equilibrium process with the minimal metal dose that triggers HγD aggregation. Using a wide variety of biophysics techniques such as turbidimetry, dynamic light scattering, fluorescence, nuclear magnetic resonance and computational methods, we obtained information on the molecular mechanisms for the formation of aggregates. Zn(II) ions bind to different regions at the protein, probably with similar affinities. This binding induces a small conformational rearrangement within and between domains and aggregates via the formation of metal bridges without any detectable unfolded intermediates. In contrast, Cu(II)-induced aggregation includes a lag time, in which the N-terminal domain partially unfolds while the C-terminal domain and parts of the N-terminal domain remain in a native-like conformation. This partially unfolded intermediate is prone to form the high-molecular weight aggregates. Our results clearly show that different external factors can promote protein aggregation following different pathways.

Site-Specific Interactions with Copper Promote Amyloid Fibril Formation for λ6aJL2-R24G.

Light-chain amyloidosis (AL) is one of the most common systemic amyloidoses, and it is characterized by the deposition of immunoglobulin light chain (LC) variable domains as insoluble amyloid fibers in vital organs and tissues. The recombinant protein 6aJL2-R24G contains λ6a and JL2 germline genes and also contains the Arg24 by Gly substitution. This mutation is present in 25% of all amyloid-associated λ6 LC cases, reduces protein stability, and increases the propensity to form amyloid fibers. In this study, it was found that the interaction of 6aJL2-R24G with Cu(II) decreases the thermal stability of the protein and accelerates the amyloid fibril formation, as observed by fluorescence spectroscopy. Isothermal calorimetry titration showed that Cu(II) binds to the protein with micromolar affinity. His99 may be one of the main Cu(II) interaction sites, as observed by nuclear magnetic resonance spectroscopy. The binding of Cu(II) to His99 induces larger fluctuations of the CDR1 and loop C″, as shown by molecular dynamics simulations. Thus, Cu(II) binding may be inducing the loss of interactions between CDR3 and CDR1, making the protein less stable and more prone to form amyloid fibers. This study provides insights into the mechanism of metal-induced aggregation of the 6aJL2-R24G protein and sheds light on the bio-inorganic understanding of AL disease.

Antimicrobial activity and structure of a consensus human ß-defensin and its comparison to a novel putative hBD10.

The spread of multidrug resistant bacteria owing to the intensive use of antibiotics is challenging current antibiotic therapies, and making the discovery and evaluation of new antimicrobial agents a high priority. The evaluation of novel peptide sequences of predicted antimicrobial peptides from different sources is valuable approach to identify alternative antibiotic leads. Two strategies were pursued in this study to evaluate novel antimicrobial peptides from the human ß-defensin family (hBD). In the first, a 32-residue peptide was designed based on the alignment of all available hBD primary structures, while in the second a putative 35-residue peptide, hBD10, was mined from the gene DEFB110. Both hBDconsensus and hBD10 were chemically synthesized, folded and purified. They showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis, but were not hemolytic on human red blood cells. The NMR-based solution structure of hBDconsensus revealed that it adopts a classical ß-defensin fold and disulfide connectivities. Even though the mass spectrum of hBD10 confirmed the formation of three disulfide bonds, it showed limited dispersion in 1 H NMR spectra and structural studies were not pursued. The evaluation of different ß-defensin structures may identify new antimicrobial agents effective against multidrug-resistant bacterial strains.

Crystal structure of 6aJL2-R24G light chain variable domain: Does crystal packing explain amyloid fibril formation?

Light chain amyloidosis is one of the most common systemic amyloidosis, characterized by the deposition of immunoglobulin light variable domain as insoluble amyloid fibrils in vital organs, leading to the death of patients. Germline λ6a is closely related with this disease and has been reported that 25% of proteins encoded by this germline have a change at position 24 where an Arg is replaced by a Gly (R24G). This germline variant reduces protein stability and increases the propensity to form amyloid fibrils. In this work, the crystal structure of 6aJL2-R24G has been determined to 2.0 Šresolution by molecular replacement. Crystal belongs to space group I212121 (PDB ID 5JPJ) and there are two molecules in the asymmetric unit. This 6aJL2-R24G structure as several related in PDB (PDB entries: 5C9K, 2W0K, 5IR3 and 1PW3) presents by crystal packing the formation of an octameric assembly in a helicoidal arrangement, which has been proposed as an important early stage in amyloid fibril aggregation. However, other structures of other protein variants in PDB (PDB entries: 3B5G, 3BDX, 2W0L, 1CD0 and 2CD0) do not make the octameric assembly, regardless their capacity to form fibers in vitro or in vivo. The analysis presented here shows that the ability to form the octameric assembly in a helicoidal arrangement in crystallized light chain immunoglobulin proteins is not required for amyloid fibril formation in vitro. In addition, the fundamental role of partially folded states in the amyloid fibril formation in vitro, is not described in any crystallographic structure published or analyzed here, being those structures, in any case examples of proteins in their native states. Those partially folded states have been recently described by cryo-EM studies, showing the necessity of structural changes in the variants before the amyloid fiber formation process starts.

Different Dynamics in 6aJL2 Proteins Associated with AL Amyloidosis, a Conformational Disease.

Light-chain amyloidosis (AL) is the most common systemic amyloidosis and is caused by the deposition of mainly insoluble immunoglobulin light chain amyloid fibrils in multiple organs, causing organ failure and eventually death. The germ-line λ6a has been implicated in AL, where a single point mutant at amino acid 24 (6aJL2-R24G) has been observed in around 25% of patient samples. Structural analysis has shown only subtle differences between both proteins; nevertheless, 6aJL2-R24G is more prone to form amyloid fibrils. To improve our understanding of the role of protein flexibility in amyloid fibril formation, we have used a combination of solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations to complement the structural insight with dynamic knowledge. Fast timescale dynamics (ps-ns) were equivalent for both proteins, but suggested exchange events for some residues. Even though most of the intermediate dynamics (µs-ms) occurred at a similar region for both proteins, the specific characteristics are very different. A minor population detected in the dispersion experiments could be associated with the formation of an off-pathway intermediate that protects from fiber formation more efficiently in the germ-line protein. Moreover, we found that the hydrogen bond patterns for both proteins are similar, but the lifetime for the mutant is significantly reduced; as a consequence, there is a decrease in the stability of the tertiary structure that extends throughout the protein and leads to an increase in the propensity to form amyloid fibers.

The human adenovirus type 5 E1B 55kDa protein interacts with RNA promoting timely DNA replication and viral late mRNA metabolism.

The E1B 55kDa produced by human adenovirus type 5 is a multifunctional protein that participates in the regulation of several steps during the viral replication cycle. Previous studies suggest this protein plays an important role in postranscriptional regulation of viral and cellular gene expression, as it is required for the selective accumulation of maximal levels of viral late mRNA in the cytoplasm of the infected cell; however the molecular mechanisms that are altered or regulated by this protein have not been elucidated. A ribonucleoprotein motif that could implicate the direct interaction of the protein with RNA was initially predicted and tested in vitro, but the interaction with RNA could not be detected in infected cells, suggesting the interaction may be weak or transient. Here it was determined that the E1B 55kDa interacts with RNA in the context of the viral infection in non-transformed human cells, and its contribution to the adenovirus replication cycle was evaluated. Using recombinant adenoviruses with amino acid substitutions or a deletion in the ribonucleoprotein motif the interaction of E1B 55kDa with RNA was found to correlate with timely and efficient viral DNA replication and viral late mRNA accumulation and splicing.

Metal-binding polymorphism in late embryogenesis abundant protein AtLEA4-5, an intrinsically disordered protein.

Late embryogenesis abundant (LEA) proteins accumulate in plants during adverse conditions and their main attributed function is to confer tolerance to stress. One of the deleterious effects of the adverse environment is the accumulation of metal ions to levels that generate reactive oxygen species, compromising the survival of cells. AtLEA4-5, a member of group 4 of LEAs in Arabidopsis, is an intrinsically disordered protein. It has been shown that their N-terminal region is able to undergo transitions to partially folded states and prevent the inactivation of enzymes. We have characterized metal ion binding to AtLEA4-5 by circular dichroism, electronic absorbance spectroscopy (UV-vis), electron paramagnetic resonance, dynamic light scattering, and isothermal titration calorimetry. The data shows that AtLEA4-5 contains a single binding site for Ni(II), while Zn(II) and Cu(II) have multiple binding sites and promote oligomerization. The Cu(II) interacts preferentially with histidine residues mostly located in the C-terminal region with moderate affinity and different coordination modes. These results and the lack of a stable secondary structure formation indicate that an ensemble of conformations remains accessible to the metal for binding, suggesting the formation of a fuzzy complex. Our results support the multifunctionality of LEA proteins and suggest that the C-terminal region of AtLEA4-5 could be responsible for antioxidant activity, scavenging metal ions under stress conditions while the N-terminal could function as a chaperone.

Synthesis, folding, structure and activity of a predicted peptide from the sea anemone Oulactis sp. with an ShKT fold.

Sea anemone venom is rich in bioactive compounds, including peptides containing multiple disulfide bridges. In a transcriptomic study on Oulactis sp., we identified the putative 36-residue peptide, OspTx2b, which is an isoform of the KV channel blocker OspTx2a (Sunanda P et al. [2018] Identification, chemical synthesis, structure and function of a new KV1 channel blocking peptide from Oulactis sp. Peptide Science, in press). As OspTx2b contains a ShK/BgK-like cysteine framework, with high amino acid sequence similarity to BgK, we were interested to investigate its structure and function. The solution structure of OspTx2b was determined using nuclear magnetic resonance spectroscopy. OspTx2b does indeed possess a BgK-like scaffold, with the same disulfide bond connectivities. The orientation of the Lys-Tyr dyad in OspTx2b is more similar to that in ShK than in BgK. However, it failed to show against a range of voltage-gated potassium channels in Xenopus oocytes and human T lymphocytes. OspTx2b also showed no growth inhibitory activity against several strains of bacteria and fungi. Having a BgK-like fold with the Lys-Tyr dyad but no BgK-like activity highlights the importance of key amino acid residues in BgK that are missing in OspTx2b. The lack of activity against the KV channels assessed in this study emphasises that the ShK/BgK scaffold is capable of supporting functional activity beyond potassium channel blockade.

Localized conformational changes trigger the pH-induced fibrillogenesis of an amyloidogenic λ light chain protein.

Solvent conditions modulate the expression of the amyloidogenic potential of proteins. In this work the effect of pH on the fibrillogenic behavior and the conformational properties of 6aJL2, a model protein of the highly amyloidogenic variable light chain λ6a gene segment, was examined. Ordered aggregates showing the ultrastructural and spectroscopic properties observed in amyloid fibrils were formed in the 2.0-8.0 pH range. At pH <3.0 a drastic decrease in lag time and an increase in fibril formation rate were found. In the 4.0-8.0 pH range there was no spectroscopic evidence for significant conformational changes in the native state. Likewise, heat capacity measurements showed no evidence for residual structure in the unfolded state. However, at pH <3.0 stability is severely decreased and the protein suffers conformational changes as detected by circular dichroism, tryptophan and ANS fluorescence, as well as by NMR spectroscopy. Molecular dynamics simulations indicate that acid-induced conformational changes involve the exposure of the loop connecting strands E and F. These results are compatible with pH-induced changes in the NMR spectra. Overall, the results indicate that the mechanism involved in the acid-induced increase in the fibrillogenic potential of 6aJL2 is profoundly different to that observed in κ light chains, and is promoted by localized conformational changes in a region of the protein that was previously not known to be involved in acid-induced light chain fibril formation. The identification of this region opens the potential for the design of specific inhibitors.

Unraveling self-assembly pathways of the 468-kDa proteolytic machine TET2.

The spontaneous formation of biological higher-order structures from smaller building blocks, called self-assembly, is a fundamental attribute of life. Although the protein self-assembly is a time-dependent process that occurs at the molecular level, its current understanding originates either from static structures of trapped intermediates or from modeling. Nuclear magnetic resonance (NMR) spectroscopy has the unique ability to monitor structural changes in real time; however, its size limitation and time-resolution constraints remain a challenge when studying the self-assembly of large biological particles. We report the application of methyl-specific isotopic labeling combined with relaxation-optimized NMR spectroscopy to overcome both size- and time-scale limitations. We report for the first time the self-assembly process of a half-megadalton protein complex that was monitored at the structural level, including the characterization of intermediate states, using a mutagenesis-free strategy. NMR was used to obtain individual kinetics data on the different transient intermediates and the formation of final native particle. In addition, complementary time-resolved electron microscopy and native mass spectrometry were used to characterize the low-resolution structures of oligomerization intermediates.

The first description of a hormone-sensitive lipase from a basidiomycete: Structural insights and biochemical characterization revealed Bjerkandera adusta BaEstB as a novel esterase.

The heterologous expression and characterization of a Hormone-Sensitive Lipases (HSL) esterase (BaEstB) from the Basidiomycete fungus Bjerkandera adusta is reported for the first time. According to structural analysis, amino acid similarities and conservation of particular motifs, it was established that this enzyme belongs to the (HSL) family. The cDNA sequence consisted of 969 nucleotides, while the gene comprised 1133, including three introns of 57, 50, and 57 nucleotides. Through three-dimensional modeling and phylogenetic analysis, we conclude that BaEstB is an ortholog of the previously described RmEstB-HSL from the phylogenetically distant fungus Rhizomucor miehei. The purified BaEstB was characterized in terms of its specificity for the hydrolysis of different acyl substrates confirming its low lipolytic activity and a noticeable esterase activity. The biochemical characterization of BaEstB, the DLS analysis and the kinetic parameters determination revealed this enzyme as a true esterase, preferentially found in a dimeric state, displaying activity under alkaline conditions and relative low temperature (pH = 10, 20°C). Our data suggest that BaEstB is more active on substrates with short acyl chains and bulky aromatic moieties. Phylogenetic data allow us to suggest that a number of fungal hypothetical proteins could belong to the HSL family.

Copper and Zinc Ions Specifically Promote Nonamyloid Aggregation of the Highly Stable Human γ-D Crystallin.

Cataract is the leading cause of blindness in the world. It results from aggregation of eye lens proteins into high-molecular-weight complexes, causing light scattering and lens opacity. Copper and zinc concentrations in cataractous lens are increased significantly relative to a healthy lens, and a variety of experimental and epidemiological studies implicate metals as potential etiological agents for cataract. The natively monomeric, ß-sheet rich human γD (HγD) crystallin is one of the more abundant proteins in the core of the lens. It is also one of the most thermodynamically stable proteins in the human body. Surprisingly, we found that both Cu(II) and Zn(II) ions induced rapid, nonamyloid aggregation of HγD, forming high-molecular-weight light-scattering aggregates. Unlike Zn(II), Cu(II) also substantially decreased the thermal stability of HγD and promoted the formation of disulfide-bridged dimers, suggesting distinct aggregation mechanisms. In both cases, however, metal-induced aggregation depended strongly on temperature and was suppressed by the human lens chaperone αB-crystallin (HαB), implicating partially folded intermediates in the aggregation process. Consistently, distinct site-specific interactions of Cu(II) and Zn(II) ions with the protein and conformational changes in specific hinge regions were identified by nuclear magnetic resonance. This study provides insights into the mechanisms of metal-induced aggregation of one of the more stable proteins in the human body, and it reveals a novel and unexplored bioinorganic facet of cataract disease.

Inhibition of Light Chain 6aJL2-R24G Amyloid Fiber Formation Associated with Light Chain Amyloidosis.

Light chain amyloidosis (AL) is a deadly disease characterized by the deposition of monoclonal immunoglobulin light chains as insoluble amyloid fibrils in different organs and tissues. Germ line λ VI has been closely related to this condition; moreover, the R24G mutation is present in 25% of the proteins of this germ line in AL patients. In this work, five small molecules were tested as inhibitors of the formation of amyloid fibrils from the 6aJL2-R24G protein. We have found by thioflavin T fluorescence and transmission electron microscopy that EGCG inhibits 6aJL2-R24G fibrillogenesis. Furthermore, using nuclear magnetic resonance spectroscopy, dynamic light scattering, and isothermal titration calorimetry, we have determined that the inhibition is due to binding to the protein in its native state, interacting mainly with aromatic residues.

Information flow and protein dynamics: the interplay between nuclear magnetic resonance spectroscopy and molecular dynamics simulations.

Proteins participate in information pathways in cells, both as links in the chain of signals, and as the ultimate effectors. Upon ligand binding, proteins undergo conformation and motion changes, which can be sensed by the following link in the chain of information. Nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations represent powerful tools for examining the time-dependent function of biological molecules. The recent advances in NMR and the availability of faster computers have opened the door to more detailed analyses of structure, dynamics, and interactions. Here we briefly describe the recent applications that allow NMR spectroscopy and MD simulations to offer unique insight into the basic motions that underlie information transfer within and between cells.

Structural basis for the inhibition of truncated islet amyloid polypeptide aggregation by Cu(II): insights into the bioinorganic chemistry of type II diabetes.

Type 2 diabetes (T2D) is one of the most common chronic diseases, affecting over 300 million people worldwide. One of the hallmarks of T2D is the presence of amyloid deposits of human islet amyloid polypeptide (IAPP) in the islets of Langerhans of pancreatic ß-cells. Recent reports indicate that Cu(II) can inhibit the aggregation of human IAPP, although the mechanism for this inhibitory effect is not clear. In this study, different spectroscopic techniques and model fragments of IAPP were employed to shed light on the structural basis for the interaction of Cu(II) with human IAPP. Our results show that Cu(II) anchors to His18 and the subsequent amide groups toward the C-terminal, forming a complex with an equatorial coordination mode 3N1O at physiological pH. Cu(II) binding to truncated IAPP at the His18 region is the key event for its inhibitory effect in amyloid aggregation. Electron paramagnetic resonance studies indicate that the monomeric Cu(II)-IAPP(15-22) complex differs significantly from Cu(II) bound to mature IAPP(15-22) fibers, suggesting that copper binding to monomeric IAPP(15-22) competes with the conformation changes needed to form ß-sheet structures, thus delaying fibril formation. A general mechanism is proposed for the inhibitory effect of copper and other imidazole-binding metal ions in IAPP amyloid formation, providing further insights into the bioinorganic chemistry of T2D.