Copper Reductase Activity and Free Radical Chemistry by Cataract-Associated Human Lens γ-Crystallins.

Cataracts are caused by high-molecular-weight aggregates of human eye lens proteins that scatter light, causing lens opacity. Metal ions have emerged as important potential players in the etiology of cataract disease, as human lens γ-crystallins are susceptible to metal-induced aggregation. Here, the interaction of Cu2+ ions with γD-, γC-, and γS-crystallins, the three most abundant γ-crystallins in the lens, has been evaluated. Cu2+ ions induced non-amyloid aggregation in all three proteins. Solution turbidimetry, sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), circular dichroism, and differential scanning calorimetry showed that the mechanism for Cu-induced aggregation involves: (i) loss of ß-sheet structure in the N-terminal domain; (ii) decreased thermal and kinetic stability; (iii) formation of metal-bridged species; and (iv) formation of disulfide-bridged dimers. Isothermal titration calorimetry (ITC) revealed distinct Cu2+ binding affinities in the γ-crystallins. Electron paramagnetic resonance (EPR) revealed two distinct Cu2+ binding sites in each protein. Spin quantitation demonstrated the reduction of γ-crystallin-bound Cu2+ ions to Cu+ under aerobic conditions, while X-ray absorption spectroscopy (XAS) confirmed the presence of linear or trigonal Cu+ binding sites in γ-crystallins. Our EPR and XAS studies revealed that γ-crystallins' Cu2+ reductase activity yields a protein-based free radical that is likely a Tyr-based species in human γD-crystallin. This unique free radical chemistry carried out by distinct redox-active Cu sites in human lens γ-crystallins likely contributes to the mechanism of copper-induced aggregation. In the context of an aging human lens, γ-crystallins could act not only as structural proteins but also as key players for metal and redox homeostasis.

Co-expression of CCT subunits hints at TRiC assembly.

The eukaryotic cytosolic chaperonin, t-complex polypeptide 1 (TCP-1) ring complex or TRiC, is responsible for folding a tenth of the proteins in the cell. TRiC is a double-ringed barrel with each ring composed of eight different CCT (chaperonin containing TCP-1) subunits. In order for the subunits to assemble together into mature TRiC, which is believed to contain one and only one of each of these subunits per ring, they must be translated from different chromosomes, correctly folded and assembled. When expressed alone in Escherichia coli, the subunits CCT4 and CCT5, interestingly, form TRiC-like homo-oligomeric rings. To explore potential subunit-subunit interactions, we co-expressed these homo-oligomerizing CCT4 and CCT5 subunits or the archaeal chaperonin Mm-Cpn (Methanococcus maripaludis chaperonin) with CCT1-8, one at a time. We found that CCT5 shifted all of the CCT subunits, with the exception of CCT6, into double-barrel TRiC-like complexes, while CCT4 only interacted with CCT5 and CCT8 to form chaperonin rings. We hypothesize that these specific interactions may be due to the formation of hetero-oligomers in E. coli, although more work is needed for validation. We also observed the interaction of CCT5 and Mm-Cpn with smaller fragments of the CCT subunits, confirming their intrinsic chaperone activity. Based on this hetero-oligomer data, we propose that TRiC assembly relies on subunit exchange with some stable homo-oligomers, possibly CCT5, as base assembly units. Eventually, analysis of CCT arrangement in various tissues and at different developmental times is anticipated to provide additional insight on TRiC assembly and CCT subunit composition.

Kinetic Stability of Long-Lived Human Lens γ-Crystallins and Their Isolated Double Greek Key Domains.

The γ-crystallins of the eye lens nucleus are among the longest-lived proteins in the human body. Synthesized in utero, they must remain folded and soluble throughout adulthood to maintain lens transparency and avoid cataracts. γD- and γS-crystallin are two major monomeric crystallins of the human lens. γD-crystallin is concentrated in the oldest lens fiber cells, the lens nucleus, whereas γS-crystallin is concentrated in the younger cells of the lens cortex. The kinetic stability parameters of these two-domain proteins and their isolated domains were determined and compared. Kinetic unfolding experiments monitored by fluorescence spectroscopy in varying concentrations of guanidinium chloride were used to extrapolate unfolding rate constants and half-lives of the crystallins in the absence of the denaturant. Consistent with their long lifespans in the lens, extrapolated half-lives for the initial unfolding step were on the timescale of years. Both proteins' isolated N-terminal domains were less kinetically stable than their respective C-terminal domains at denaturant concentrations predicted to disrupt the domain interface, but at low denaturant concentrations, the relative kinetic stabilities were reversed. Cataract-associated aggregation has been shown to proceed from partially unfolded intermediates in these proteins; their extreme kinetic stability likely evolved to protect the lens from the initiation of aggregation reactions. Our findings indicate that the domain interface is the source of significant kinetic stability. The gene duplication and fusion event that produced the modern two-domain architecture of vertebrate lens crystallins may be the origin of their high kinetic as well as thermodynamic stability.

Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography.

Cyanobacteria are photosynthetic organisms responsible for ~25% of the organic carbon fixation on earth. A key step in carbon fixation is catalyzed by ribulose bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme in the biosphere. Applying Zernike phase-contrast electron cryo-tomography and automated annotation, we identified individual RuBisCO molecules and their assembly intermediates leading to the formation of carboxysomes inside Syn5 cyanophage infected cyanobacteria Synechococcus sp. WH8109 cells. Surprisingly, more RuBisCO molecules were found to be present as cytosolic free-standing complexes or clusters than as packaged assemblies inside carboxysomes. Cytosolic RuBisCO clusters and partially assembled carboxysomes identified in the cell tomograms support a concurrent assembly model involving both the protein shell and the enclosed RuBisCO. In mature carboxysomes, RuBisCO is neither randomly nor strictly icosahedrally packed within protein shells of variable sizes. A time-averaged molecular dynamics simulation showed a semi-liquid probability distribution of the RuBisCO in carboxysomes and correlated well with carboxysome subtomogram averages. Our structural observations reveal the various stages of RuBisCO assemblies, which could be important for understanding cellular function.

Structure of the human TRiC/CCT Subunit 5 associated with hereditary sensory neuropathy.

The human chaperonin TRiC consists of eight non-identical subunits, and its protein-folding activity is critical for cellular health. Misfolded proteins are associated with many human diseases, such as amyloid diseases, cancer, and neuropathies, making TRiC a potential therapeutic target. A detailed structural understanding of its ATP-dependent folding mechanism and substrate recognition is therefore of great importance. Of particular health-related interest is the mutation Histidine 147 to Arginine (H147R) in human TRiC subunit 5 (CCT5), which has been associated with hereditary sensory neuropathy. In this paper, we describe the crystal structures of CCT5 and the CCT5-H147R mutant, which provide important structural information for this vital protein-folding machine in humans. This first X-ray crystallographic study of a single human CCT subunit in the context of a hexadecameric complex can be expanded in the future to the other 7 subunits that form the TRiC complex.

Accurate model annotation of a near-atomic resolution cryo-EM map.

Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo-EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata. The functional interpretation of any structural features in the model and its utilization for future studies can be made in the context of its measure of uncertainty. We applied this protocol to the 3.3-Å map of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly. With this protocol, we identify and annotate previously undescribed molecular interactions between capsid subunits that are crucial to maintain stability in the absence of cementing proteins or cross-linking, as occur in other bacteriophages.

An Internal Disulfide Locks a Misfolded Aggregation-prone Intermediate in Cataract-linked Mutants of Human γD-Crystallin.

Considerable mechanistic insight has been gained into amyloid aggregation; however, a large number of non-amyloid protein aggregates are considered "amorphous," and in most cases, little is known about their mechanisms. Amorphous aggregation of γ-crystallins in the eye lens causes cataract, a widespread disease of aging. We combined simulations and experiments to study the mechanism of aggregation of two γD-crystallin mutants, W42R and W42Q: the former a congenital cataract mutation, and the latter a mimic of age-related oxidative damage. We found that formation of an internal disulfide was necessary and sufficient for aggregation under physiological conditions. Two-chain all-atom simulations predicted that one non-native disulfide in particular, between Cys(32) and Cys(41), was likely to stabilize an unfolding intermediate prone to intermolecular interactions. Mass spectrometry and mutagenesis experiments confirmed the presence of this bond in the aggregates and its necessity for oxidative aggregation under physiological conditions in vitro Mining the simulation data linked formation of this disulfide to extrusion of the N-terminal ß-hairpin and rearrangement of the native ß-sheet topology. Specific binding between the extruded hairpin and a distal ß-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation.

Aggregation of Trp > Glu point mutants of human gamma-D crystallin provides a model for hereditary or UV-induced cataract.

Numerous mutations and covalent modifications of the highly abundant, long-lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native-state polymers are commonly found in opaque lenses. The ßγ-crystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV-B irradiation. We mimicked this class of oxidative damage using Trp→Glu point mutants of human γD-crystallin. Such substitutions may represent a model of UV-induced photodamage-introduction of a charged group into the hydrophobic core generating "denaturation from within." The effects of Trp→Glu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold-W42E and W130E-yielded robust aggregation of partially unfolded intermediates at 37°C and pH 7. The αB-crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N-terminal vs C-terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 20°C below the melting temperature of the native ß-sheets. These features are most consistent with domain-swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization.

Resolution and Probabilistic Models of Components in CryoEM Maps of Mature P22 Bacteriophage.

CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and it can be useful to estimate the resolution in specific molecular components of a large assembly. In this study, we present procedures to 1) estimate the resolution in subcomponents by gold-standard Fourier shell correlation (FSC); 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting; and 3) build probabilistic models that combine high-accuracy priors (such as crystallographic structures) with medium-resolution cryoEM densities. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-standard FSC show the highest resolution in the coat region (7.6 Å), whereas other components are at slightly lower resolutions: portal (9.2 Å), hub (8.5 Å), tailspike (10.9 Å), and needle (10.5 Å). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Probabilistic models for these subcomponents provide new insights, to our knowledge, and structural information when taking into account uncertainty given the limitations of the observed density.

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.

Structural Mechanisms of Mutant Huntingtin Aggregation Suppression by the Synthetic Chaperonin-like CCT5 Complex Explained by Cryoelectron Tomography.

Huntington disease, a neurodegenerative disorder characterized by functional deficits and loss of striatal neurons, is linked to an expanded and unstable CAG trinucleotide repeat in the huntingtin gene (HTT). This DNA sequence translates to a polyglutamine repeat in the protein product, leading to mutant huntingtin (mHTT) protein aggregation. The aggregation of mHTT is inhibited in vitro and in vivo by the TCP-1 ring complex (TRiC) chaperonin. Recently, a novel complex comprised of a single type of TRiC subunit has been reported to inhibit mHTT aggregation. Specifically, the purified CCT5 homo-oligomer complex, when compared with TRiC, has a similar structure, ATP use, and substrate refolding activity, and, importantly, it also inhibits mHTT aggregation. Using an aggregation suppression assay and cryoelectron tomography coupled with a novel computational classification method, we uncover the interactions between the synthetic CCT5 complex (∼ 1 MDa) and aggregates of mutant huntingtin exon 1 containing 46 glutamines (mHTTQ46-Ex1). We find that, in a similar fashion to TRiC, synthetic CCT5 complex caps mHTT fibrils at their tips and encapsulates mHTT oligomers, providing a structural description of the inhibition of mHTTQ46-Ex1 by CCT5 complex and a shared mechanism of mHTT inhibition between TRiC chaperonin and the CCT5 complex: cap and contain.

Wild-type human γD-crystallin promotes aggregation of its oxidation-mimicking, misfolding-prone W42Q mutant.

Non-native protein conformers generated by mutation or chemical damage template aggregation of wild-type, undamaged polypeptides in diseases ranging from amyotrophic lateral sclerosis to cancer. We tested for such interactions in the natively monomeric human eye lens protein γd-crystallin, whose aggregation leads to cataract disease. The oxidation-mimicking W42Q mutant of γd-crystallin formed non-native polymers starting from a native-like state under physiological conditions. Aggregation occurred in the temperature range 35-45 °C, in which the mutant protein began to lose the native conformation of its N-terminal domain. Surprisingly, wild-type γd-crystallin promoted W42Q polymerization in a catalytic manner, even at mutant concentrations too low for homogeneous nucleation to occur. The presence of wild-type protein also downshifted the temperature range of W42Q aggregation. W42Q aggregation required formation of a non-native intramolecular disulfide bond but not intermolecular cross-linking. Transient WT/W42Q binding may catalyze this oxidative misfolding event in the mutant. That a more stable variant in a mixture can specifically promote aggregation of a less stable one rationalizes how extensive aggregation of rare damaged polypeptides can occur during the course of aging.

Identifying the assembly pathway of cyanophage inside the marine bacterium using electron cryo-tomography.

Advances in electron cryo-tomography open up a new avenue to visualize the 3-D internal structure of a single bacterium before and after its infection by bacteriophages in its native environment, without using chemical fixatives, fluorescent dyes or negative stains. Such direct observation reveals the presence of assembly intermediates of the bacteriophage and thus allows us to map out the maturation pathway of the bacteriophage inside its host.

Biochemical characterization of mutants in chaperonin proteins CCT4 and CCT5 associated with hereditary sensory neuropathy.

Hereditary sensory neuropathies are a class of disorders marked by degeneration of the nerve fibers in the sensory periphery neurons. Recently, two mutations were identified in the subunits of the eukaryotic cytosolic chaperonin TRiC, a protein machine responsible for folding actin and tubulin in the cell. C450Y CCT4 was identified in a stock of Sprague-Dawley rats, whereas H147R CCT5 was found in a human Moroccan family. As with many genetically identified mutations associated with neuropathies, the underlying molecular basis of the mutants was not defined. We investigated the biochemical properties of these mutants using an expression system in Escherichia coli that produces homo-oligomeric rings of CCT4 and CCT5. Full-length versions of both mutant protein chains were expressed in E. coli at levels approaching that of the WT chains. Sucrose gradient centrifugation revealed chaperonin-sized complexes of both WT and mutant chaperonins, but with reduced recovery of C450Y CCT4 soluble subunits. Electron microscopy of negatively stained samples of C450Y CCT4 revealed few ring-shaped species, whereas WT CCT4, H147R CCT5, and WT CCT5 revealed similar ring structures. CCT5 complexes were assayed for their ability to suppress aggregation of and refold the model substrate γd-crystallin, suppress aggregation of mutant huntingtin, and refold the physiological substrate ß-actin in vitro. H147R CCT5 was not as efficient in chaperoning these substrates as WT CCT5. The subtle effects of these mutations are consistent with the homozygous disease phenotype, in which most functions are carried out during development and adulthood, but some selective function is lost or reduced.

Protruding knob-like proteins violate local symmetries in an icosahedral marine virus.

Marine viruses play crucial roles in shaping the dynamics of oceanic microbial communities and in the carbon cycle on Earth. Here we report a 4.7-Å structure of a cyanobacterial virus, Syn5, by electron cryo-microscopy and modelling. A Cα backbone trace of the major capsid protein (gp39) reveals a classic phage protein fold. In addition, two knob-like proteins protruding from the capsid surface are also observed. Using bioinformatics and structure analysis tools, these proteins are identified to correspond to gp55 and gp58 (each with two copies per asymmetric unit). The non 1:1 stoichiometric distribution of gp55/58 to gp39 breaks all expected local symmetries and leads to non-quasi-equivalence of the capsid subunits, suggesting a role in capsid stabilization. Such a structural arrangement has not yet been observed in any known virus structures.

The ßγ-crystallins: native state stability and pathways to aggregation.

The ßγ-crystallins are among the most stable and long-lived proteins in the human body. With increasing age, however, they transform to high molecular weight light-scattering aggregates, resulting in cataracts. This occurs despite the presence in the lens of high concentrations of the a-crystallin chaperones. Aggregation of crystallins can be induced in vitro by a variety of stresses, including acidic pH, ultraviolet light, oxidative damage, heating or freezing, and specific amino acid substitutions. Accumulating evidence points to the existence of specific biochemical pathways of protein: protein interaction and polymerization. We review the methods used for studying crystallin stability and aggregation and discuss the sometimes counterintuitive relationships between factors that favor native state stability and those that favor non-native aggregation. We discuss the behavior of ßγ-crystallins in mixtures and their chaperone ability; the consequences of missense mutations and covalent damage to the side-chains; and the evolutionary strategies that have shaped these proteins. Efforts are ongoing to reveal the nature of cataractous crystallin aggregates and understand the mechanisms of aggregation in the context of key models of protein polymerization: amyloid, native-state, and domain-swapped. Such mechanistic understanding is likely to be of value for the development of therapeutic interventions and draw attention to unanswered questions about the relationship between a protein's native state stability and its transformation to an aggregated state.

Group II archaeal chaperonin recognition of partially folded human γD-crystallin mutants.

The features in partially folded intermediates that allow the group II chaperonins to distinguish partially folded from native states remain unclear. The archaeal group II chaperonin from Methanococcus Mauripaludis (Mm-Cpn) assists the in vitro refolding of the well-characterized ß-sheet lens protein human γD-crystallin (HγD-Crys). The domain interface and buried cores of this Greek key conformation include side chains, which might be exposed in partially folded intermediates. We sought to assess whether particular features buried in the native state, but absent from the native protein surface, might serve as recognition signals. The features tested were (a) paired aromatic side chains, (b) side chains in the interface between the duplicated domains of HγD-Crys, and (c) side chains in the buried core which result in congenital cataract when substituted. We tested the Mm-Cpn suppression of aggregation of these HγD-Crys mutants upon dilution out of denaturant. Mm-Cpn was capable of suppressing the off-pathway aggregation of the three classes of mutants indicating that the buried residues were not recognition signals. In fact, Mm-Cpn recognized the HγD-Crys mutants better than (wild-type) WT and refolded most mutant HγD-Crys to levels twice that of WT HγD-Crys. This presumably represents the increased population or longer lifetimes of the partially folded intermediates of the mutant proteins. The results suggest that Mm-Cpn does not recognize the features of HγD-Crys tested-paired aromatics, exposed domain interface, or destabilized core-but rather recognizes other features of the partially folded ß-sheet conformation that are absent or inaccessible in the native state of HγD-Crys.

Two novel proteins of cyanophage Syn5 compose its unusual horn structure.

The marine cyanophage Syn5 can be propagated to a high titer in the laboratory on marine photosynthetic Synechococcus sp. strain WH8109. The purified particles carry a novel slender horn structure projecting from the vertex opposite the tail vertex. The genome of Syn5 includes a number of genes coding for novel proteins. Using immune-electron microscopy with gold-labeled antibodies, we show that two of these novel proteins, products of genes 53 and 54, are part of the horn structure. A third novel protein, the product of gene 58, is assembled onto the icosahedral capsid lattice. Characterization of radioactively labeled precursor procapsids by sucrose gradient centrifugation shows that there appear to be three classes of particles-procapsids, scaffold-deficient procapsids, and expanded capsids. These lack fully assembled horn appendages. The horn presumably assembles onto the virion just before or after DNA packaging. Antibodies raised to the recombinant novel Syn5 proteins did not interfere with phage infectivity, suggesting that the functions of these proteins are not directly involved in phage attachment or infection of the host WH8109. The horn structure may represent some adaption to the marine environment, whose function will require additional investigation.