Biophysical properties of gammaC-crystallin in human and mouse eye lens: the role of molecular dipoles.

The eye lens is packed with soluble crystallin proteins, providing a lifetime of transparency and light refraction. gamma-Crystallins are major components of the dense, high refractive index central regions of the lens and generally have high solubility, high stability and high levels of cysteine residues. Human gammaC belongs to a group of gamma-crystallins with a pair of cysteine residues at positions 78 and 79. Unlike other gamma-crystallins it has relatively low solubility, whereas mouse gammaC, which has the exposed C79 replaced with arginine, and a novel mouse splice variant, gammaCins, are both highly soluble. Furthermore, human gammaC is extremely stable, while the mouse orthologs are less stable. Evolutionary pressure may have favoured stability over solubility for human gammaC and the reverse for the orthologs in the mouse. Mutation of C79 to R79, in human gammaC, greatly increased solubility, however, neither form produced crystals. Remarkably, when the human gammaD R36S crystallization cataract mutation was mimicked in human gammaC-crystallin, the solubility of gammaC was dramatically increased, although it still did not crystallize. The highly soluble mouse gammaC-crystallin did crystallize. Its X-ray structure was solved and used in homology modelling of human gammaC, and its mutants C79R and R36S. The human gammaD R36S mutant was also modelled from human gammaD coordinates. Molecular dynamics simulation of the six molecules in the solution state showed that the human gammaCs differed from gammaDs in domain pairing, behaviour that correlates with interface sequence changes. When the fluctuations of the calculated molecular dipoles, for the six structures, over time were analysed, characteristic patterns for soluble gammaC and gammaD proteins were observed. Individual sequence changes that increase or decrease solubility correlated well with changes in the magnitude and direction of these dipoles. It is suggested that changes in surface residues have allowed adaptation for the differing needs of human and mouse lenses.

Mutation of interfaces in domain-swapped human betaB2-crystallin.

The superfamily of eye lens betagamma-crystallins is highly modularized, with Greek key motifs being used to form symmetric domains. Sequences of monomeric gamma-crystallins and oligomeric beta-crystallins fold into two domains that pair about a further conserved symmetric interface. Conservation of this assembly interface by domain swapping is the device adopted by family member betaB2-crystallin to form a solution dimer. However, the betaB1-crystallin solution dimer is formed from an interface used by the domain-swapped dimer to form a tetramer in the crystal lattice. Comparison of these two structures indicated an intriguing relationship between linker conformation, interface ion pair networks, and higher assembly. Here the X-ray structure of recombinant human betaB2-crystallin showed that domain swapping was determined by the sequence and not assembly conditions. The solution characteristics of mutants that were designed to alter an ion pair network at a higher assembly interface and a mutant that changed a proline showed they remained dimeric. X-ray crystallography showed that the dimeric mutants did not reverse domain swapping. Thus, the sequence of betaB2-crystallin appears well optimized for domain swapping. However, a charge-reversal mutation to the conserved domain-pairing interface showed drastic changes to solution behavior. It appears that the higher assembly of the betagamma-crystallin domains has exploited symmetry to create diversity while avoiding aggregation. These are desirable attributes for proteins that have to exist at very high concentration for a very long time.

Lengsin is a survivor of an ancient family of class I glutamine synthetases re-engineered by evolution for a role in the vertebrate lens.

Lengsin is a major protein of the vertebrate eye lens. It belongs to the hitherto purely prokaryotic GS I branch of the glutamine synthetase (GS) superfamily, but has no enzyme activity. Like the taxon-specific crystallins, Lengsin is the result of the recruitment of an ancient enzyme to a noncatalytic role in the vertebrate lens. Cryo-EM and modeling studies of Lengsin show a dodecamer structure with important similarities and differences with prokaryotic GS I structures. GS homology regions of Lengsin are well conserved, but the N-terminal domain shows evidence of dynamic evolutionary changes. Compared with birds and fish, most mammals have an additional exon corresponding to part of the N-terminal domain; however, in human, this is a nonfunctional pseudoexon. Genes related to Lengsin are also present in the sea urchin, suggesting that this branch of the GS I family, supplanted by GS II enzymes in vertebrates, has an ancient role in metazoans.

Urochordate betagamma-crystallin and the evolutionary origin of the vertebrate eye lens.

A refracting lens is a key component of our image-forming camera eye; however, its evolutionary origin is unknown because precursor structures appear absent in nonvertebrates. The vertebrate betagamma-crystallin genes encode abundant structural proteins critical for the function of the lens. We show that the urochordate Ciona intestinalis, which split from the vertebrate lineage before the evolution of the lens, has a single gene coding for a single domain monomeric betagamma-crystallin. The crystal structure of Ciona betagamma-crystallin is very similar to that of a vertebrate betagamma-crystallin domain, except for paired, occupied calcium binding sites. The Ciona betagamma-crystallin is only expressed in the palps and in the otolith, the pigmented sister cell of the light-sensing ocellus. The Ciona betagamma-crystallin promoter region targeted expression to the visual system, including lens, in transgenic Xenopus tadpoles. We conclude that the vertebrate betagamma-crystallins evolved from a single domain protein already expressed in the neuroectoderm of the prevertebrate ancestor. The conservation of the regulatory hierarchy controlling betagamma-crystallin expression between organisms with and without a lens shows that the evolutionary origin of the lens was based on co-option of pre-existing regulatory circuits controlling the expression of a key structural gene in a primitive light-sensing system.

gammaN-crystallin and the evolution of the betagamma-crystallin superfamily in vertebrates.

The beta and gamma crystallins are evolutionarily related families of proteins that make up a large part of the refractive structure of the vertebrate eye lens. Each family has a distinctive gene structure that reflects a history of successive gene duplications. A survey of gamma-crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of gammaN-crystallin, an evolutionary bridge between the beta and gamma families. In all species examined, gammaN-crystallins have a hybrid gene structure, half beta and half gamma, and thus appear to be the 'missing link' between the beta and gamma crystallin lineages. Overall, there are four major classes of gamma-crystallin: the terrestrial group (including mammalian gammaA-F); the aquatic group (the fish gammaM-crystallins); the gammaS group; and the novel gammaN group. Like the evolutionarily ancient beta-crystallins (but unlike the terrestrial gammaA-F and aquatic gammaM groups), both the gammaS and gammaN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, gammaN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the gamma-crystallins, also in the retina. Although well conserved throughout vertebrate evolution, gammaN in primates has apparently undergone major changes and possible loss of functional expression.

Sulfur in human crystallins.

Molecular models of human gamma-crystallins and the 'alpha-crystallin domain' of human alphaA-crystallin have been built based on available related X-ray crystal structures. The accessibilities of the component cysteine, methionine and tryptophan side chains in the crystallin models have been calculated. The reactivities of these cysteines, which are oxidised in cataract, are assessed based on their known modifications and within the context of their location within the 3D models.

Crystal structure of truncated human betaB1-crystallin.

Crystallins are long-lived proteins packed inside eye lens fiber cells that are essential in maintaining the transparency and refractive power of the eye lens. Members of the two-domain betagamma-crystallin family assemble into an array of oligomer sizes, forming intricate higher-order networks in the lens cell. Here we describe the 1.4 angstroms resolution crystal structure of a truncated version of human betaB1 that resembles an in vivo age-related truncation. The structure shows that unlike its close homolog, betaB2-crystallin, the homodimer is not domain swapped, but its domains are paired intramolecularly, as in more distantly related monomeric gamma-crystallins. However, the four-domain dimer resembles one half of the crystallographic bovine betaB2 tetramer and is similar to the engineered circular permuted rat betaB2. The crystal structure shows that the truncated betaB1 dimer is extremely well suited to form higher-order lattice interactions using its hydrophobic surface patches, linker regions, and sequence extensions.

Polydispersity of a mammalian chaperone: mass spectrometry reveals the population of oligomers in alphaB-crystallin.

The quaternary structure of the polydisperse mammalian chaperone alphaB-crystallin, a member of the small heat-shock protein family, has been investigated by using electrospray mass spectrometry. The intact assemblies give rise to mass spectra that are complicated by the overlapping of charge states from the different constituent oligomers. Therefore, to determine which oligomers are formed by this protein, tandem mass spectrometry experiments were performed. The spectra reveal a distribution, primarily of oligomers containing 24-33 subunits, the relative populations of which were quantified, to reveal a dominant species being composed of 28 subunits. Additionally, low levels of oligomers as small as 10-mers and as large as 40-mers were observed. Interpretation of the tandem mass spectral data was confirmed by simulating and summing spectra arising from the major individual oligomers. The ability of mass spectrometry to quantify the relative populations of particular oligomeric states also revealed that, contrary to the dimeric associations observed in other small heat-shock proteins, there is no evidence for any stable substructures of bovine alphaB-crystallin isolated from the lens.

High-resolution X-ray crystal structures of human gammaD crystallin (1.25 A) and the R58H mutant (1.15 A) associated with aculeiform cataract.

Several human cataracts have been linked to mutations in the gamma crystallin gene. One of these is the aculeiform cataract, which is caused by an R58H mutation in gammaD crystallin. We have shown previously that this cataract is caused by crystallization of the mutant protein, which is an order of magnitude less soluble than the wild-type. Here, we report the very high-resolution crystal structures of the mutant and wild-type proteins. Both proteins crystallize in the same space group and lattice. Thus, a strict comparison of the protein-protein and protein-water intermolecular interactions in the two crystal lattices is possible. Overall, the differences between the mutant and wild-type structures are small. At position 58, the mutant protein loses the direct ion-pair intermolecular interaction present in the wild-type, due to the differences between histidine and arginine at the atomic level; the interaction in the mutant is mediated by water molecules. Away from the mutation site, the mutant and wild-type lattice structures differ in the identity of side-chains that occupy alternate conformations. Since the interactions in the crystal phase are very similar for the two proteins, we conclude that the reduction in the solubility of the mutant is mainly due to the effect of the R58H mutation in the solution phase. The results presented here are also important as they are the first high-resolution X-ray structures of human gamma crystallins.

The X-ray crystal structure of human gamma S-crystallin C-terminal domain.

gammaS-crystallin is a major human lens protein found in the outer region of the eye lens, where the refractive index is low. Because crystallins are not renewed they acquire post-translational modifications that may perturb stability and solubility. In common with other members of the betagamma-crystallin superfamily, gammaS-crystallin comprises two similar beta-sheet domains. The crystal structure of the C-terminal domain of human gammaS-crystallin has been solved at 2.4 A resolution. The structure shows that in the in vitro expressed protein, the buried cysteines remain reduced. The backbone conformation of the "tyrosine corner" differs from that of other betagamma-crystallins because of deviation from the consensus sequence. The two C-terminal domains in the asymmetric unit are organized about a slightly distorted 2-fold axis to form a dimer with similar geometry to full-length two-domain family members. Two glutamines found in lattice contacts may be important for short range interactions in the lens. An asparagine known to be deamidated in human cataract is located in a highly ordered structural region.