Orval A. Bateman

Crystal Structures of α-Crystallin Domain Dimers of αB-Crystallin and Hsp20

Small heat shock proteins (sHsps) are a family of large and dynamic oligomers highly expressed in long-lived cells of muscle, lens and brain. Several family members are upregulated during stress, and some are strongly cytoprotective. Their polydispersity has hindered high-resolution structure analyses, particularly for vertebrate sHsps. Here, crystal structures of excised alpha-crystallin domain from rat Hsp20 and that from human alphaB-crystallin show that they form homodimers with a shared groove at the interface by extending a beta sheet. However, the two dimers differ in the register of their interfaces. The dimers have empty pockets that in large assemblies will likely be filled by hydrophobic sequence motifs from partner chains. In the Hsp20 dimer, the shared groove is partially filled by peptide in polyproline II conformation. Structural homology with other sHsp crystal structures indicates that in full-length chains the groove is likely filled by an N-terminal extension. Inside the groove is a symmetry-related functionally important arginine that is mutated, or its equivalent, in family members in a range of neuromuscular diseases and cataract. Analyses of residues within the groove of the alphaB-crystallin interface show that it has a high density of positive charges. The disease mutant R120G alpha-crystallin domain dimer was found to be more stable at acidic pH, suggesting that the mutation affects the normal dynamics of sHsp assembly. The structures provide a starting point for modelling higher assembly by defining the spatial locations of grooves and pockets in a basic dimeric assembly unit. The structures provide a high-resolution view of a candidate functional state of an sHsp that could bind non-native client proteins or specific components from cytoprotective pathways. The empty pockets and groove provide a starting model for designing drugs to inhibit those sHsps that have a negative effect on cancer treatment.

Polydispersity of a mammalian chaperone: Mass spectrometry reveals the population of oligomers in αB-crystallin

The quaternary structure of the polydisperse mammalian chaperone αB-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 αB-crystallin isolated from the lens.

High-resolution X-ray Crystal Structures of Human γD Crystallin (1.25 Å) and the R58H Mutant (1.15 Å) 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.

Urochordate βγ-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 [1]. The vertebrate βγ-crystallin genes encode abundant structural proteins critical for the function of the lens [2]. 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 βγ-crystallin. The crystal structure of Ciona βγ-crystallin is very similar to that of a vertebrate βγ-crystallin domain, except for paired, occupied calcium binding sites. The Ciona βγ-crystallin is only expressed in the palps and in the otolith, the pigmented sister cell of the light-sensing ocellus. The Ciona βγ-crystallin promoter region targeted expression to the visual system, including lens, in transgenic Xenopus tadpoles. We conclude that the vertebrate βγ-crystallins evolved from a single domain protein already expressed in the neuroectoderm of the prevertebrate ancestor. The conservation of the regulatory hierarchy controlling βγ-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.

Crystal structure of truncated human βB1-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 βγ‐crystallin family assemble into an array of oligomer sizes, forming intricate higher‐order networks in the lens cell. Here we describe the 1.4 Å resolution crystal structure of a truncated version of human βB1 that resembles an in vivo age‐related truncation. The structure shows that unlike its close homolog, βB2‐crystallin, the homodimer is not domain swapped, but its domains are paired intramolecularly, as in more distantly related monomeric γ‐crystallins. However, the four‐domain dimer resembles one half of the crystallographic bovine βB2 tetramer and is similar to the engineered circular permuted rat βB2. The crystal structure shows that the truncated βB1 dimer is extremely well suited to form higher‐order lattice interactions using its hydrophobic surface patches, linker regions, and sequence extensions.

γN‐crystallin and the evolution of the βγ‐crystallin superfamily in vertebrates

The β and γ 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 γ‐crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of γN‐crystallin, an evolutionary bridge between the β and γ families. In all species examined, γN‐crystallins have a hybrid gene structure, half β and half γ, and thus appear to be the ‘missing link’ between the β and γ crystallin lineages. Overall, there are four major classes of γ‐crystallin: the terrestrial group (including mammalian γA–F); the aquatic group (the fish γM‐crystallins); the γS group; and the novel γN group. Like the evolutionarily ancient β‐crystallins (but unlike the terrestrial γA–F and aquatic γM groups), both the γS and γN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, γN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the γ‐crystallins, also in the retina. Although well conserved throughout vertebrate evolution, γN in primates has apparently undergone major changes and possible loss of functional expression.

The stability of human acidic β-crystallin oligomers and hetero-oligomers

Abstract Crystallins are bulk structural proteins of the eye lens that have to last a life time. They gradually become modified with age, denature and form light scattering centres. High thermodynamic and kinetic stability of the crystallins enables them to resist unfolding and delay cataract. Here we have made recombinant human βA1-, βA3-, and βA4-crystallins. The βA3-crystallin formed higher oligomers that lead to precipitation at ambient temperature. Heat-induced precipitation of βA3-crystallin was compared with human and calf βB2-crystallins, showing that the human proteins start to precipitate above 50°C while the calf βB2-crystallin stays in solution even when unfolded. The stabilities of these human acidic β-crystallin homo-oligomers have been estimated by measuring their unfolding in urea at neutral pH. βA3/1/βB1 and βA4/βB1-crystallin hetero-oligomers have been prepared from homo-oligomers by subunit exchange. The resolution of the methodology used was insufficient to detect a stabilization of the βA4-crystallin subunit in the hetero-oligomer, the βA1-crystallin subunit was clearly stabilized by its interaction with βB1-crystallin. Circular dichroism and fluorescence spectroscopies show that homo-dimer surface tryptophans become buried in the βA3/1/βB1-crystallin hetero-dimer concomitant with changes in polypeptide chain conformation.

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

The β and γ 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 γ‐crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of γN‐crystallin, an evolutionary bridge between the β and γ families. In all species examined, γN‐crystallins have a hybrid gene structure, half β and half γ, and thus appear to be the ‘missing link’ between the β and γ crystallin lineages. Overall, there are four major classes of γ‐crystallin: the terrestrial group (including mammalian γA–F); the aquatic group (the fish γM‐crystallins); the γS group; and the novel γN group. Like the evolutionarily ancient β‐crystallins (but unlike the terrestrial γA–F and aquatic γM groups), both the γS and γN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, γN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the γ‐crystallins, also in the retina. Although well conserved throughout vertebrate evolution, γN in primates has apparently undergone major changes and possible loss of functional expression.

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

The superfamily of eye lens βγ‐crystallins is highly modularized, with Greek key motifs being used to form symmetric domains. Sequences of monomeric γ‐crystallins and oligomeric β‐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 βB2‐crystallin to form a solution dimer. However, the βB1‐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 βB2‐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 βB2‐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 βγ‐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.

γN-crystallin and the evolution of the βγ-crystallin superfamily in vertebrates: γN-crystallin

The β and γ 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 γ‐crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of γN‐crystallin, an evolutionary bridge between the β and γ families. In all species examined, γN‐crystallins have a hybrid gene structure, half β and half γ, and thus appear to be the ‘missing link’ between the β and γ crystallin lineages. Overall, there are four major classes of γ‐crystallin: the terrestrial group (including mammalian γA–F); the aquatic group (the fish γM‐crystallins); the γS group; and the novel γN group. Like the evolutionarily ancient β‐crystallins (but unlike the terrestrial γA–F and aquatic γM groups), both the γS and γN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, γN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the γ‐crystallins, also in the retina. Although well conserved throughout vertebrate evolution, γN in primates has apparently undergone major changes and possible loss of functional expression.