Christine Slingsby

Crystal structure and assembly of a eukaryotic small heat shock protein.

The 2.7 Å structure of wheat HSP16.9, a member of the small heat shock proteins (sHSPs), indicates how its α-crystallin domain and flanking extensions assemble into a dodecameric double disk. The folding of the monomer and assembly of the oligomer are mutually interdependent, involving strand exchange, helix swapping, loose knots and hinged extensions. In support of the chaperone mechanism, the substrate-bound dimers, in temperature-dependent equilibrium with higher assembly forms, have unfolded N-terminal arms and exposed conserved hydrophobic binding sites on the α-crystallin domain. The structure also provides a model by which members of the sHSP protein family bind unfolded substrates, which are involved in a variety of neurodegenerative diseases and cataract formation.

Structure and function of the small heat shock protein/α-crystallin family of molecular chaperones

Publisher Summary The goal of this chapter is to clarify the diversity within the heat shock proteins (sHsps) family and to describe evidence indicating that sHsps have many different substrates and affect a wide range of cellular functions. The diversity of sHsp structure and expression patterns is immense, and their activities in vivo may involve multiple mechanisms. The sHsps and the structurally related vertebrate eye lens α-crystallins are the poor cousins in the family of molecular chaperones, and remain the least understood both structurally and functionally. The chaperone model for sHsp function provides a basic framework to explain the many proposed sHsp/protein interactions and potential functions. The diversity of the sHsp family, however, indicates that care must be taken in generalizing biochemical properties and activities across different family members. Nonetheless, the chapter has a firmer structural foundation on which to design future experiments to build a biochemical mechanism of action.

X-ray analysis of the eye lens protein γ-II crystallin at 1·9 Å resolution*

We report the X-ray structure analysis and refinement at 1·9 A resolution of calf γ-II crystallin, a lens-specific protein. The sequence of Croft (1972) has been modified to give a polypeptide chain of 174 residues (cf. 165). The protein has a symmetrical, hierarchical structure of two globular domains each comprising two similar “Greek key” motifs, consecutive along the polypeptide chain, and related by a pseudo 2-fold axis. The two domains pack together with a single connection and are related by a further pseudo 2-fold axis which bisects the angle between the intra-domain dyads. Forty-two pairs of Cα positions for the two most similar motifs have root-mean-square separation at best fit of 0·69 A. The N and C-terminal domains gave root-mean-square separation of 0·89 A for 82 pairs of Cα atoms at best fit. In each domain the two Greek key motifs form a pair of four-stranded antiparallel β-pleated sheets, each sheet composed of three stands from one motif and one from the other. The sheets pack together in a wedge shape, closed at the top by the loops connecting the third and fourth strands of each motif. The two strands of each motif form an extended β-hairpin which is folded on to the β-sheet. The packing of each motif into the globular domains involves a staggered bilayer of side-chains between each pair of β-sheets which does not preserve the pseudo 2-fold axes observed in the Cα position topology. In the core of each domain there are interactions between polarizable aromatic groups and sulphur-containing residues which may contribute to stability and may also serve to protect aromatic side-chains from ultraviolet light damage in the lens. At the surface of the molecule over half the ionic side-chains are closely paired, which probably stabilizes the tertiary fold and may reduce the water bound. Crystal lattice interactions are described which may be similar to those occurring in vivo in the lens between crystallins. Seven cysteine residues have been identified in the structure and these may have a role in the thermodynamic stability of the molecule, its intermolecular interactions under the normal reducing conditions of the lens, and also in the aggregation and cross-linking which occur in some forms of cataract. Three of these residues, Cys18, Cys23 and Cys74, form a cluster in the N-terminal domain. The high-resolution data from relatively aged crystals suggest that a disulphide bond exists between Cys18 and Cys23 under appropriate oxidizing conditions. Cys15 is very exposed, is involved in a crystal lattice interaction with arginine, and could form an intermolecular disulphide in solution when oxidized.

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.

Lens Crystallins and Their Microbial Homologs: Structure, Stability, and Function

Referee: Franz Schmid, Biochemicshes Laboratorium, Universitaet Bayeuth, D-95440 Bayeuth, Germany abg-Crystallins are the major protein components in the vertebrate eye lens — a as a molecular chaperone and b and g as structural proteins. Surprisingly, the latter two share some structural characteristics with a number of microbial stress proteins. The common denominator is not only the Greek key topology of their polypeptide chains but also their high intrinsic stability, which, in certain microbial crystallin homologs, is further enhanced by high-affinity Ca2+-binding. Recent studies of natural and mutant vertebrate bg-crystallins as well as spherulin 3a from Physarum polycephalum and Protein S from Myxococcus xanthus allowed the correlation of structure and stability of crystallins to be elucidated in some detail. From the thermo-dynamic point of view, stability increments come from (1) local interactions involved in the close packing of the cooperative units, (2) the all-b secondary structure of the Greek-key motif, (3) intramolecular interactions between domains, (4) intermolecular domain interactions, including 3D domain swapping and (v) excluded volume effects due to “molecular crowding” at the high cellular protein concentrations. Apart from these contributions to the Gibbs free energy of stability, significant kinetic stabilization originates from the high activation energy barrier determining the rate of unfolding from the native to the unfolded state. From the functional point of view, the high stability is responsible for the long-term transparency of the eye lens, on the one hand, and the stress resistance of the microorganisms in their dormant state on the other. Local structural perturbations due to chemical modification, wrong protein interactions, or other irreversible processes may lead to protein aggregation. A leading cataract hypothesis is that only after a-crystallin, a member of the small heat-shock protein family, is titrated out does pathological opacity occur. Understanding the structural basis of protein stability in the healthy eye lens is the route to solve the enormous medical and economical problem of cataract.

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.

Eye-lens proteins: The three-dimensional structure of β-crystallin predicted from monomeric γ-crystallin

Recent sequence determinations [l-4] have demonstrated that two of the three classes of mammalian lens-specific proteins, the pand y-crystallins, form a single superfamily of proteins, accounting for >50% of the protein in the mammalian lens. show such a strong internal homology except for some residues at particular features in the 711 fold which do display a 4-fold repeat [ 121. There is, however, a general conservation of functional types of residue between the 4 folding motifs of the structure.

Crystal structure of R120G disease mutant of human αB-crystallin domain dimer shows closure of a groove.

Small heat shock proteins form large cytosolic assemblies from an “α-crystallin domain” (ACD) flanked by sequence extensions. Mutation of a conserved arginine in the ACD of several human small heat shock protein family members causes many common inherited diseases of the lens and neuromuscular system. The mutation R120G in αB-crystallin causes myopathy, cardiomyopathy and cataract. We have solved the X-ray structure of the excised ACD dimer of human αB R120G close to physiological pH and compared it with several recently determined wild-type vertebrate ACD dimer structures. Wild-type excised ACD dimers have a deep groove at the interface floored by a flat extended “bottom sheet.” Solid-state NMR studies of large assemblies of full-length αB-crystallin have shown that the groove is blocked in the ACD dimer by curvature of the bottom sheet. The crystal structure of R120G ACD dimer also reveals a closed groove, but here the bottom sheet is flat. Loss of Arg120 results in rearrangement of an extensive array of charged interactions across this interface. His83 and Asp80 on movable arches on either side of the interface close the groove by forming two new salt bridges. The residues involved in this extended set of ionic interactions are conserved in Hsp27, Hsp20, αA- and αB-crystallin sequences. They are not conserved in Hsp22, where mutation of the equivalent of Arg120 causes neuropathy. We speculate that the αB R120G mutation disturbs oligomer dynamics, causing the growth of large soluble oligomers that are toxic to cells by blocking essential processes.