Circular permutation of betaB2-crystallin changes the hierarchy of domain assembly.

The betagamma-crystallins form a superfamily of eye lens proteins comprised of multiple Greek motifs that are symmetrically organized into domains and higher assemblies. In the betaB2-crystallin dimer each polypeptide folds into two similar domains that are related to monomeric gamma-crystallin by domain swapping. The crystal structure of the circularly permuted two-domain betaB2 polypeptide shows that permutation converts intermolecular domain pairing into intramolecular pairing. However, the dimeric permuted protein is, in fact, half a native tetramer. This result shows how the sequential order of domains in multi-domain proteins can affect quaternary domain assembly.     

The N-terminal domain of betaB2-crystallin resembles the putative ancestral homodimer

betagamma-crystallins from the eye lens are proteins consisting of two similar domains joined by a short linker. All three-dimensional structures of native proteins solved so far reveal similar pseudo-2-fold pairing of the domains reflecting their presumed ancient origin from a single-domain homodimer. However, studies of engineered single domains of members of the betagamma-crystallin superfamily have not revealed a prototype ancestral solution homodimer. Here we report the 2.35 A X-ray structure of the homodimer of the N-terminal domain of rat betaB2-crystallin (betaB2-N). The two identical domains pair in a symmetrical manner very similar to that observed in native betagamma-crystallins, where N and C-terminal domains (which share approximately 35% sequence identity) are related by a pseudo-2-fold axis. betaB2-N thus resembles the ancestral prototype of the betagamma-crystallin superfamily as it self-associates in solution to form a dimer with an essentially identical domain interface as that between the N and C domains in betagamma-crystallins, but without the benefit of a covalent linker. The structure provides further evidence for the role of two-domain pairing in stabilising the protomer fold. These results support the view that the betagamma-crystallin superfamily has evolved by a series of gene duplication and fusion events from a single-domain ancestor capable of forming homodimers.     

Specificity of alphaA-crystallin binding to destabilized mutants of betaB1-crystallin

To elucidate the structural and energetic basis of attractive protein interactions in the aging lens, we investigated the binding of destabilized mutants of betaB1-crystallin to the lens chaperones, alpha-crystallins. We show that the mutations enhance the binding affinity to alphaA- but not alphaB-crystallin at physiological temperatures. Complex formation disrupts the dimer interface of betaB1-crystallin consistent with the binding of a monomer. Binding isotherms obtained at increasing concentrations of betaB1-crystallin deviate from a classic binding equilibrium and display cooperative-like behavior. In the context of betaB1-crystallin unfolding equilibrium, these characteristics are reflective of the concentration-dependent change in the population of a dimeric intermediate that has low affinity to alphaA-crystallin. In the lens, where alpha-crystallin binding sites are not regenerated, this may represent an added mechanism to maintain lens transparency.     

Structural and Mechanical Hierarchies in the α-Crystallin Domain Dimer of the Hyperthermophilic Small Heat Shock Protein Hsp16.5

In biological systems, proteins rarely act as isolated monomers. Association to dimers or higher oligomers is a commonly observed phenomenon. As an example, small heat shock proteins form spherical homo-oligomers of mostly 24 subunits, with the dimeric α-crystallin domain as the basic structural unit. The structural hierarchy of this complex is key to its function as a molecular chaperone. In this article, we analyze the folding and association of the basic building block, the α-crystallin domain dimer, from the hyperthermophilic archaeon Methanocaldococcus jannaschii Hsp16.5 in detail. Equilibrium denaturation experiments reveal that the α-crystallin domain dimer is highly stable against chemical denaturation. In these experiments, protein dissociation and unfolding appear to follow an “all-or-none” mechanism with no intermediate monomeric species populated. When the mechanical stability was determined by single-molecule force spectroscopy, we found that the α-crystallin domain dimer resists high forces when pulled at its termini. In contrast to bulk denaturation, stable monomeric unfolding intermediates could be directly observed in the mechanical unfolding traces after the α-crystallin domain dimer had been dissociated by force. Our results imply that for this hyperthermophilic member of the small heat shock protein family, assembly of the spherical 24mer starts from folded monomers, which readily associate to the dimeric structure required for assembly of the higher oligomer.     

Unfolding crystallins: the destabilizing role of a beta-hairpin cysteine in betaB2-crystallin by simulation and experiment

The thermodynamic and kinetic stabilities of the eye lens family of betagamma-crystallins are important factors in the etiology of senile cataract. They control the chance of proteins unfolding, which can lead to aggregation and loss of transparency. betaB2-Crystallin orthologs are of low stability and comprise two typical betagamma-crystallin domains, although, uniquely, the N-terminal domain has a cysteine in one of the conserved folded beta-hairpins. Using high-temperature (500 K) molecular dynamics simulations with explicit solvent on the N-terminal domain of rodent betaB2-crystallin, we have identified in silico local flexibility in this folded beta-hairpin. We have shown in vitro using two-domain human betaB2-crystallin that replacement of this cysteine with a more usual aromatic residue (phenylalanine) results in a gain in conformational stability and a reduction in the rate of unfolding. We have used principal components analysis to visualize and cluster the coordinates from eight separate simulated unfolding trajectories of both the wild-type and the C50F mutant N-terminal domains. These data, representing fluctuations around the native well, show that although the mutant and wild-type appear to behave similarly over the early time period, the wild type appears to explore a different region of conformational space. It is proposed that the advantage of having this low-stability cysteine may be correlated with a subunit-exchange mechanism that allows betaB2-crystallin to interact with a range of other beta-crystallin subunits.     

Analysis of betaB1-crystallin unfolding equilibrium by spin and fluorescence labeling: evidence of a dimeric intermediate

A central step in understanding lens aging is to characterize the thermodynamic stability of its proteins and determine the consequences of changes in the primary sequence on their folding equilibria. For this purpose, destabilized mutations were introduced in betaB1-crystallin targeting the domain interface within the fold of a subunit. Global unfolding was monitored by tryptophan fluorescence while concomitant structural changes at the dimer interface were monitored by fluorescence and spin labels. Both spectral probes report explicit evidence of multi-state unfolding equilibrium. The biphasic nature of the unfolding curves was more pronounced at higher protein concentration. Distinct shifts in the midpoint of the second transition reflect the population of a dimeric intermediate. This intermediate may be a critical determinant for the life-long stability of the beta-crystallins and has important consequences on interactions with alpha-crystallin.     

Expression of human topoisomerase I with a partial deletion of the linker region yields monomeric and dimeric enzymes that respond differently to camptothecin

Human topoisomerase I is a 765-residue protein composed of four major domains as follows: the unconserved and highly charged NH(2)-terminal domain, a conserved core domain, the positively charged linker region, and the highly conserved COOH-terminal domain containing the active site tyrosine. Previous studies of the domain structure revealed that near full topoisomerase I activity can be reconstituted in vitro by fragment complementation between recombinant polypeptides approximating the core and COOH-terminal domains. Here we demonstrate that deletion of linker residues Asp(660) to Lys(688) yields an active enzyme (topo70DeltaL) that purifies as both a monomer and a dimer. The dimer is shown to result from domain swapping involving the COOH-terminal and core domains of the two subunits. The monomeric form is insensitive to the anti-tumor agent camptothecin and distributive during in vitro plasmid relaxation assays, whereas the dimeric form is camptothecin-sensitive and processive. However, the addition of camptothecin to enzyme/DNA mixtures causes enhancement of SDS-induced breakage by both monomeric and dimeric forms of the mutant enzyme. The similarity of the dimeric form to the wild type enzyme suggests that some structural feature of the dimer is providing a surrogate linker. Yeast cells expressing topo70DeltaL were found to be insensitive to camptothecin.     

Unfolding of human lens recombinant betaB2- and gammaC-crystallins

betaB2- and gammaC-crystallins belong to the betagamma-crystallin superfamily and have very similar structures. Molecular spectroscopic techniques such as UV-visible absorption, circular dichroism, and fluorescence indicate they have similar biophysical properties. Their structures are characterized by the presence of two domains consisting of four Greek key motifs. The only difference is the connecting peptide of the two domains, which is flexible in gamma-crystallin but extended in beta-crystallin; thus, an intradomain association and a monomer are formed in gamma-crystallin and an interdomain association and a dimer are formed in beta-crystallin. The difference may be reflected in the thermodynamic stability. In the present study, we calculated the standard free-energy by equilibrium unfolding transition in guanidine hydrochloride using three spectroscopic parameters: absorbance at 235nm, Trp fluorescence intensity at 320nm, and far-UV circular dichroism at 223nm. Global analyses indicate that both dimeric betaB2- and monomeric gammaC-crystallins are a better fit to a three-state model than to a two-state model. In terms of standard free-energy, deltaG(0)(H(2)O,i) both betaB2-crystallin and gammaC-crystallin are stable proteins and dimeric betaB2-crystallin is more stable than the monomeric gammaC-crystallin. The significance of the thermodynamic stability for betaB2- and gammaC-crystallins may be related to their functions in the lens.     

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.     

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 α-crystallin domain from rat Hsp20 and that from human αB-crystallin show that they form homodimers with a shared groove at the interface by extending a β 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 αB-crystallin interface show that it has a high density of positive charges. The disease mutant R120G α-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.     

Three-dimensional structure of α-crystallin domain dimers of human small heat shock proteins HSPB1 and HSPB6

Small heat shock proteins (sHSPs) are a family of evolutionary conserved ATP-independent chaperones. These proteins share a common architecture defined by a signature α-crystallin domain (ACD) flanked by highly variable N- and C-terminal extensions. The ACD, which has an immunoglobulin-like fold, plays an important role in sHSP assembly. This domain mediates dimer formation of individual protomers, which then may assemble into larger oligomers. In vertebrate sHSPs, the dimer interface is formed by the symmetrical antiparallel pairing of two β-strands (β7), generating an extended β-sheet on one face of the ACD dimer. Recent structural studies of isolated ACDs from a number of vertebrate sHSPs suggest a variability in the register of the β7/β7 strand interface, which may, in part, give rise to the polydispersity often associated with the full-length proteins. To further analyze the structure of ACD dimers, we have employed a combination of X-ray crystallography and solution small-angle X-ray scattering (SAXS) to study the ACD-containing fragments of human HSPB1 (HSP27) and HSPB6 (HSP20). Unexpectedly, the obtained crystal structure of the HSPB1 fragment does not reveal the typical β7/β7 dimers but, rather, hexamers formed by an asymmetric contact between the β4 and the β7 strands from adjacent ACDs. Nevertheless, in solution, both ACDs form stable dimers via the symmetric antiparallel interaction of β7 strands. Using SAXS, we show that it is possible to discriminate between different putative registers of the β7/β7 interface, with the results indicating that, under physiological conditions, there is only a single register of the strands for both proteins.     

Formation of betaA3/betaB2-crystallin mixed complexes: involvement of N- and C-terminal extensions.

The sequence extensions of the beta-crystallin subunits have been suggested to play an important role in the oligomerization of these eye lens proteins. This, in turn, may contribute to maintaining lens transparency and proper light refraction. In homo-dimers of the betaA3- and betaB2-crystallin subunits, these extensions have been shown by (1)H-NMR spectroscopy to be solvent-exposed and highly flexible. In this study, we show that betaA3- and betaB2-crystallins spontaneously form mixed betaA3/betaB2-crystallin complexes, which, from analytical ultracentrifugation experiments, are dimeric at low concentrations (<1 mg ml(-1)) and tetrameric at higher protein concentrations. (1)H-NMR spectroscopy reveals that in the betaA3/betaB2-crystallin tetramer, the N-terminal extensions of betaA3-crystallin remain water-exposed and flexible, whereas both N- and C-terminal extensions of betaB2-crystallin lose their flexibility. We conclude that both extensions of betaB2-crystallin are involved in protein-protein interactions in the betaA3/betaB2-crystallin hetero-tetramer. The extensions may stabilize and perhaps promote the formation of this mixed complex.     

Deamidation in human lens betaB2-crystallin destabilizes the dimer

Two major determinants of the transparency of the lens are protein-protein interactions and stability of the crystallins, the structural proteins in the lens. betaB2 is the most abundant beta-crystallin in the human lens and is important in formation of the complex interactions of lens crystallins. betaB2 readily forms a homodimer in vitro, with interacting residues across the monomer-monomer interface conserved among beta-crystallins. Due to their long life spans, crystallins undergo an unusually large number of modifications, with deamidation being a major factor. In this study the effects of two potential deamidation sites at the monomer-monomer interface on dimer formation and stability were determined. Glutamic acid substitutions were constructed to mimic the effects of previously reported deamidations at Q162 in the C-terminal domain and at Q70, its N-terminal homologue. The mutants had a nativelike secondary structure similar to that of wild type betaB2 with differences in tertiary structure for the double mutant, Q70E/Q162E. Multiangle light scattering and quasi-elastic light scattering experiments showed that dimer formation was not interrupted. In contrast, equilibrium unfolding and refolding in urea showed destabilization of the mutants, with an inflection in the transition of unfolding for the double mutant suggesting a distinct intermediate. These results suggest that deamidation at critical sites destabilizes betaB2 and may disrupt the function of betaB2 in the lens.     

Biophysical Analysis of the MHR Motif in Folding and Domain Swapping of the HIV Capsid Protein C-Terminal Domain

Infection by human immunodeficiency virus (HIV) depends on the function, in virion morphogenesis and other stages of the viral cycle, of a highly conserved structural element, the major homology region (MHR), within the carboxyterminal domain (CTD) of the capsid protein. In a modified CTD dimer, MHR is swapped between monomers. While no evidence for MHR swapping has been provided by structural models of retroviral capsids, it is unknown whether it may occur transiently along the virus assembly pathway. Whatever the case, the MHR-swapped dimer does provide a novel target for the development of anti-HIV drugs based on the concept of trapping a nonnative capsid protein conformation. We have carried out a thermodynamic and kinetic characterization of the domain-swapped CTD dimer in solution. The analysis includes a dissection of the role of conserved MHR residues and other amino acids at the dimerization interface in CTD folding, stability, and dimerization by domain swapping. The results revealed some energetic hotspots at the domain-swapped interface. In addition, many MHR residues that are not in the protein hydrophobic core were nevertheless found to be critical for folding and stability of the CTD monomer, which may dramatically slow down the swapping reaction. Conservation of MHR residues in retroviruses did not correlate with their contribution to domain swapping, but it did correlate with their importance for stable CTD folding. Because folding is required for capsid protein function, this remarkable MHR-mediated conformational stabilization of CTD may help to explain the functional roles of MHR not only during immature capsid assembly but in other processes associated with retrovirus infection. This energetic dissection of the dimerization interface in MHR-swapped CTD may also facilitate the design of anti-HIV compounds that inhibit capsid assembly by conformational trapping of swapped CTD dimers.     

Crystal Structure of HypA, a Nickel-Binding Metallochaperone for [NiFe] Hydrogenase Maturation

HypA is one of the auxiliary proteins involved in the maturation of [NiFe] hydrogenases. By an unknown mechanism, HypA functions as a metallochaperone in the insertion of the Ni atom into hydrogenases. We have determined the crystal structures of HypA from Thermococcus kodakaraensis KOD1 in both monomeric and dimeric states. The structure of the HypA monomer consists of Ni- and Zn-binding domains. The relative arrangement of the two metal-binding domains has been shown to be associated with local conformations of the conserved Ni-binding motif, suggesting a communication between the Ni- and Zn-binding sites. The HypA dimer has been shown to be stabilized by unexpected domain swapping through archaea-specific linker helices. In addition, the hexameric structure of HypA is formed in the crystal packing. Several hydrogen bonds and hydrophobic interactions stabilize the hexamer interface. These findings suggest the functional diversity of HypA proteins.     

Domain interaction sites of human lens betaB2-crystallin

betaB2-crystallin, the major component of beta-crystallin, is a dimer at low concentrations but can form oligomers under physiological conditions. The interaction domains have been speculated to be the beta-sheets, each of which is formed by two or more beta-strands. betaB2-crystallin consists of 16 beta-strands, 8 in the N-terminal domain and 8 in the C-terminal domain. Domain interaction sites may be removed by destroying the beta-strands, which can be done by site-specific mutations, substituting the beta-formers (Val, Phe, Leu) with Glu or Asn, strong beta-breakers. We have cloned the following beta-strand-deleted mutants, I20E, L34E, V54E, V60E, V73E, L97E, I109E, I124E, V144E, V152E, L162E, L165E, and V187E and their corresponding X --> Asn mutants. We also made two mutants, V46E and V129E, that were not on the beta-strand as controls. Disruption of protein-protein interactions was screened by a mammalian two-hybrid system assay. Protein-protein interactions decreased for all beta-strand-deleted mutants except I20E, L34E, and L162E mutants; this effect was not seen in the two mutant controls, V46E and V129E. The sequences around Val-54, Val-60, Val-73, and Leu-97 in the N-terminal region and Ile-109, Ile-124, Val-144, Val-152, Leu-165, and Val-187 in the C-terminal region that formed beta-strands appear to be important in dimerization. Some selected mutant proteins that showed strong (V46E and V129E) and reduced (V60E, V144E, V60N, and V144N) interactions were expressed in bacterial culture and were studied with spectroscopy and chromatography. The V60E and V144E mutants were found to be partially unfolded and incapable of forming a complete dimer.     

Crystal structure of human glyoxalase I--evidence for gene duplication and 3D domain swapping.

The zinc metalloenzyme glyoxalase I catalyses the glutathione-dependent inactivation of toxic methylglyoxal. The structure of the dimeric human enzyme in complex with S-benzyl-glutathione has been determined by multiple isomorphous replacement (MIR) and refined at 2.2 A resolution. Each monomer consists of two domains. Despite only low sequence homology between them, these domains are structurally equivalent and appear to have arisen by a gene duplication. On the other hand, there is no structural homology to the 'glutathione binding domain' found in other glutathione-linked proteins. 3D domain swapping of the N- and C-terminal domains has resulted in the active site being situated in the dimer interface, with the inhibitor and essential zinc ion interacting with side chains from both subunits. Two structurally equivalent residues from each domain contribute to a square pyramidal coordination of the zinc ion, rarely seen in zinc enzymes. Comparison of glyoxalase I with other known structures shows the enzyme to belong to a new structural family which includes the Fe2+-dependent dihydroxybiphenyl dioxygenase and the bleomycin resistance protein. This structural family appears to allow members to form with or without domain swapping.     

Crystal structure of human filamin C domain 23 and small angle scattering model for filamin C 23-24 dimer

Filamin C is a dimeric, actin-binding protein involved in organization of cortical cytoskeleton and of the sarcomere. We performed crystallographic, small-angle X-ray scattering and analytical ultracentrifugation experiments on the constructs containing carboxy-terminal domains of the protein (domains 23-24 and 19-21). The crystal structure of domain 23 of filamin C showed that the protein adopts the expected immunoglobulin (Ig)-like fold. Small-angle X-ray scattering experiments performed on filamin C tandem Ig-like domains 23 and 24 reveal a dimer that is formed by domain 24 and that domain 23 has little interactions with itself or with domain 24, while the analytical ultracentrifugation experiments showed that the filamin C domains 19-21 form elongated monomers in diluted solutions.     

The X-ray structure of a mutant eye lens beta B2-crystallin with truncated sequence extensions.

beta-Crystallins are oligomeric eye lens proteins that are related to monomeric gamma-crystallins by domain swapping: like gamma-crystallins, they are comprised of two similar domains but they differ in having long sequence extensions. beta B2, a major component of beta-crystallin oligomers, self-associates to a homodimer in solution. In two crystal structures of native beta B2, the protein is a 222-symmetric tetramer of eight domains. It has previously been shown that a mutant of rat beta B2-crystallin, in which the bulk of the N- and C-terminal sequence extensions has been deleted, assembles into dimers and tetramers. Here we present the 3.0 A resolution X-ray structure of the tetramer, beta B2 delta NC1. The mutant tetramer has a very similar set of domain interactions to the native structure. However, the structures differ in the relative orientation of the two sets of four domains. The paired N- and C-terminal domain interface, which is at the heart of the dimer structure, is very similar to the native structure. However, the truncation of the C-terminal extension removes an important tryptophan residue, which prevents the extension from acting as a (non-covalent) linker, as it does in native beta B2. There is a knock-on structural effect that removes a contact between extension and covalent linker, and this appears to cause a small twist in the linker that is amplified into a 20 degrees rotation between sets of paired domains.     

Regions Outside the α-Crystallin Domain of the Small Heat Shock Protein Hsp26 Are Required for Its Dimerization

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones. They form homo-oligomers, composed of mostly 24 subunits. The immunoglobulin-like α-crystallin domain, which is flanked by N- and C-terminal extensions, is the most conserved element in sHsps. It is assumed to be the dimeric building block from which the sHsp oligomers are assembled.Hsp26 from Saccharomyces cerevisiae is a well-characterized member of this family. With a view to study the structural stability and oligomerization properties of its α-crystallin domain, we produced a series of α-crystallin domain constructs. We show that a minimal α-crystallin domain can, against common belief, be monomeric and stably folded. Elongating either the N- or the C-terminus of this minimal α-crystallin domain with the authentic extensions leads to the formation of dimeric species. In the case of N-terminal extensions, their population is dependent on the presence of the complete so-called Hsp26 “middle domain”. For the C-terminal extensions, the presence of the conserved IXI motif of sHsps is necessary and sufficient to induce dimerization, which can be inhibited by increasing ionic strength. Dimerization does not induce major changes in secondary structure of the Hsp26 α-crystallin domain. A thermodynamic analysis of the monomeric and dimeric constructs revealed that dimers are not significantly stabilized against thermal and chemical denaturation in comparison to monomers, supporting our notion that dimerization is not a prerequisite for the formation of a well-folded Hsp26 α-crystallin domain.