NMR spectroscopy of alpha-crystallin. Insights into the structure, interactions and chaperone action of small heat-shock proteins

The subunit molecular mass of alpha-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved 1H NMR spectrum is observed for alpha-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, alpha A- and alpha B-crystallin. These extensions are not involved in interactions with other proteins (e.g. beta- and gamma-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of alpha A-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic alpha-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the alpha A-crystallin subunit maintains its flexibility whereas the alpha B-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the 'substrate' protein. The conformation of 'substrate' proteins when they interact with alpha-crystallin has been probed by 1H NMR spectroscopy and it is concluded that alpha-crystallin interacts with 'substrate' proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the 1H and 31P NMR spectra of alpha-crystallin in the presence of increasing concentrations of urea, it is proposed that alpha-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of alpha-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with 'substrate' proteins during the chaperone action.     

Structural alterations of alpha-crystallin during its chaperone action

The small heat-shock protein, alpha-crystallin, has chaperone ability whereby it stabilises proteins under stress conditions. In this study, alterations in the structure of alpha-crystallin during its interaction with a variety of substrate proteins (insulin, alpha-lactalbumin, ovotransferrin and serum albumin) under stress conditions have been examined using visible absorption, 31P-NMR and 1H-NMR and fluorescence spectroscopy. The fluorescence and 31P-NMR data imply that during the chaperone action of alpha-crystallin under reducing conditions, there is a slight increase in hydrophilicity of its N-terminal region and an alteration in flexibility of its C-terminal region, but overall, alpha-crystallin does not undergo a gross structural change. The fluorescence data suggest that substrate proteins interact with alpha-crystallin in a molten globule or intermediately folded state. The same conclusion is made from 1H-NMR spectroscopic monitoring of the interaction of alpha-crystallin with substrate proteins, e.g. the insulin B chain. The stoichiometry of interaction between alpha-crystallin and the various substrate proteins reveals that steric factors are important in determining the efficiency of interaction between the two proteins, i.e. on a molar subunit basis, alpha-crystallin is a more efficient chaperone protein with smaller substrate proteins. Comparison is also made between the high-molecular-mass (HMM) complexes formed between alpha-crystallin and ovotransferrin when reduced and heat stressed. Under heating conditions, fluorescence spectroscopy indicates that the HMM complex has a greater exposure of hydrophobicity to solution than that formed by reduction. Furthermore, in interacting with heated ovotransferrin, the C-terminal extension of the alphaB-crystallin subunit preferentially loses its flexibility suggesting that it is involved in stabilising bound ovotransferrin. By contrast, this extension is only partially reduced in flexibility in the HMM complex formed after reduction of ovotransferrin. The functional role of the C-terminal extensions in the chaperone action and the overall quaternary structure of alpha-crystallin is discussed.     

Solution conformations and interactions of alpha and beta subunits of the Oxytricha nova telomere binding protein: investigation by Raman spectroscopy

Solution conformations of the alpha and beta subunits of the Oxytricha nova telomere binding protein have been investigated by Raman spectroscopy. Raman spectra have also been obtained for a deletion mutant of the beta subunit, betaC232, which retains the N-terminal domain that is active in ternary complex (alpha:beta:DNA) formation but lacks the C-terminal domain that is active in catalyzing guanine quadruplex formation. The Raman spectra show that alpha, beta, and betaC232 are rich in beta-strand secondary structure ( approximately 40-50%) and turns. The Raman signature of the C-terminal 153 amino acids of beta, generated by subtracting the spectrum of betaC232 (residues 1-232) from that of the full subunit, indicates that the domain active in guanine quadruplex formation contains less beta-strand secondary structure and more irregular structure than the domain active in alpha:beta:DNA formation. Raman markers also provide information about the environments and orientations of several key side chains, including tryptophan residues in N- and C-terminal domains of the beta subunit. Both alpha and beta denature between 30 and 40 degrees C, as evidenced by large changes in Raman bands diagnostic of main chain conformation and side chain environments. The Raman spectrum of an equimolar alpha/beta mixture exhibits no evidence of specific interaction between the subunits; further, the denaturation profile of this mixture is indistinguishable from the sum of denaturation profiles of the constituent subunits, consistent with the absence of appreciable interaction between alpha and beta throughout the range 0-50 degrees C. The present results provide insights into the solution conformations of the Oxytricha telomere binding protein subunits and serve as the basis for future study of subunit interactions with telomeric DNA.     

Studies of the denaturation patterns of bovine alpha-crystallin using an ionic denaturant, guanidine hydrochloride and a non-ionic denaturant, urea

The effects of non-ionic and ionic denaturation and denaturation/renaturation on the native structure of alpha-crystallin at room temperature were examined. Native alpha-crystallin, at concentrations above and below the previously reported critical micelle concentration (CMC) range, was denatured by varying concentrations of urea and guanidine hydrochloride. The resulting denatured samples were examined by gel filtration fast performance liquid chromatography (FPLC), circular dichroism spectropolarimetry (CD), and transmission electron microscopy. Elution peak samples from gel filtration chromatography with sufficiently high concentrations were examined for subunit composition by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The studies presented herein demonstrate that the denaturation and renaturation of alpha-crystallin via non-ionic urea denaturation results in different renaturation species, depending upon the initial concentration of alpha-crystallin which is denatured and the concentration of urea, including certain species which, by gel filtration FPLC, have an apparent molecular weight greater than the native 800 kD aggregate. Transmission electron microscopy has also demonstrated the existence of a high molecular weight aggregate form for denatured samples. Ionic dissociation, in contrast, proceeds much in the same manner above and below the CMC range, the major difference occurring at 2 M guanidine hydrochloride. alpha B-crystallin is preferentially removed from the native alpha-crystallin aggregate upon treatment with 2 M guanidine hydrochloride indicating, once again, differences between the two subunits. Above and below the CMC range, dissociation with guanidine hydrochloride appears to plateau after 4 M guanidine hydrochloride as indicated by the presence of two apparent homotetrameric species and no further dissociation of these species with increasing guanidine hydrochloride concentrations. CD demonstrates that some secondary structure, which is lost with lower concentrations of alpha-crystallin, is still present when concentrations of alpha-crystallin, well above the critical micelle concentration range, are treated with high concentrations of urea at room temperature. In contrast, concentrations both above and below the CMC range demonstrate a significant loss of secondary structure upon treatment with 2 M guanidine hydrochloride. Finally, ionic denaturation and subsequent renaturation results in the formation of a species which is functionally incapable of protecting gamma-crystallin from heat-induced aggregation.     

alpha-Crystallin polymers and polymerization: the view from down under

Several models have been proposed for the quaternary structure of alpha-crystallin. Some suggest the subunits are arranged in concentric shells. Others propose that the subunits are in a micelle-like arrangement. However, none is able to satisfactorily account for all observations on the protein and the quaternary structure of alpha-crystallin remains to be established. In this review, factors contributing to the assembly and polymerization are examined in order to evaluate the different models. Consideration of the variations in particle size and molecular weight under different conditions leads to the conclusion that alpha-crystallin cannot be a micelle or a layered structure. Instead, it is suggested that the protein may be assembled from a 'monomeric' unit comprising eight subunits arranged in two tetramers with cyclic symmetry. The octameric unit is proposed to be disc-like particle with a diameter of 9.5 nm and a height of 3 nm. The larger particles, chains and sheet-like structures commonly observed are assembled from the octamers. Structural predictions indicate that the polypeptide may be folded into three independent domains which have different roles in the structural organization and functions of the protein. It is suggested that the tetramers are stabilized through interactions involving the second domain (residues 64-104) while assembly into the octamers and higher polymers requires hydrophobic interactions involving the N-terminal domain. Deletion of parts of this domain by site directed mutagenesis revealed that residues 46-63 play a critical role in the assembly. Current research aims to identify the specific amino acids involved.     

High-resolution crystal structures of human hemoglobin with mutations at tryptophan 37beta: structural basis for a high-affinity T-state,

The high-resolution X-ray structures of the deoxy forms of four recombinant hemoglobins in which Trp37(C3)beta is replaced with Tyr (betaW37Y), Ala (betaW37A), Glu (betaW37E), or Gly (betaW37G) have been refined and analyzed with superposition methods that partition mutation-induced perturbations into quaternary structure changes and tertiary structure changes. In addition, a new cross-validation statistic that is sensitive to local changes in structure (a "local Rfree" parameter) was used as an objective measure of the significance of the tertiary structure changes. No significant mutation-induced changes in tertiary structure are detected at the mutation site itself for any of the four mutants studied. Instead, disruption of the intersubunit contacts associated with Trp37(C3)beta results in (1) a change in quaternary structure at the alpha1beta2 interface, (2) alpha subunit tertiary structure changes that are centered at Asp94(G1)alpha-Pro95(G2)alpha, (3) beta subunit tertiary structure changes that are located between residues Asp99(G1)beta and Asn102(G4)beta, (4) increased mobility of the alpha subunit COOH-terminal dipeptide, and (5) shortening of the Fe-Nepsilon2His(F8) bond in the alpha and beta subunits of the betaW37G and betaW37E mutants. In each case, the magnitude of the change in a particular structural parameter increases in the order betaW37Y < betaW37A < betaW37E approximately betaW37G, which corresponds closely to the degree of functional disruption documented in the preceding papers.     

Quaternary structure of bovine alpha-crystallin: influence of temperature

The tertiary and quaternary structure of alpha-crystallin is still a matter of controversy. We have characterized the native alpha-crystallin quaternary structure by isolating it at the in vivo temperature and solvent conditions. It can be represented by a distribution of expanded particles with a weight average molar mass of 550,000 g/mol. On decreasing (to 4 degrees C) or increasing (up to 50 degrees C) the temperature, the size distribution increases to larger particles. Only at lower temperatures (4 degrees C), a stable population of particles is obtained with weight average molar mass of 700,000 g/mol. In all conditions, alpha-crystallin behaves as a very expanded particle with a maximum hydrodynamic volume of 3.15 ml/g. The transitions in quaternary structure are rather slow: it takes several hours to evolve from a population of aggregates, characteristic for given solvent conditions, to another distribution in size and quaternary structure on changing the environment. The quaternary structure of alpha-crystallin is an uncharacteristic parameter of the particle: a broad distribution of values can be obtained on changing the environment. Any realistic model should include this property. Our studies favor an open loose structure, where peptides can be added or removed without drastic changes of secondary and tertiary structure of the peptides.     

alpha-crystallin stabilizes actin filaments and prevents cytochalasin-induced depolymerization in a phosphorylation-dependent manner

alpha-crystallin, a major lens protein of approximately 800 kDa with subunits of about 20 kDa has previously been shown to act as a chaperone protecting other proteins from stress-induced damage and to share sequence similarity with small heat-shock proteins, sHsp. It is now demonstrated that this chaperone effect extends to protection of the intracellular matrix component actin. It was found that the powerful depolymerization effect of cytochalasin D could be almost completely blocked by alpha-crystallin, alpha A-crystallin or alpha B-crystallin. However, phosphorylation of alpha-crystallin markedly decreased its protective effect. It is suggested that phosphorylation of alpha-crystallin may contribute to changes in actin structure observed during cellular remodeling that occurs with the terminal differentiation of a lens epithelial cell to a fiber cell and contributes to cellular remodeling in other cell types that contain alpha-crystallin species. This communication presents biochemical evidence clearly demonstrating that alpha-crystallin is involved in actin polymerization-depolymerization dynamics. It is also shown that alpha-crystallin prevented heat-induced aggregation of actin filaments. alpha-crystallin was found to stabilize actin polymers decreasing dilution-induced depolymerization rates up to twofold while slightly decreasing the critical concentration from 0.23 microM to 0.18 microM. Similar results were found with either alpha-crystallin or its purified subunits alpha A-crystallin and alpha B-crystallin. In contrast to the experiments with cytochalasin D, phosphorylation had no effect. There does not appear to be an interaction between alpha-crystallin and actin monomers since the effect of alpha-crystallin in enhancing actin polymerization does not become apparent until some polymerization has occurred. Examination of the stoichiometry of the alpha-crystallin effect indicates that 2-3 alpha-crystallin monomers/actin monomer give maximum actin polymer stabilization.     

Structure changes in hemoglobin upon deletion of C-terminal residues, monitored by resonance Raman spectroscopy

Loss of C-terminal residues in hemoglobin raises oxygen affinity and reduces both cooperativity and the Bohr effect. These functional changes are expected from the loss of C-terminal salt bridges, which are seen crystallographically to stabilize the T quaternary structure. Ultraviolet resonance Raman (UVRR) difference spectroscopy confirms that the strength of the T state contacts is diminished when the C-terminal and also the penultimate residues are removed chemically. Deoxy minus CO difference signals arising from the Trpbeta37-Aspalpha94 and Tyralpha42-Aspbeta99 H bonds at the alpha1 beta2 subunit interface are diminished, and at pH 9, the difference spectra reveal a shift to the R quaternary structure. These effects are small for desHisbeta146 Hb and large for desArgalpha141 Hb, consistent with the order of functional changes. In addition, the H bond between the A and E helices is strengthened by removal of Argalpha141 and is further strengthened when the effector molecule IHP (inositol hexaphosphate) is added to deoxy-desArgalpha141 Hb or when its pH is lowered to 5.8. This effect is attributed to the loss of the C-terminal anchor of the alpha chain H helix, which supports the F and A helices. The beta chain is not as sensitive because it has extra F-H interhelix H bonds. Removal of both Hisbeta146 and Tauyrbeta145 produce UVRR changes which are intermediate between desHisbeta146 and desArgalpha141 Hb, although the functional consequences are greater than for desArgalpha141 Hb. Removal of Tyralpha140 as well as Argalpha141 abolishes cooperative binding as well as the Bohr effect, and the UVRR difference signals are also lost, suggesting that quaternary constraints are removed in both the T and the R states. When the approximately 220 cm-1 iron-histidine stretching vibration of the deoxy-proteins is examined, using Raman excitation in resonance with the heme Soret band, the frequency is observed to diminish toward that of deoxyHb A (215 cm-1) as the pH is lowered and IHP is added and to increase toward a completely relaxed value (223 cm-1) as the pH is raised to 9. The relaxation is in the same order as the functional perturbations: desHisbeta146 < desArgalpha141 < desHisbeta146-Tyrbeta145 < desArgalpha141-Tyralpha140. However, even desArgalpha141-Tyralpha140 Hb shows significant reduction in the Fe-His frequency as IHP is added at low pH. The Fe-His frequency is sensitive to both tertiary and quaternary structure changes and is a global indicator of forces at the heme. The order of affinity changes can be understood on the basis of the number of stabilizing H bonds between the F and H helices. Titration curves of the Fe-His frequency against pH are not sigmoidal, consistent with a multiplicity of contributions to the Bohr effect.     

Molecular cloning of a putative cyclic nucleotide-gated ion channel cDNA from Limulus polyphemus

Cyclic nucleotide-gated channels have been proposed to mediate the electrical response to light in the ventral photoreceptor cells of the horseshoe crab, Limulus polyphemus. However, a cyclic nucleotide-gated channel has not been identified from Limulus. We have cloned a putative full-length cyclic nucleotide-gated channel cDNA by screening cDNA libraries constructed from Limulus brain using a probe developed from Limulus ventral eye nerves. The putative full-length cDNA was derived from two overlapping partial cDNA clones. The open reading frame encodes 905 amino acids; the sequence shows 44% identity to that of the alpha subunit of the bovine rod cyclic GMP-gated channel over the region containing the transmembrane domains and the cyclic nucleotide binding domain. This Limulus channel has a novel C-terminal region of approximately 200 amino acids, containing three putative Src homology domain 3 binding motifs and a putative coiled-coil domain. The possibility that this cloned channel is the same as that detected previously in excised patches from the photoreceptive membrane of Limulus ventral photoreceptors is discussed in terms of its sequence and its expression in the ventral eye nerves.     

High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis. Implications for substrate activation in pyruvate decarboxylases

The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 A resolution and refined to a crystallographic R-factor of 16.2% and Rfree of 19.7%. The subunit consists of three domains, all of the alpha/beta type. Two of the subunits form a tight dimer with an extensive interface area. The thiamin diphosphate binding site is located at the subunit-subunit interface, and the cofactor, bound in the V conformation, interacts with residues from the N-terminal domain of one subunit and the C-terminal domain of the second subunit. The 2-fold symmetry generates the second thiamin diphosphate binding site in the dimer. Two of the dimers form a tightly packed tetramer with pseudo 222 symmetry. The interface area between the dimers is much larger in pyruvate decarboxylase from Z. mobilis than in the yeast enzyme, and structural differences in these parts result in a completely different packing of the subunits in the two enzymes. In contrast to other pyruvate decarboxylases, the enzyme from Z. mobilis is not subject to allosteric activation by the substrate. The tight packing of the dimers in the tetramer prevents large rearrangements in the quaternary structure as seen in the yeast enzyme and locks the enzyme in an activated conformation. The architecture of the cofactor binding site and the active site is similar in the two enzymes. However, the x-ray analysis reveals subtle but significant structural differences in the active site that might be responsible for variations in the biochemical properties in these enzymes.     

Structure-function studies on small heat shock protein oligomeric assembly and interaction with unfolded polypeptides

The small heat shock protein (smHSP) and alpha-crystallin genes encode a family of 12-43-kDa proteins which assemble into large multimeric structures, function as chaperones by preventing protein aggregation, and contain a conserved region termed the alpha-crystallin domain. Here we report on the structural and functional characterization of Caenorhabditis elegans HSP16-2, a 16-kDa smHSP produced only under stress conditions. A combination of sedimentation velocity, size exclusion chromatography, and cross-linking analyses on wild-type HSP16-2 and five derivatives demonstrate that the N-terminal domain but not most of the the C-terminal extension which follows the alpha-crystallin domain is essential for the oligomerization of the smHSP into high molecular weight complexes. The N terminus of HSP16-2 is found to be buried within complexes which can accommodate at least an additional 4-kDa of heterologous sequence per subunit. Studies on the interaction of HSP16-2 with fluorescently-labeled and radiolabeled actin and tubulin reveal that this smHSP possesses a high affinity for unfolded intermediates which form early on the aggregation pathway, but has no apparent substrate specificity. Furthermore, both wild-type and C-terminally-truncated HSP16-2 can function as molecular chaperones by suppressing the thermally-induced aggregation of citrate synthase. Taken together, our data on HSP16-2 and a unique 12.6-kDa smHSP we have recently characterized demonstrate that multimerization is a prerequisite for the interaction of smHSPs with unfolded protein as well as for chaperone activity.     

A model of the quaternary structure of enolases, based on structural and evolutionary analysis of the octameric enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pHo) for activity is 8.1-8.2, and the Km for 2-PGA is approximately 0.67 mM. Using the dimeric Calpha structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86-96) and the loop between helix L and strand 1 (HL-S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL-S1 loop is truncated by 4-6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL-S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.     

Erythrocyte passive potassium flux is increased in patients with ischemic coronary disease (ICD) and in subjects with family history of ICD

The subunit of catalase HPII from Escherichia coli is 753 residues in length and contains a core of approximately 500 residues, with high structural similarity to all other heme catalases. To this core are added extensions of approximately 80 and 180 residues at the N- and C-termini, respectively. The tetrameric structure is made up of a pair of interwoven dimers in which 90 N-terminal residues of each subunit are inserted through a loop formed by the hinge region linking the beta-barrel and alpha-helical domains of the adjacent subunit. A high concentration of proline residues is found in the vicinity of the overlap regions. To study the influence of the extended regions on folding and subunit association of HPII, a diversity of modifications have been introduced. Removal of the complete C-terminal domain or the N-terminal extension, either separately or together, effectively creating a small subunit catalase, resulted in no enzyme accumulation. Systematic truncations showed that only nine C-terminal residues (Ile745 to Ala753) could be removed without significantly affecting the accumulation of active enzyme. Removal or even conservative replacements of the side chain of Arg744 significantly reduced the accumulation of active enzyme despite this residue interacting only with the C-terminal domain. Removal of as few as 18 residues from the N-terminus also reduced accumulation of active enzyme. Changes to other residues in the protein, including residues in the heme binding pocket, also reduced the accumulation of active protein without substantially affecting the enzyme specific activity. Implications of these data for the interdependence of subunit folding and subunit-subunit interactions are discussed.     

Reduced chaperone-like activity of alpha A(ins)-crystallin, an alternative splicing product containing a large insert peptide

alpha-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several non-lenticular tissues. Like other members of the small heat shock protein family, alpha-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major alpha A- and alpha B-subunits, alpha-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, alpha A(ins)-crystallin. This polypeptide is identical to normal alpha A-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat alpha A- and alpha A(ins)-crystallin in Escherichia coli. The multimeric particles formed by alpha A(ins) are larger and more disperse than those of alpha A, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with alpha A, the alpha A(ins)-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of beta low-crystallin. Our experiments indicate that alpha A(ins)-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant alpha A(ins)-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.