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Nat Struct Mol Biol. Author manuscript; available in PMC 2010 October 20.
Published in final edited form as:
PMCID: PMC2957905
NIHMSID: NIHMS238352

Solid-state NMR and SAXS studies provide a structural basis for the activation of αB-crystallin oligomers

Abstract

The small heat shock protein αB-crystallin (αB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric αB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of ~121° between the planes of the β-sandwich formed by α-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with β4 and β8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with β3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit αB oligomer with tetrahedral symmetry.

Small heat shock proteins (sHSPs) help to maintain protein homeostasis by interacting with unfolded, aggregated or misfolded proteins to prevent cell damage13. The ATP-independent chaperone αB-crystallin (αB, 20 kDa, 175 residues) is a paradigm example4. αB was originally found in the eye-lens as the B-subunit of α-crystallin, a protein essential for maintaining eye-lens transparency. In recent years, the list of biological roles for αB has grown substantially, including involvement in the regulation of the ubiquitin-proteasome pathway as well as apoptosis5. Dysfunctions of αB in humans are associated with the occurrence of neurodegenerative diseases like Alzheimer’s disease, Alexander’s disease, myopathies and cataracts611. In the brain of patients with Alexander’s disease, the insoluble cell fraction contains protein fibers (Rosenthal fibers) coprecipitated with αB phosphorylated at Ser59, whereas unphosphorylated αB remains in the soluble fraction7. A missense mutation, R120G, in αB is associated with desmin-related cardiomyopathy8,9. The mutations D140N and Q151X are associated with congenital cataracts and myopathy, respectively10,11. A decreased concentration of αB in the cerebrospinal fluid was found to be associated with multiple sclerosis; therapeutic application of αB was shown to reverse the typical symptoms in mice by an involvement in the regulation of caspase 3 levels in the apoptotic pathway12,13. Hypoxia, a condition often found in diseased states that include cancer and ischemia, induces the overexpression of αB14. Hypoxia is accompanied by acidosis, and it has been shown that αB binds cardiac-muscle proteins such as desmin and titin more tightly at acidic pH, presumably to confer protection to these proteins under conditions of cardiac ischemia14.

As is common for most sHSPs, αB is a polydisperse, supramolecular complex with a variable number of subunits (~24–32) (ref. 15) whose inherent dynamics is an important feature and prerequisite of its function. For decades, high-resolution structural studies on functional, full-length αB and mammalian sHSPs in general have been confounded by this polydisperse nature. αB contains a conserved, ~90-residue α-crystallin domain with an immunoglobulin fold, flanked by variable N- and C-terminal extensions. Intermolecular interactions of the N and C termini with the α-crystallin domain are responsible for the formation of the higher-order oligomers16. The segment containing the phosphorylation site Ser59 (Ser59-Trp60-Phe61), for example, was found to alter the oligomerization behavior if mutated or inverted17. Crystal structures of monodisperse sHSPs (Supplementary Figs. 1a,b and 2) and, after deposition of these coordinates, structures of short constructs comprising the conserved α-crystallin domain of rat HSP20 obtained at pH 6.5 and human αB obtained at pH 9.0 became available1821. In most of these structures, the α-crystallin domain forms a dimer of 7-stranded β-sandwiches (β2–β9). From previous NMR studies, it is known that residues 64–152 of αB form a 6-stranded β-sandwich and a dimer interface comprised of an antiparallel arrangement of the long β6+7 strands from two protomers22. Electron spin resonance (EPR) analysis using spin labels had given a preliminary indication of this type of arrangement in the dimer interface as well23. First indications of the oligomer structure came from electron microscopy studies, which showed that αB has a variable quaternary structure with a roughly spherical shape and a central cavity24. More recently, single-particle reconstruction from negative-stain electron microscopy at 20-Å resolution showed that a dominant population of αB forms a hollow sphere-like particle with 24 subunits and tetrahedral symmetry25.

Here, we have performed structural investigations on human full-length αB-crystallin (HspB5) using a combination of magic angle spinning (MAS) solid-state NMR, small-angle X-ray scattering (SAXS) and computational methods to determine the structure of the α-crystallin domain dimer within αB oligomers at pH 7.5 as well as its intersubunit interactions involving parts of the N- and C-terminal regions. The C-terminal residues Arg157–Arg163, including the IXI motif, bind into a presumed substrate binding groove, suggesting an autoinhibitory function. NMR spectra reveal a modulation of this interaction by pH changes, providing a structural mechanism for the known pH-dependent activation of αB.

RESULTS

Atomic-level structure of the homodimeric α-crystallin domain

We performed the MAS NMR investigations on full-length human αB (Fig. 1a and Supplementary Fig. 2) precipitated by PEG at pH 7.5. Initial solution- and solid-state NMR studies provided resonance assignments for the α-crystallin domain and parts of the C terminus, defining the secondary structure22 (Supplementary Fig. 2). We expanded resonance assignments to 139 of 175 residues using a novel assignment procedure26 (BMRB 16391). NMR spectra collected at pH 7.5 reveal a structurally homogeneous part of the protein comprising α-crystallin domain residues 71–150, with the exception of Ser76, Val77 and Asn78 in the β3 strand, which each show two backbone 15N signals. We performed structure calculations27,28 for residues 69–150 using 540 inter-residue NMR-derived distance restraints per protomer (Table 1 and Supplementary Table 1). We obtained structure-defining restraints from a variety of spectra (see Supplementary Table 2). Intramolecular Cα-Cα and Hα-Hα correlations define the topology of two antiparallel β-sheets, β3-β9-β8 and β4-β5-β6+7 (Supplementary Fig. 3). We obtained the Cα-Cα restraints from 3D NCACX spectra29 of 13C/15N-labeled αB made from 1,3– and 2-[13C]–labeled glycerol (Supplementary Fig. 3.) and the Hα-Hα restraints from CHHC spectra30, using a sample containing 20% 13C/15N-labeled and 80% unlabeled protein, using deuteration at the exchangeable sites to suppress sequential transfer. The homodimer is unambiguously defined by 26 intermolecular correlations derived from oligomers prepared from mixtures of differently labeled αB, using either 2-[13C]– or 1,3-[13C]–labeled glycerol and 14NH4Cl for expression mixed with αB made from 13C-depleted glucose and 15NH4Cl (Supplementary Fig. 3). All 13C-15N cross-peaks observed in spectra recorded from these samples reflect intermolecular correlations. The dimer interface is comprised of the antiparallel β6+7 strands from each protomer, with Glu117 adjacent to the two-fold axis. This topology is consistent with solution NMR data from the homodimeric αB10.1 construct (residues 64–152)22.

Figure 1
Structure of the αB-crystallin core domain dimer and its intermolecular interactions. (a) Domain architecture of small heat-shock proteins consists of a proline- and phenylalanine-rich N-terminal segment, a conserved α-crystallin domain ...
Table 1
Structure statistics for the α-crystallin dimer

The calculations yielded a curved dimer of the α-crystallin domain β-sandwich as the recurring building block of the oligomer (PDB 2KLR). The dimer is shown in Figure 1b–d together with interdimer interactions involving residues 59–61 (β2a) and 157–163 (IXI motif). The backbone r.m.s. deviation for the ten lowest-energy structures in the ensemble is 1.36 Å for the dimer (0.87 Å if only the secondary structure is considered) and 1.14 Å when superposing monomers (Table 1 and Supplementary Table 1). A distinctive feature of the dimer is its curvature, with an average angle of ~121° between the two β4-β5-β6+7 planes (Fig. 2a). The concave face is notable for its network of ionic interactions, especially across the dimer interface (Fig. 2b). In particular, the residue pairs Glu99-His119 and Glu117-His101 form potentially pH-dependent electrostatic interactions on both sides of the dimer interface that may contribute to the observed curvature (Fig. 2b). Intermolecular cation-π interactions between Phe118 and Arg116′, identified using the program CAPTURE31, may contribute to dimer stability. The Hsp20 α-crystallin domain has a similar array of histidines and glutamates and is curved in a crystal structure solved at pH 6.5 (ref. 21) (Fig. 2c,d). In contrast, an X-ray structure of the human αB α-crystallin domain determined at pH 9.0 is virtually flat21, implying a pH dependence for this feature. A key difference between our curved dimer and the flat dimer observed at pH 9.0 involves Arg107, which forms a salt bridge across the dimer interface with Asp80′ (Fig. 1d), whereas in the X-ray structure21 (PDB 2WJ7), Arg107 forms an intramolecular salt bridge with Glu88. The curved shape of the dimer is consistent with SAXS data obtained on the excised α-crystallin domain dimer αB10.1 (ref. 22) in solution (Supplementary Fig. 4). Monomers from the X-ray structures of isolated mammalian α-crystallin domains21 (PDB 2WJ7 and 2WJ5) superimpose with high accuracy (r.m.s. deviation 1.4 Å, Supplementary Fig. 1c), except for the tip of the loop connecting β5 and β6+7.

Figure 2
Curvature of the α-crystallin domain dimer. (a) Side view of the αB-crystallin dimer (solid-state NMR, pH 7.5), showing the curvature of the extended β-sheet formed by the dimer interface. (b) Concave face of β4-β5-β6+7 ...

Intermolecular contacts within αB oligomers

The MAS NMR spectra contain multiple NMR signals, indicating diverse environments, for the putative β2 strand observed in other sHSPs, which we instead call heterogeneity region 1 (HR1, residues Glu67–Leu70)22. We could not extract a consistent set of long-range restraints for HR1. In a recent X-ray structure of the α-crystallin domain from αB21, two of five protomers in the unit cell contain a β2 strand that aligns antiparallel to β3 of the same protomer. In our spectra, residues that precede HR1 (Ser59, Trp60, and Phe61) show intermolecular contacts to β3 (see Supplementary Fig. 3f,h), indicating a ‘β2a’ strand that is involved in the formation of quaternary structure. At opposite edges of the dimer, a putative substrate binding site32,33 formed by the groove between β4 and β8 contains small pockets that can accommodate aliphatic side chains (Fig. 3a). Indeed, the NMR data reveal intermolecular contacts between the IXI motif (Ile159-Pro160-Ile161) and residues of β4 and β8 (Leu89–Val93 and Ile133–Ser138; Fig. 3b and Supplementary Fig. 2g,h). We used 12 intermolecular 13C-15N constraints to dock the C-terminal residues to the α-crystallin domain using Sybil (Tripos). In the resulting model, residues Arg157–Arg163 cross the binding site in an extended conformation, with the side chains of Ile159 and Ile161 residing in the two prominent hydrophobic pockets (Fig. 3a).

Figure 3
A substrate binding site of αB-crystallin. (a) Model of the C-terminal IXI motif binding to a hydrophobic substrate binding groove of αB. A docking study was performed on the basis of 12 restraints from solid-state NMR data. The C-terminal ...

Model of a 24-mer αB-crystallin oligomer

The approach we have used to determine the structures shown in this study is shown schematically in Supplementary Figure 5. Using the dimer structure and intermolecular interactions obtained from MAS solid-state NMR and the shape of αB determined from SAXS, interactive modeling with Chimera34,35 and energy minimization with XPLOR-NIH36 allowed us to obtain a structural model of a representative αB oligomer.

SAXS data obtained at pH 7.5 for oligomeric αB in solution were used to calculate six bead models, assuming tetrahedral symmetry, consistent with previous electron microscopy studies25. χ2 versus normalized spatial discrepancy plots generated with DAMAVER37 indicate high similarity among the six reconstructions and a reasonable fit with the experimental SAXS curve (Supplementary Fig. 6a). We averaged the initial six bead models and transformed them into a volumetric map (Fig. 4a) that has a diameter of 14.5–16 nm. The individual bead models (Supplementary Fig. 6b) generated using the program GASBOR38 have extrusions emanating from the central body, which we interpret as flexible C-terminal residues 166–175 of the oligomer39. In individual models, these extrusions create a maximum diameter Dmax of 19 nm. The cited range of αB oligomers is 24–32 subunits15, and a heterogeneous mixture exists in solution. Reasoning that larger species are assembled by combining fragments of 24-mers, we undertook to model the smallest species. αB69–175 has been shown to form dimers and trimers40, indicating that the N-terminal domain is crucial for the formation of higher-order assemblies. Hence, the C-terminal domain alone is not responsible for the formation of higher-order oligomers. Accordingly, adjacent dimers in the recurring hexameric substructure (Fig. 4, red and blue) were connected by satisfying the observed C-terminal interactions. Using P432 symmetry, an oligomer may contain 24 or 48 subunits, with the asymmetric unit being the monomer or the dimer, respectively. Using P23 symmetry, the symmetry operations allow 12, 24 or 36 subunits in the oligomer, with the smallest asymmetric unit being the monomer, dimer or three monomers (trimer), respectively. We chose P23 symmetry because of both its congruence with our data (that is, a dimer is the smallest building block) and its agreement with the recently reported electron microscopy structure25. We therefore constructed an atomic model of a 24-mer on the basis of the dimer structure, the intermolecular N- and C-terminal interactions observed by NMR and the volumetric data, applying P23 symmetry (Fig. 4a,b). We kept the core of each dimeric unit rigid, whereas we considered the loop connecting the α-crystallin domain with the IXI motif, docked in the β4/β8 substrate binding site, to be flexible. An electron density calculated at 20-Å resolution from the resulting molecular oligomer model (Fig. 4b) shows a similar molecular organization to that of the recently reported electron microscopy structure25. We consider this solution as one species in a heterogeneous ensemble of oligomers. Variable conformations of the flexible C-terminal extensions (residues Lys166–Lys175) and variable intermolecular interactions may contribute to the observed heterogeneity.

Figure 4
Molecular organization of the oligomer. (a) A model of the αB oligomer is shown within the average volumetric map generated from SAXS data. Six bead models calculated using P23 symmetry with the program GASBOR were averaged using the program DAMAVER. ...

pH-dependent activation of αB chaperone function

The activation of cellular protection systems against stress is often associated with a decrease of pH2,41. For example, αB binds cardiac-muscle proteins actin and desmin more strongly at acidic pH, preventing damage to these proteins during cardiac ischemia accompanied by acidification14. We investigated the effect of pH on αB and particularly on a substrate binding site between β4 and β8 (Fig. 5a) by NMR and SAXS. In 13C-13C correlation spectra collected at pH 7.5 (Fig. 5b–d), we observed single cross-peaks for Ile133 (β8), Ile159 and Ile161. At pH 6.5, the involved isoleucines and threonines (Thr132 and Thr134 in β8 and Thr158 and Thr162 of the C terminus) show chemical-shift perturbations or doubling of intraresidual cross-peaks (for example, at ~59–63 p.p.m., ~36–39 p.p.m. or 26 p.p.m. in F1 and at 69–72.5 p.p.m. or 9.5–27 p.p.m. in F2), indicating a pH-dependent change in the substrate binding groove (Figs. 3a and and5a).5a). The radius of gyration (Rg) determined by SAXS is 5.4 nm at pH 7.5 and 6.7 nm at pH 6.5; Dmax increases from 19 nm at pH 7.5 to 30 nm at pH 6.5 (Supplementary Fig. 7a,b), indicating a substantial change in quaternary structure of the αB oligomer. Such a change may be predominantly due to release of the IXI motif from the β4/β8 site.

Figure 5
pH-dependent modulation of a substrate binding site. (a) Substrate binding site between β4 and β8 of αB (red ribbons) and the C terminus from an adjacent dimer (blue ribbon). The isoleucine and threonine residues that show chemical-shift ...

Our results provide a structural explanation for the autoinhibitory function of the C-terminal IXI motif and its role in balancing homeostasis of active and inactive binding sites under varying cellular conditions14. At physiological pH conditions, the IXI motif binds nearly quantitatively into the substrate binding groove, as indicated by the strong restraints and the single set of chemical shifts observed for Thr132, Thr134, Ile159 and Ile161 at pH 7.5. Thus, hydrophobic substrates must compete with the C terminus for binding, whereas this interaction is modulated at lower pH, very likely exposing a population of binding sites in the oligomer. The role of the IXI motif in regulating the chaperone activity is supported by the effects of mutating both isoleucines (Ile159 and Ile161) to glycine, which substantially enhances the chaperone activity of αB in vitro16, and by the truncation ΔGlu151, which causes myofibrillar myopathies11, presumably by generating a constitutively active form with exposed hydrophobic surfaces and without the highly soluble C-terminal tail.

Cellular conditions thus modulate the dynamic structure of αB to adjust its functional role according to physiological needs. We have shown that a drop in pH results in observable changes in a substrate binding site of αB. Protein phosphorylation is another way to modulate protein function. Along these lines, the observed intermolecular interactions involving residues Ser59-Trp60-Phe61 (β2a) in one dimer and β3 in another contribute to a structural basis for understanding the effects of phosphorylation at Ser59 (ref. 7), which is also expected to alter the oligomerization behavior of αB17.

DISCUSSION

The polydisperse human αB oligomer has been refractory to structural analysis for decades. Structures have been solved for isolated α-crystallin domains from human αB and rat HSP20 (ref. 21) and for three sHSPs that form monodisperse multimers1820 (Supplementary Figs. 1a,b and 2) and electron spin-label studies have been performed on several sHSPs23. These reveal a conserved β-sandwich topology but do not explain the nature of heterogeneity in the native oligomer. Here, application of MAS NMR and SAXS to αB provides four new insights. First, the independently determined MAS NMR structure reveals a highly curved dimer as the fundamental building block of αB oligomers at pH 7.5; an array of histidines and glutamates on the concave face of the dimer may constitute a pH switch that controls the curvature and, ultimately, the quaternary structure (Fig. 2a,b). Second, experimentally observed distance restraints and chemical-shift changes among residues in the N or C termini and the α-crystallin domain provide atomic-level details of the quaternary structure of αB oligomers and its modulation upon activation. Third, NMR signal heterogeneity observed for residues Ser59–Leu70 indicate multiple environments for this segment within oligomers. Notably, this segment is important for the oligomerization of αB and in the formation of hetero-oligomers with related sHSPs17. Fourth, SAXS measurements reveal large increases in both the Rg and Dmax with a drop in pH from 7.5 to 6.5. We propose that release of the C-terminal IXI motif from the substrate binding cleft contributes to the more expanded oligomeric structure, activating αB as a chaperone.

In conclusion, application of solid-state NMR and SAXS has provided high- and intermediate-resolution structural information on a dynamic protein assembly under conditions in which its biological function is known to be modulated. αB’s chaperone function is implicated in diseases including multiple sclerosis, cancer, neurodegenerative diseases and cardiomyopathy. These and other diseases are connected to imbalanced protein homeostasis, making the responsible proteins potential therapeutic targets. The ability to obtain structural information on large dynamic protein assemblies while maintaining the ability to control relevant solution conditions such as pH represents a milestone for understanding both the function and dysfunction of sHSPs, in particular αB, and may help to guide future structure-based drug design efforts.

METHODS

Methods and any associated references are available in the online version of the paper at http://www.nature.com/nsmb/.

Supplementary Material

Supplemental material

Acknowledgments

The authors thank K. Rehbein and A. Diehl for sample preparation and discussions and D. Svergun for fruitful discussions concerning the SAXS experiments. This work was funded by US National Institutes of Health grant 1R01EY017370. SAXS data on αB10.1 was collected at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The Stanford Synchrotron Radiation Lightsource Structural Molecular Biology Program is supported by the US Department of Energy, Office of Biological and Environmental Research, and by the US National Institutes of Health, National Center for Research Resources, Biomedical Technology Program. SAXS data collection for full-length αB was supported by European Community–European Molecular Biology Laboratory Hamburg Outstation; Deutsches Elektronen-Synchrotron Hamburg X33 beamline.

Footnotes

Accession codes. Biological Magnetic Resonance Bank: Chemical shifts have been deposited with accession code 16391. Protein Data Bank: Atomic coordinates and NMR restraints for the dimer, including noncrystallographic symmetry restraints for the hexamer have been deposited with accession code 2KLR.

Note: Supplementary information is available on the Nature Structural & Molecular Biology website.

AUTHOR CONTRIBUTIONS

S.J. contributed to all aspects of the manuscript; P.R. performed solution NMR experiments and helped to write the manuscript; B.B. performed structure calculations; S.M. did solid-state NMR and SAXS measurements as well as data analysis; R.K. contributed to modeling of C-terminal intermolecular interactions; J.R.S. prepared samples; V.A.H. contributed to assignment strategies, was involved in structure calculations and helped write the manuscript; R.E.K. contributed to the interpretation of results and wrote the manuscript; B.J.v.R. contributed to solid-state NMR measurements, discussed the results and helped to write the manuscript; H.O. designed experimental strategies, contributed to the interpretation of results and wrote the manuscript.

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/.

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