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

Abstract

The small heat shock protein alphaB-crystallin (alphaB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric alphaB 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 approximately 121 degrees between the planes of the beta-sandwich formed by alpha-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 beta4 and beta8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with beta3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit alphaB oligomer with tetrahedral symmetry.

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 and a C-terminal region that contains the conserved IXI motif. The heterogeneous region 1 (HR1), the IXI motif, the flexible C-terminal residues Lys166–Lys175 and three disease-related mutations involved in cataract and myopathy (R120G, D140N and Gln151-stop) are indicated. (b) Ribbon representation of the lowest-energy solid-state NMR structure of the αB-crystallin dimer (shown in red and blue, PDB 2KLR) and its intermolecular interactions with N- and C-terminal regions of other protomers (shown in gray, labeled C-ter and β2a). The side chains of residues Asp109, Arg116, Phe118 and Arg120 at the dimer interface are shown, and the sites of known disease-associated mutations D140N and Q151X are indicated separately. (c) Ionic interactions between Asp80′ and Arg107 across the dimer interface in full- length αB and correlations of Asp80′ Cγ with Ile114 Cβ and Arg107 Cβ and Cγ from a 2D ¹³C-¹³C correlation spectrum recorded from 2-[¹³C]-glycerol–labeled αB. (d) Stereo view of a superposition of the ten lowest-energy structures, showing the intermolecular interactions with N- and C-terminal regions in red and blue, respectively; see main text for details.

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 sheet of αB dimer (solid-state NMR, pH 7.5). A superposition of two conformers taken from the NMR ensemble is shown. (c) Side view of the α-crystallin domain dimer from HSP20 (X-ray, pH 6.5). (d) Concave face of β4-β5-β6+7 plane of HSP20 (X-ray, pH 6.5), with multiple side chain conformations according to PDB 2WJ5 (ref. 21).

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 sequence Pro155-Glu156-Arg157-Thr158-Ile159-Pro160-Ile161-Thr162-Arg163 is shown as sticks. In the surface plot, yellow represents hydrophobic residues; green, hydrophilic residues. (b) Contour plot of the amide-methyl region of a 2D TEDOR spectrum recorded from differentially labeled αB; the sample was obtained using a mixture of protomers expressed from 1,3-[¹³C]-glycerol/¹⁴NH₄Cl and ¹³C-depleted glucose/¹⁵NH₄Cl. Cross-peaks between amide nitrogens and methyl groups are observed for Ser135, Leu137, Lys90 and Val91 (on the α-crystallin domain), with Ile159, Ile161 and Thr162 (on the C terminus) defining the binding interface between the IXI motif and the hydrophobic groove formed by β4 and β8. Cross-peaks between the amide nitrogens of Val81 and Lys121 with the side chain of Ile114 are also observed.

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. The volumetric data (shown as light gray surface) were generated from the average bead model using the software SITUS. The volumetric SAXS data were fitted with αB homodimers (shown as red and blue ribbons) using P₂₃ symmetry and NMR-derived intermolecular distance constraints in an energy minimization protocol using the program XPLOR-NIH. (b) Predicted electronic density map calculated at 20-Å resolution from the molecular oligomer model using the program CHIMERA,. (c) Arrangement of three dimers, connected by their C termini.

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 perturbation or peak doubling are shown as sticks. (b,c) Superposition of contour plots measured at pH 7.5 (blue) and pH 6.5 (red), showing the spectral region where cross-peaks between Cδ, Cγ1, Cγ2, Cα and Cβ of isoleucines (b) and threonines (c) are visible. Cross-peaks involving Thr158, Ile159, Ile161 and Thr162 in the IXI motif and Thr132, Ile133 and Thr134 in the substrate binding site are underlined and show peak doubling or chemical-shift perturbations upon a pH decrease from 7.5 to 6.5. (d) Cross-sections along F₂ from 2D ¹³C-¹³C spectra recorded at pH 7.5 (blue) and pH 6.5 (red), extracted at the Cα resonance of Thr134 (60.2 p.p.m.) and the Cγ2 resonance of Ile133 (18.3 p.p.m.), respectively.