Binding determinants of the small heat shock protein, αB-crystallin: recognition of the 'IxI' motif

Abstract

Small heat shock proteins (sHSPs) play a central role in protein homeostasis under conditions of stress by binding partly unfolded, aggregate-prone proteins and keeping them soluble. Like many sHSPs, the widely expressed human sHSP, αB-crystallin ('αB'), forms large polydisperse multimeric assemblies. Molecular interactions involved in both sHSP function and oligomer formation remain to be delineated. A growing database of structural information reveals that a central conserved α-crystallin domain (ACD) forms dimeric building blocks, while flanking N- and C-termini direct the formation of larger sHSP oligomers. The most commonly observed inter-subunit interaction involves a highly conserved C-terminal 'IxI/V' motif and a groove in the ACD that is also implicated in client binding. To investigate the inherent properties of this interaction, peptides mimicking the IxI/V motif of αB and other human sHSPs were tested for binding to dimeric αB-ACD. IxI-mimicking peptides bind the isolated ACD at 22°C in a manner similar to interactions observed in the oligomer at low temperature, confirming these interactions are likely to exist in functional αB oligomers.

Figure 1

Overlay of ¹H–¹⁵N HSQC spectra of ¹⁵N αB-ACD collected at 22°C, in the absence (black) and at saturating concentrations (five-fold) of αB-IxI peptide (red).

Figure 2

αB-ACD peaks titrate to multiple resonances at saturating concentrations of αB-IxI peptide. Three distinct resonances are observed for the amide of L94 in the presence of αB-IxI peptide (upper left arrows), while a single dominant peak is observed in the presence of the HSPB2-VxI peptide. The resonance for T132 also titrates to multiple positions in the presence of αB-IxI peptide (lower left), while a single dominant peak is observed in the presence of the HSPB2-VxI peptide. Spectra of the peptide-free and peptide-saturated ACD are shown in black and red, respectively.

Figure 3

Histogram of calculated CSPs (black) for the αB-ACD in the presence of saturating concentrations of αB-IxI peptide. Positions where CSPs could not be determined due to exchange behaviour (and therefore have very large chemical shifts) are indicated in grey. Secondary structure elements are shown above.

Figure 4

The chemical shift perturbations due to αB-IxI peptide binding map to the β4/β8 groove on the edge of the ACD. (Top) CSPs are mapped onto a secondary structure representation of the αB-ACD dimer (green and grey) (2klr). The most affected peaks (CSP>0.16) are shown in magenta on one monomer (green) and map to the β4/β8 groove and β4/5 loop. Residues where CSPs could not be quantitated due to exchange over large chemical shifts are shown in red. (Bottom) Surface representation of the αB-ACD dimer (2klr) rotated 45° with the same residue colouring as above. The C-terminal αB sequence PERTIPITREEK (white sticks) is shown as modelled previously with ssNMR restraints to illustrate the congruence between the chemical shifts observed for binding outside the oligomeric context and the structure observed in the context of αB oligomer.

Figure 5

Mutation of residue S135 in the β4/β8 groove disrupts IxI peptide binding. (A) Selected resonances from ¹H–¹⁵N HSQC of WT ACD (left) and the mutant S135Q ACD (right) in the absence and presence of αB-IxI peptide are compared. Spectra collected in the absence of peptide are shown in black. Spectra collected in the presence of 6-fold αB-IxI peptide (red) for the WT and S135Q ACD and 10-fold (green) peptide for S135Q are shown. In the S135Q mutant, residues K92 from the β4 strand and R120 (which is close to and behaves similarly to perturbed residues on the β3 strand) show a decrease in peptide-dependent chemical shift perturbation. The titration vector for the resonance of K92 in the WT spectra is shown as a grey arrow. (B) Peptide-induced CSPs observed for resonances of L94 and I133 are compared, with the same conditions and colouring as in (A).

Figure 6

Comparison of ¹⁵N chemical shift between the αB-ACD in the context of the oligomer observed by ssNMR and isolated αB-ACD in solution. (¹⁵N_{solid state}−¹⁵N_{solution state}) differences were measured using the solution-state values observed both in the absence of peptide (red bars) and in the presence of saturating αB-IxI peptide (black bars). Note the greatly reduced values for (¹⁵N_{solid state}-¹⁵N_{solution state}) in the presence of peptide in the β4 strand and β4/5 loop (residues 89–98) and the β8 strand (residues 133–137). Positions where (¹⁵N_{solid state}−¹⁵N_{solution state}) could not be determined are assigned the value of 0.0 p.p.m. for clarity. For residues where multiple states were observed in the peptide saturated spectrum, the position of the most intense peak in the saturated spectrum was used.