Insights into the domains required for dimerization and assembly of human alphaB crystallin

Abstract

Protein pin array technology was used to identify subunit-subunit interaction sites in the small heat shock protein (sHSP) alphaB crystallin. Subunit-subunit interaction sites were defined as consensus sequences that interacted with both human alphaA crystallin and alphaB crystallin. The human alphaB crystallin protein pin array consisted of contiguous and overlapping peptides, eight amino acids in length, immobilized on pins that were in a 96-well ELISA plate format. The interaction of alphaB crystallin peptides with physiological partner proteins, alphaA crystallin and alphaB crystallin, was detected using antibodies and recorded using spectrophotometric absorbance. Five peptide sequences including 37LFPTSTSLSPFYLRPPSF54 in the N terminus, 75FSVNLDVK82)(beta3), 131LTITSSLS138 (beta8) and 141GVLTVNGP148 (beta9) that form beta strands in the conserved alpha crystallin core domain, and 155PERTIPITREEK166 in the C-terminal extension were identified as subunit-subunit interaction sites in human alphaB crystallin using the novel protein pin array assay. The subunit-subunit interaction sites were mapped to a three-dimensional (3D) homology model of wild-type human alphaB crystallin that was based on the crystal structure of wheat sHSP16.9 and Methanococcus jannaschi sHSP16.5 (Mj sHSP16.5). The subunit-subunit interaction sites identified and mapped onto the homology model were solvent-exposed and had variable secondary structures ranging from beta strands to random coils and short alpha helices. The subunit-subunit interaction sites formed a pattern of hydrophobic patches on the 3D surface of human alphaB crystallin.

Figure 1.

(A,left) Protein pin array and ELISA assay identified interactive sequences of human αB crystallin. Sequential 8-mer peptides were synthesized on the individual tips of derivatized polyethylene pins arranged in a 96-well microtiter plate format. Each pin contained nanomoles of a specific eight-amino-acid in length peptide immobilized on it and consecutive peptides were offset by two amino acids. All peptides were covalently bonded to the surface of the plastic pins. The first peptide was ₁MDIAIHHP₈, and the last peptide was ₁₆₈PAVTAAPK₁₇₅ for the human αB crystallin. In all, 84 peptides corresponding to the 175-amino-acid primary sequence of human αB crystallin were synthesized and immobilized on 84 individual pins of the pin array (Table 2). (B,center) ELISA plate result of the negative control for interactions between human myo-globin and the human αB crystallin peptide library. Blue coloration indicates a positive interaction, and a clear solution indicates no interaction. (C,right) ELISA plate result for the interactions between human αB crystalline and the peptides of the human αB crystallin protein pin array. Blue coloration indicates a positive interaction; a clear solution indicates no interaction.

Figure 2.

A plot of the αB crystallin peptide vs. absorbance when human αA crystallin or human αB crystallin was used as ligand in the human αB crystallin protein pin array. Patterns of absorbance indicates the interactive sequences in human αB crystallin. Absorbance maxima were observed at three sequences in the α crystallin core domain and one in each of the extensions of the C and N termini. The X-axis lists the 8-mer peptides in order on the 84 pins of the protein pin array. The Y-axis is the absorbance measured at 405 nm in each well of the ELISA plate when αA crystallin and αB crystallin were screened using an ELISA-based colorimetric method. Interactions between human αA crystallin and the peptides of the human αB crystallin protein pin array were detected in a similar way. The height of the vertical bars represents the absorbance for each peptide and is proportional to the interaction of that peptide with the ligand protein (human αA crystallin or human αB crystallin). Absorbance readings in the range of 0.00–0.05 were observed in the absence of any ligand and were considered baseline noise. Solid black bars represent the absorbance for human αB crystallin at room temperature; diagonal striped bars represent the absorbance recorded when human αA crystallin was used as the ligand. At room temperature, interactions were not observed at every peptide, and there was a distinct pattern to the interactions. Wells corresponding to interactive peptides were blue and are the taller bars in the plot. Wells without color are short bars or no bars. Peptides that had tall bars for both the human αA crystallin and human αB crystallin experiments were selected as the sequences involved in subunit–subunit interactions, and are identified with arrows and brackets. The predicted secondary structure of human αB crystallin (~, helix; bars, β strands; black lines, unstructured stretch or loop) is at the top of the figure. The figure is divided into three structural regions. Sequences in the left (pink) were in the N-terminal extension. Sequences in the C-terminal extension are to the right (light blue), and sequences in the α crystallin core domain are in the center (dark blue).

Figure 3.

Sequence alignment, secondary structure elements, and interactive sequences for human αB crystallin, sHSP16.5, and sHSP16.9. The observed secondary structure of wheat sHSP16.9 and Methanococcus jannaschi sHSP16.5 along with the predicted secondary structure of human αB crystallin (~,α helix;-, β strand) are shown. The interactive sequences in the dodecameric quaternary structure of wheat HSP16.9 are in green boxes; the interactive sequences for the Mj sHSP16.5 24-mer structure are in blue boxes. The interactive domains identified in human αB crystallin using protein pin arrays are in orange boxes and corresponded with the interactive sequences observed in the crystal structures of Mj sHSP16.5 and wheat sHSP16.9. In the crystal structure of wheat sHSP16.9, the α helix in the N terminus interacted with β4 and β7 in the α crystallin core domain.

Figure 4.

Superposition of the monomeric subunits of Mj sHSP16.5 (blue), wheat sHSP16.9 (green), and human αB crystallin (red). The 3D coordinates of the three structures were superimposed yielding a final RMSD of 3.25 Å. All three structures have the same basic topology. The hydrophobic N terminus is largely helical (except in Mj sHSP16.5, where the N terminus is unresolved in the X-ray structure). An immunoglobulin-like α crystallin core domain consists of β strands (11 in Mj sHSP16.5, eight in wheat sHSP16.9, and seven in αB crystallin) that form two anti-parallel β sheets connected by a flexible loop. The C-terminal extension is highly charged and unstructured. Note that the C-terminal extension is unresolved in the X-ray structure of Mj sHSP16.5.

Figure 5.

(A,top) Mapping of the interactive sequences to the 3D computer model of human αB crystallin. All five binding regions (one in the N terminus, one in the C terminus, and three in the α crystallin core domain) are solvent-exposed in the monomeric subunit. Interactive sites identified by the protein pin arrays are in color and coded as follows: blue, acidic; red, basic; green, hydrophobic; orange, hydrophilic. (B,bottom) A space-filling representation of human αB crystallin using the same colors as in A.