Environmentally induced reversible conformational switching in the yeast cell adhesion protein alpha-agglutinin

Abstract

The yeast cell adhesion protein alpha-agglutinin is expressed on the surface of a free-living organism and is subjected to a variety of environmental conditions. Circular dichroism (CD) spectroscopy shows that the binding region of alpha-agglutinin has a beta-sheet-rich structure, with only approximately 2% alpha-helix under native conditions (15-40 degrees C at pH 5.5). This region is predicted to fold into three immunoglobulin-like domains, and models are consistent with the CD spectra as well as with peptide mapping and site-specific mutagenesis. However, secondary structure prediction algorithms show that segments comprising approximately 17% of the residues have high alpha-helical and low beta-sheet potential. Two model peptides of such segments had helical tendencies, and one of these peptides showed pH-dependent conformational switching. Similarly, CD spectroscopy of the binding region of alpha-agglutinin showed reversible conversion from beta-rich to mixed alpha/beta structure at elevated temperatures or when the pH was changed. The reversibility of these changes implied that there is a small energy difference between the all-beta and the alpha/beta states. Similar changes followed cleavage of peptide or disulfide bonds. Together, these observations imply that short sequences of high helical propensity are constrained to a beta-rich state by covalent and local charge interactions under native conditions, but form helices under non-native conditions.

Fig. 1.

Effect of pH on far-UV CD of α-agglutinin^20–351. (A) Basic pH: Spectra were taken at 25°C in 30 mM sodium acetate at pH 5.5 (solid line), or in 100 mM sodium phosphate at pH 8.5 (dashed line). The spectrum of the reconstituted sample pH (8.5→5.5), which had been preincubated in 100 mM sodium phosphate at pH 8.5 for 2 h and 30 mM sodium acetate at pH 5.5 for 30 min, was measured at 25°C in 30 mM sodium acetate at pH 5.5 (dotted line). (Inset) Secondary structure content calculated by curve fitting: (open bar) β-sheet; (solid bar) α-helix; (hatched bar) aperiodic structures. Error bars: see text. All spectra contained ∼16% turn (not shown). (B) Acidic pH: Spectra were measured at 25°C in 30 mM sodium acetate at pH 5.5 (solid line); 30 mM sodium acetate at pH 3.5 (dotted line); or 100 mM sodium phosphate at pH 1.5 (dashed line). (Inset) Secondary structure content calculated by curve fitting.

Fig. 2.

Effect of temperature on far-UV CD of α-agglutinin^20–351. Samples were equilibrated at various temperatures for 20 min and spectra were measured in 30 mM sodium acetate at pH 5.5 at 15°, 25°, 35°, and 45°C (solid line); 55°C (dotted line); or 65°C (dashed line). (Inset) Secondary structure content at different temperatures calculated by curve fitting: (open bar) β-sheet; (solid bar) α-helix; (hatched bar) aperiodic structures. Error bars: see text. All spectra contained ∼16% turn (not shown).

Fig. 3.

Effect of DTT and brief heat treatment at 100°C on far-UV CD of α-agglutinin^20–351. Spectra were taken at 25°C in 30 mM sodium acetate at pH 5.5; without any treatment (solid line); with 10 mM DTT treatment (dotted line); with heat treatment at 100°C for 5 min (dashed line); or DTT treatment plus heat treatment at 100°C for 5 min (dash/dot line). (Inset) Secondary structure content calculated by curve fitting: (open bar) β-sheet;; (solid bar) α-helix; (hatched bar) aperiodic structures. Error bars: see text. All spectra contained ∼16% turn (not shown).

Fig. 4.

Three-dimensional orientation of two peptides of high helical propensity derived from the proposed β-strands regions of α-agglutinin^20–351. Model of domains II and III of α-agglutinin, with peptide I (C–C` strand region of domain II) shown in cyan and peptide II (E–F strand region of domain III) shown in yellow. The models were based on similarity to the CD2/CD4 subfamily of the Ig superfamily, and were tested by peptide mapping and site-directed mutagenesis (Lipke et al. 1995; Grigorescu et al. 2000). The peptide sequences are shown in single letter code, and their helical propensities are summarized in Table 1.

Fig. 5.

Effect of TFE on far-UV CD of peptide I at different pH values. Spectra were measured in 10 mM sodium phosphate at pH 2–10 in 10% TFE (solid line); 20% TFE (dotted line); 40% TFE (dashed line), or 60% TFE (dash/dot line). The spectra below pH 3 were similar to one another, as were those above 8. The spectra at pH 3, 5, and 7 are shown.

Fig. 6.

Effect of TFE on far-UV CD of peptide II. Spectra were measured in 10 mM sodium phosphate at pH 2–10 in 0% TFE (dash/dot line); 10% TFE (solid line); 20% TFE (dotted line); 40% TFE (dashed line), or 60% TFE (dash/dot/dot line). Because the spectra at all pH values were similar, only the curves at pH 5 are shown. (Inset) Molar ellipticity at 222 mn versus concentration of TFE.