Evolutionary remodeling of βγ-crystallins for domain stability at cost of Ca2+ binding

Abstract

The topologically similar βγ-crystallins that are prevalent in all kingdoms of life have evolved for high innate domain stability to perform their specialized functions. The evolution of stability and its control in βγ-crystallins that possess either a canonical (mostly from microorganisms) or degenerate (principally found in vertebrate homologues) Ca2+-binding motif is not known. Using equilibrium unfolding of βγ-crystallin domains (26 wild-type domains and their mutants) in apo- and holo-forms, we demonstrate the presence of a stability gradient across these members, which is attained by the choice of residues in the (N/D)(N/D)XX(S/T)S Ca2+-binding motif. The occurrence of a polar, hydrophobic, or Ser residue at the 1st, 3rd, or 5th position of the motif is likely linked to a higher domain stability. Partial conversion of a microbe-type domain (with a canonical Ca2+-binding motif) to a vertebrate-type domain (with a degenerate Ca2+-binding motif) by mutating serine to arginine/lysine disables the Ca2+-binding but significantly augments its stability. Conversely, stability is compromised when arginine (in a vertebrate-type disabled domain) is replaced by serine (as a microbe type). Our results suggest that such conversions were acquired as a strategy for desired stability in vertebrate members at the cost of Ca2+-binding. In a physiological context, we demonstrate that a mutation such as an arginine to serine (R77S) mutation in this motif of γ-crystallin (partial conversion to microbe-type), implicated in cataracts, decreases the domain stability. Thus, this motif acts as a "central tuning knob" for innate as well as Ca2+-induced gain in stability, incorporating a stability gradient across βγ-crystallin members critical for their specialized functions.

FIGURE 1.

A, amino acid sequence alignment of βγ-crystallin domains studied. Residues shown in gray are the signature sequence of Greek key motif, and Ca²⁺-binding (N/D)(N/D)XX(S/T)S motif is highlighted. B, schematic representation of the βγ-crystallin domains present in proteins from their respective species. C, depiction of topology of a vertebrate (N-terminal domain of γ-crystallin, Protein Data Bank code 4GCR) and a microbial (Clostrillin, Protein Data Bank code 3IAJ) crystallin, demonstrating the structural similarity of both types of domains.

FIGURE 2.

Isothermal titration calorigram of Ca²⁺ binding to Vibrillin (A), Centillin WT (B), Centillin F75D (C), Centillin R36S, F75D, (D) and Clostrillin T41K (E). The concentrations of protein and Ca²⁺ used in ITC experiments were 100 μm and 10 mm, respectively. The protein in sample cell was titrated with Ca²⁺ that was loaded in a syringe with 80 injections of 3 μl each from a stock of 10 mm CaCl₂. The K_d values are listed in supplemental Table S1.

FIGURE 3.

Stability gradient across βγ-crystallin domains. Equilibrium unfolding of βγ-crystallin domains in the absence or presence of CaCl₂. Change in the λ_max and best fits in the two-state unfolding process with [GdmCl] of Vibrillin (A and B), Flavollin (C and D), Clostrillin (E and F), and Centillin (G and H) proteins. There is a gradual increase in stability (or stability gradient) of the selected domains in apo-form; similar gradient is also seen in terms of gain of stability by Ca²⁺. F_N and F_U represent the fractions at native and unfolded states, respectively.

FIGURE 4.

Stability change in a βγ-crystallin domain with minor change in microenvironment of Ca²⁺-binding motif. Equilibrium unfolding of Clostrillin mutants (Thr to Ser) and Flavollin mutants (Ser to Thr) in the absence or presence of CaCl₂. Changes in λ_max with [GdmCl] concentration are as follows: A, Clostrillin T41S; B, Clostrillin T82S; and C, Clostrillin T41S,T82S; compared with Clostrillin WT. Best fits at two-state transitions and fractions of unfolded states for Clostrillin mutants are as follows: D, Clostrillin T41S; E, Clostrillin T82S, and F, Clostrillin T41S,T82S. Changes in wavelength maxima with increasing [GdmCl] of Flavollin S40T (G), Flavollin S81T (H), and Flavollin S40T,S81T (I) are shown.

FIGURE 5.

Control of domain stability via (N/D)(N/D)XX(S/T)S motif. Equilibrium unfolding of Clostrillin W39D, Flavollin D38V, Centillin R34V, and Centillin F75D mutants. Comparison of changes in the λ_max and their best fits in to two-state unfolding process: Clostrillin W39D (A and E), flavollin D38V (B and F), Centillin R34V (C and G), and Centillin F75D (D and H) mutants with [GdmCl] in the absence or presence of CaCl₂. The negligible change in the c½ value was observed in the holo-states of Clostrillin W39D and Flavollin D38V (1.3 and 2.1 m) as compared with apo-form (1.1 and 2.0 m), respectively. The significant change in the c½ value was observed in the holo-state of Centillin F75D (4.8 m) as compared with its apo-form (3.3 m). The significant change in the c½ value was also observed in Centillin R34V (2.7 m) as compared with Centillin WT (2.3 m).

FIGURE 6.

Augmentation of stability upon conversion of a functional Ca²⁺-binding motif to disabled motif. Equilibrium unfolding of (Thr or Ser to Arg) mutants in the absence or presence of CaCl₂. Changes in λ_max with [GdmCl] concentration are as follows: A, Clostrillin T41R; B, Clostrillin T82R; C, Clostrillin T41R,T82R, and D, Centillin S78R compared with Clostrillin WT and Centillin WT, respectively. Best fit and fractions of unfolded states for the above mutants are as follows: E, Clostrillin T41R; F, Clostrillin T82R; G, Clostrillin T41R, T82R, and H, Centillin S78R. The data were fitted in two-state transitions.

FIGURE 7.

Attenuation of stability upon conversion of a naturally disabled Ca²⁺-binding motif to functional motif. Equilibrium unfolding of Clostrillin T41K, Centillin R36S,F75D, nitrollin R50S,R95S mutants. Comparison of changes in the λ_max and their best fits in to two-state unfolding process as follows: A and B, Clostrillin T41K; C and D, Centillin R36S,F75D; E and F, and nitrollin R50S,R95S mutants with [GdmCl] in the absence or presence of CaCl₂. The c½ value of Clostrillin T41K was comparable with the holo-form of Clostrillin WT. The significant change in the c½ value in the holo-state of Centillin F75D mutant (4.8 m) was lost in Centillin R36S,F75D mutant (2.3 m). Similarly c½ value decreases in nitrollin R50S,R95S (Arg to Ser) (2.8 m) as compared with nitrollin WT (3.3 m).

FIGURE 8.

Comparison of stability of N-terminal domain of lens γB-crystallin and its cataract-related R77S mutant. A, location of Arg-77 in the crystal structure of N-terminal domain of γ-crystallin (Protein Data Bank code 1hko) in 2nd Greek key motif as marked in figure. B, changes in λ_max with [GdmCl] concentration of γB-WT and R77S mutant. C, change in ratios of fluorescence intensity at fixed wavelengths (360 and 320 nm) with increasing concentrations of denaturant (GdmCl) of N-terminal domain of γB-crystallin and its R77S mutant. D, best fit and fractions of unfolded states for the γB-WT (single domain) and R77S mutant. The c½ value 3.0 m, ΔG decreased from 8.3 (γB WT) to 2.2 (γB-R77S mutant) kcal/mol. The data were fitted in a two-state transition.

FIGURE 9.

A, domain stability controlled by the choice of residues in the ¹(N/D)(N/D)XX(S/T)S⁶ motif. Changes in transition midpoint (Δc½) of βγ-crystallin domains calculated as the difference between c½ of mutant and its WT protein for both apo- (gray) and holo (lines)-forms (Table 1). B, schematic representation demonstrating the (N/D)(N/D)XX(S/T)S motif as the stability tuning knob in βγ-crystallins. Hydrophobic residue at X¹ position augments the stability, although a polar residue turns off the stability. Ser residue at the 5th position in the canonical motif is favored and is responsible for the higher stability. Polar residue at the 1st position enhanced the stability. Arg at the 5th position in noncanonical motifs (in vertebrate members) provides more stability to a domain.