Substrate Binding to Alpha Crystallin Chaperone Prevents Metal-ion Binding

Abstract

Purpose: : Alpha crystallins are molecular chaperones, but also bind Cu²⁺ (and Zn²⁺) ions, which enhances their chaperone activity. Moreover, sequestration of Cu²⁺ prevents metal-ion mediated oxidations. In the lens, α-crystallins also bind substrate proteins such as γ-crystallins, and free α-crystallin is progressively depleted as the lens ages. It is not known how the depletion of free α-crystallin affects Cu²⁺ (and Zn²⁺) binding. Here we investigate (a) the spectroscopic changes accompanying Cu²⁺ binding to human αA-crystallin (HAA), and (b) the Cu²⁺ (and Zn²⁺) binding ability of HAA in the chaperone complex with human γD-crystallin (HGD).

Methods: : The HAA-Cu²⁺ complex was made by reacting CuCl₂ with HAA and purified by passing it through a Superose-6 size-exclusion chromatography (SEC) column. Absorption, circular dichroism (CD) and Raman spectroscopic studies were carried out. HAA-HGD complexes were made by incubating mixtures of HAA and HGD (2:1, w/w) at 50°C and 55°C. Cu²⁺ and Zn²⁺ ion concentrations were determined by using an extrinsic dye 4-(2-pyridylazo) resorcinol (PAR).

Results: : (a) The HAA-Cu²⁺ complex shows two absorption bands around 370 and 600 nm. The species with an absorption band ~370 nm is unstable and precipitates, but a stable complex containing ~0.5 mole of tightly bound Cu²⁺ per mole HAA monomer is obtained by SEC or extensive dialysis. The CD spectrum of the HAA-Cu²⁺ complex shows a positive band (~575 nm, d-d transition), and a negative band (~300 nm, charge-transfer). The dissociation constant (K_d) of the HAA-Cu²⁺ obtained from CD studies is 1.7x10^-12 M. Raman spectral changes of the Cu²⁺ complex suggest that Cu²⁺ is liganded to His residues in HAA through the N_π and N_τ atoms. (b) Binding of HGD, and of Cu²⁺ (and Zn²⁺) ions to HAA are inversely related: Higher the amount of HGD bound, lower the amount of bound metal ions.

Conclusions: : 1) Spectroscopic characterization of the stable HAA-Cu²⁺ complex shows detailed electronic and molecular structure 2) The chaperone complexes of α-crystallins show reduced Cu²⁺ (and Zn²⁺) binding ability.

Keywords: chaperones • crystallins • protein structure/function