In vitro unfolding of yeast multicopper oxidase Fet3p variants reveals unique role of each metal site

Abstract

Fet3p from Saccharomyces cerevisiae is a multicopper oxidase (MCO) that contains 3 cupredoxin-like beta-barrel domains and 4 copper ions located in 3 distinct metal sites (T1 in domain 3, T2, and the binuclear T3 at the interface between domains 1 and 3). To better understand how protein structure and stability is defined by cofactor coordination in MCO proteins, we assessed thermal unfolding of apo and metallated forms of Fet3p by using spectroscopic and calorimetric methods in vitro (pH 7). We find that unfolding reactions of apo and different holo forms of Fet3p are irreversible reactions that depend on the scan rate. The domains in apo-Fet3p unfold sequentially [thermal midpoint (T(m)) of 45 degrees C, 62 degrees C, and 72 degrees C; 1 K/min]. Addition of T3 imposes strain in the apo structure that results in coupled domain unfolding and low stability (T(m) of 50 degrees C; 1 K/min). Further inclusion of T2 (i.e., only T1 absent) increases overall stability by approximately 5 degrees C but unfolding remains coupled in 1 step. Introduction of T1, producing fully-loaded holo-Fet3p (or in the absence of T2), results in stabilization of domain 3, which uncouples unfolding of the domains; unfolding of domain 2 occurs first along with Cu-site perturbations (T(m) 50-55 degrees C; 1 K/min), followed by unfolding of domains 1 and 3 ( approximately 65-70 degrees C; 1 K/min). Our results suggest that there is a metal-induced tradeoff between overall protein stability and metal coordination in members of the MCO family.

Fig. 1.

Schematic diagram of Fet3p topology and Cu sites. The β-strands of the 3 domains are shown in different colors to indicate boundaries. The 3 Cu sites [T1 (Cu1), blue; T2 (Cu4), green; T3 (Cu2 and Cu3), yellow; T2 and T3 form TNC] and the coordinating side chains are indicated. The side-chain labels are color-coded based on the domain origin.

Fig. 2.

Thermal unfolding of WT apo-Fet3p probed by CD at 210 nm (Middle), Trp fluorescence (ratio F₃₃₁/F₃₅₅), ANS fluorescence (at 510 nm) (Top), and DSC (Bottom). Scan rates were 0.5, 1.0, and 1.5 K/min.

Fig. 3.

Thermal unfolding of WT, T2D, T1D, T1D/T2D and apo-Fet3p variants (top to bottom) probed by CD at 210 nm (open circles), Trp fluorescence (ratio F₃₃₁/F₃₅₅; red), and absorption at 330 nm (yellow) (Center). For WT holo, oxidase activity (open triangles) and 610-nm absorption (filled triangles) are shown. CD (circles) and DSC (solid line) profiles are overlaid at Right. Scan rate in all experiments shown was 1.0 K/min. For additional mutant data see Fig. S2.

Fig. 4.

Thermal unfolding of WT holo-Fet3p probed by CD at 210 nm (Middle), Trp fluorescence (ratio F₃₃₁/F₃₅₅), ANS fluorescence (at 510 nm) (Top), and DSC (Bottom). Scan rates were 0.5, 1.0, and 1.5 K/min.