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 ␤-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°C, 62°C, and 72°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°C; 1 K/min). Further inclusion of T2 (i.e., only T1 absent) increases overall stability by Ϸ5°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°C; 1 K/min), followed by unfolding of domains 1 and 3 (Ϸ65-70°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.spectroscopy ͉ calorimetry ͉ cupredoxin ͉ copper-binding protein

Potassium and the K+/H+ Exchanger Kha1p Promote Binding of Copper to ApoFet3p Multi-copper Ferroxidase

“…Alkali cations and metal ions affect protein function and stability through site-specific binding (14,34). However, our elemental analysis of purified holo-Fet3p did not detect stably bound K ϩ , which is consistent with a previous report on crystal structure of Fet3p (55).…”

“…Methods for purification of Fet3p lacking its C-terminal transmembrane domain was prepared as described (33) as was apoFet3p (34). GST was expressed in BY4741 yeast strain using p415-GPD vector (26) and purified using glutathione (GSH)-agarose (Thermo Scientific).…”

Role of CTR4 in the Virulence of Cryptococcus neoformans

“…The importance of copper to pathogenesis is also suggested by an essential role for the copper-containing cytochrome c oxidase (12) in this obligate aerobe as well as a role in virulence (10), which is dependent on a vesicular chloride channel and proton pump for copper metalation and virulence (13, 14). In addition, copper homeostasis may play a more widespread role in virulence through its effects on Fe + acquisition (15), as copper is a substrate for the high-affinity Fe + transporter Fet3 (16). The importance of copper in chemotherapeutics is suggested by recent studies proposing microplusin, a copper-chelating antimicrobial peptide, as a potential therapy against C. neoformans (17).…”

The Tat Substrate CueO Is Transported in an Incomplete Folding State

“…The assembly of type 1 copper likely requires similar flexibility. Thermal unfolding has been studied before with the multicopper oxidase Fet3p from yeast, which behaved differently, showing sequential unfolding of the domains, albeit also in these studies the domain-connecting role of type 3 copper has been recognized (45). Yeast does not possess a Tat system, and Fet3p has a predicted eukaryotic Sec signal peptide and a C-terminal membrane anchor that places this enzyme on the outer surface of the yeast cytoplasmic membrane.…”