On the role of local unfolding and catalysis modulation

GONZALEZ LEBRERO RM; SANTIAGO MARTINEZ GACHE; ROMAN EA; AGUEDA PLACENTI; GONZALEZ FLECHA FL

Puerto Varas

Workshop; First Latin American Workshop on Artificial Intelligence for Protein Design; 2023

First Latin American Workshop on Artificial Intelligence for Protein Design (AI4PD).

P-ATPases constitute a family of membrane proteins that couple ATP hydrolysis to thetransport of solutes across biological membranes, forming a phosphorylated intermediateduring their reaction cycle. These proteins share a transmembrane domain through which thesubstrate is transported. Also share cytoplasmic domains that consists of a catalyticmodulator domain (A or "actuator") and an ATP binding and hydrolysis domain (ATPBD or“ATP binding domain") comprised by the N ("nucleotide binding") and P ("phosphorylation")subdomains. ATPBD crystallographic structures reveal different conformational statesrelated to open-closed movements which may be involved in coupling nucleotide hydrolysisto substrate transport. In our laboratory, we purified the ATPBD of a Cu+transporting PATPase from the hyperthermophile archaea Archaeoglobus fulgidus. We characterized theeffect of substrate concentration and temperature on its steady state ATPase activityobserving it could be described by a Michaelis-Menten equation and obtaining Km and Vmaxas a function of temperature. Additionally, we examined the effect of temperature on itsintrinsic fluorescence observing a conformational transition occurring within the temperaturerange where this protein is catalytically active. A simple 3 states steady-state kinetic modelexplains the activity dependence on substrate concentration, its optimal working temperatureand intrinsic fluorescence changes. Furthermore, since we hypothesized that there is an openclose transition involved in catalysis, we studied the role of local unfolding (“cracking”) onATPBD activity and found that low urea concentrations induce an increase in ATPase activityand shift the optimal working temperature to lower values. Altogether these results suggestthat, in this case, optimal working temperature is, at least partially, modulated by aconformational change.