Pb²⁺ as Modulator of Protein–Membrane Interactions

Abstract

Lead is a potent environmental toxin that mimics the effects of divalent metal ions, such as zinc and calcium, in the context of specific molecular targets and signaling processes. The molecular mechanism of lead toxicity remains poorly understood. The objective of this work was to characterize the effect of Pb2+ on the structure and membrane-binding properties of C2α. C2α is a peripheral membrane-binding domain of Protein Kinase Cα (PKCα), which is a well-documented molecular target of lead. Using NMR and isothermal titration calorimetry (ITC) techniques, we established that C2α binds Pb2+ with higher affinity than its natural cofactor, Ca2+. To gain insight into the coordination geometry of protein-bound Pb2+, we determined the crystal structures of apo and Pb2+-bound C2α at 1.9 Å and 1.5 Å resolution, respectively. A comparison of these structures revealed that the metal-binding site is not pre-organized and that rotation of the oxygen-donating sidechains is required for the metal coordination to occur. Remarkably, we found that holodirected and hemidirected coordination geometries for the two Pb2+ ions coexist within a single protein molecule. Using protein-to-membrane Förster resonance energy transfer (FRET) spectroscopy, we demonstrated that Pb2+ displaces Ca2+ from C2α in the presence of lipid membranes through the high-affinity interaction with the membrane-unbound C2α. In addition, Pb2+ associates with phosphatidylserine-containing membranes and thereby competes with C2α for the membrane-binding sites. This process can contribute to the inhibitory effect of Pb2+ on the PKCα activity.

Cellular and molecular interactions of phosphoinositides and peripheral proteins

“…Recently, the C2 domain of PKCα was shown to bind Pb 2+ with high affinity, which can compete with Ca 2+ for an interaction with the C2 domain (Morales et al 2011). Further structural studies using NMR revealed that Pb 2+ and PI(4,5)P 2 synergistically enhanced each others affinity for the C2 domain (Morales et al 2012).…”

Cd2+as a Ca2+Surrogate in Protein–Membrane Interactions: Isostructural but Not Isofunctional

“…The CSP pattern in the low-concentration Cd 2+ regime is very similar to that observed previously for the binding of two Ca 2+ ions to C2α. 16 This suggests that no less than two Cd 2+ ions with similar affinities bind to the loop region. We used the fluorescence emission spectra of native tryptophan residues in C2α to estimate the affinity of Cd 2+ to the high-affinity CMBL site.…”

“…This is in sharp contrast with Pb 2+ ions, whose coordination geometry in protein-bound state is different from that of Ca 2+ , yet their ability to drive the C2α-membrane association is comparable. 16 …”

Non-intercalative, Deoxyribose Binding of Boric Acid to Calf Thymus DNA

“…Several reports in literature detail ITC-based quantitative measurements of interactions between biological macromolecules, such as proteins [30][31][32][33][34][35] or DNA [36][37][38][39] and metal ions. In this work, ITC was utilized to characterize the thermodynamics of boric acid binding to ct-DNA.…”