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.

Background and Purpose Vertebral hemangiomas are benign vascular lesions that are almost always incidentally found in the spine. Their classic typical hyperintense appearance on T1- and T2-weighted MR images is diagnostic. Unfortunately, not all hemangiomas have the typical appearance and can mimic metastases on routine MRI. These are generally referred to as atypical hemangiomas and can result in misdiagnosis and ultimately to additional imaging, biopsy and unnecessary costs. Our objective is to assess the utility of DCE-MRI perfusion in distinguishing vertebral atypical hemangiomas and malignant vertebral metastases. We hypothesize that permeability and vascular density will be increased in metastases compared to atypical hemangiomas. Materials and Methods Consecutive patients from 2011-2015 with confirmed diagnoses of atypical hemangiomas and spinal metastases from breast and lung carcinoma with available DCE-MRI were analyzed. Time-intensity curves were qualitatively compared among the groups. Perfusion parameters, plasma volume (Vp) and permeability constant (Ktrans), were quantified using an extended Toft’s two-compartment pharmacokinetic model. Statistical significance was tested using Mann-Whitney U test. Results Qualitative inspection of DCE-MRI time-intensity curves demonstrated differences in signal intensity and morphology, between metastases and atypical hemangiomas. Quantitative analysis of Vp and Ktrans perfusion parameters showed a significantly higher values in metastatic lesions when compared to atypical hemangiomas (p<0.001). Conclusions Our data demonstrate that Vp and Ktrans perfusion parameters and qualitative inspection of contrast enhancement curves can be used to differentiate atypical hemangiomas from vertebral metastatic lesions. This works highlights the benefits of adding perfusion maps to conventional sequences that can improve diagnostic accuracy.

Due to its favorable spectroscopic properties, Cd2+ is frequently used as a probe of Ca2+ sites in proteins. We investigate the ability of Cd2+ to act as a structural and functional surrogate of Ca2+ in protein–membrane interactions. C2 domain from protein kinase Cα (C2α) was chosen as a paradigm for the Ca2+-dependent phosphatidylserine-binding peripheral membrane domains. We identified the Cd2+-binding sites of C2α using NMR spectroscopy, determined the 1.6 Å crystal structure of Cd2+-bound C2α, and characterized metal-ion-dependent interactions between C2α and phospholipid membranes using fluorescence spectroscopy and ultracentrifugation experiments. We show that Cd2+ forms a tight complex with the membrane-binding loops of C2α but is unable to support its membrane-binding function. This is in sharp contrast with Pb2+, which is almost as effective as Ca2+ in driving the C2α-membrane association process. Our results provide the first direct evidence for the specific role of divalent metal ions in mediating protein–membrane interactions, have important implications for metal substitution studies in proteins, and illustrate the potential diversity of functional responses caused by toxic metal ions.

Ca2+-responsive C2 domains are peripheral membrane modules that target their host proteins to anionic membranes upon binding Ca2+ ions. Several C2-domain containing proteins, such as Protein Kinase C (PKC) isoenzymes, have been identified as molecular targets of Pb2+, a known environmental toxin. We demonstrated previously that the C2 domain from PKCα (C2α) binds Pb2+ with high affinity and undergoes membrane insertion in the Pb2+-complexed form. The objective of this work was to determine the effect of phosphatidylinostiol-4,5-biphosphate (PIP2) on the C2α-Pb2+ interactions. Using Nuclear Magnetic Resonance (NMR) experiments, we show that Pb2+ and PIP2 synergistically enhance each other’s affinity to C2α. Moreover, the affinity of C2α to PIP2 increases upon progressive saturation of the metal-binding sites. Combining the NMR data with the results of protein-to-membrane Förster Resonance Energy Transfer (FRET) and vesicle sedimentation experiments, we demonstrate that PIP2 can influence two aspects of C2α-Pb2+-membrane interactions: the affinity of C2α to Pb2+, and the association of Pb2+ with the anionic sites on the membrane. Both factors may contribute to the toxic effect of Pb2+ resulting from the aberrant modulation of PKCα activity. Finally, we propose a mechanism for Pb2+ outcompeting Ca2+ from the membrane-bound C2α.

Ca-dependent conserved-region 2 (C2) domains target their host signaling proteins to anionic membranes. The Ca-binding event is a prerequisite for membrane association. Here, we investigate multiscale metal-ion-dependent dynamics of the C2 domain of protein kinase Cα (C2α) using NMR spectroscopy. Interactions with metal ions attenuate microsecond-timescale motions of the loop regions, indicating that preorganization of the metal-binding loops occurs before membrane insertion. Binding of a full complement of Ca ions has a profound effect on the millisecond-timescale dynamics of the N- and C-terminal regions of C2α. We propose that Ca binding allosterically destabilizes the terminal regions of C2α and thereby facilitates the conformational rearrangement necessary for full membrane insertion and activation of protein kinase Cα.

Purpose: We conducted a randomized phase II multicenter clinical trial to test the hypothesis that physiologic MRI-based radiotherapy (RT) dose escalation would improve the outcome of patients with poor prognosis head and neck cancer. Patients and Methods: MRI was acquired at baseline and at RT fraction 10 to create low blood volume/apparent diffusion coefficient maps for RT boost subvolume definition in gross tumor volume. Patients were randomized to receive 70 Gy (standard RT) or 80 Gy to the boost subvolume (RT boost) with concurrent weekly platinum. The primary endpoint was disease-free survival (DFS) with significance defined at a one-sided 0.1 level, and secondary endpoints included locoregional failure (LRF), overall survival (OS), comparison of adverse events and patient reported outcomes (PRO). Results: Among 81 randomized patients, neither the primary endpoint of DFS (HR = 0.849, P = 0.31) nor OS (HR = 1.19, P = 0.66) was significantly improved in the RT boost arm. However, the incidence of LRF was significantly improved with the addition of the RT boost (HR = 0.43, P = 0.047). Two-year estimates [90% confidence interval (CI)] of the cumulative incidence of LRF were 40% (27%–53%) in the standard RT arm and 18% (10%–31%) in the RT boost arm. Two-year estimates (90% CI) for DFS were 48% (34%–60%) in the standard RT arm and 57% (43%–69%) in the RT boost arm. There were no significant differences in toxicity or longitudinal differences seen in EORTC QLQ30/HN35 subscales between treatment arms in linear mixed-effects models. Conclusions: Physiologic MRI-based RT boost decreased LRF without a significant increase in grade 3+ toxicity or longitudinal PRO differences, but did not significantly improve DFS or OS. Additional improvements in systemic therapy are likely necessary to realize improvements in DFS and OS.

Cd 2+ and Pb 2+ are xenobiotic heavy metal ions that use ionic mimicry to interfere with the cellular function of biomacromolecules. Using a combination of SAXS, electron microscopy, FRET, and solution NMR spectroscopy, we demonstrate that treatment with Cd 2+ and Pb 2+ causes self-assembly of protein kinase C regulatory domains that peripherally associate with membranes. The self-assembly process successfully competes with ionic mimicry and is mediated by conserved protein regions that are distinct from the canonical Ca 2+ -binding motifs of protein kinase C. The ability of protein oligomers to interact with anionic membranes is enhanced compared to the monomeric species. Our findings suggest that metal-ion-dependent peripheral membrane domains can be utilized for generating protein−metal-ion nanoclusters and serve as biotemplates for the design of sequestration agents. Communication pubs.acs.org/biochemistry

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