Dataset Information

Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

ABSTRACT: To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9)TKE(12) sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ?5, from K(d)?=?25±6 nM to K(d)?=?5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d)?=?0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ?(as)(P-O) and ?(s)(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ?(as)(UO(2))(2+) vibration (from 923 cm(-1) to 908 cm(-1)) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.

SUBMITTER: Pardoux R

PROVIDER: S-EPMC3411679 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

Pardoux Romain R Sauge-Merle Sandrine S Lemaire David D Delangle Pascale P Guilloreau Luc L Adriano Jean-Marc JM Berthomieu Catherine C

PloS one 20120803 8

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitr ...[more]

PMID: 22870263

Similar Datasets

Project description:The EF hand, a helix-loop-helix structure, is one of the most common motifs found in animal genomes, and EF-hand Ca(2+)-binding proteins (EFCaBPs) are widely distributed throughout the cell. However, researchers remain confounded by a lack of understanding of how peptide sequences code for specific functions and by uncertainty about the molecular mechanisms that enable EFCaBPs to distinguish among many diverse cellular targets. Such knowledge could define the roles of EFCaBPs in health and disease and ultimately enable control or even design of Ca(2+)-dependent functions in medicine and biotechnology. In this Account, we describe our structural and biochemical research designed to understand the sequence-to-function relationship in EFCaBPs. The first structural goal was to define conformational changes induced by binding Ca(2+), and our group and others established that solution NMR spectroscopy is well suited for this task. We pinpointed residues critical to the differences in Ca(2+) response of calbindin D(9k) and calmodulin (CaM), homologous EFCaBPs from different functional classes, by using direct structure determination with site-directed mutagenesis and protein engineering. Structure combined with biochemistry provided the foundation for identifying the fundamental mechanism of cooperativity in the binding of Ca(2+) ions: this cooperativity provides EFCaBPs with the ability to detect the relatively small changes in concentration that constitute Ca(2+) signals. Using calbindin D(9k) as a model system, studies of the structure and fast time scale dynamics of each of the four ion binding states in a typical EF-hand domain provided direct evidence that site-site communication lowers the free energy cost of reorganization for binding the second ion. Our work has also extended models of how EFCaBPs interact with their cellular targets. We determined the unique dimeric architecture of S100 proteins, a specialized subfamily of EFCaBPs found exclusively in vertebrates. We described the implications for how these proteins transduce signals and went on to characterize interactions with peptide fragments of important cellular targets. Studies of the CaM homolog centrin revealed novel characteristics of its binding of Ca(2+) and its interaction with its cellular target Kar1. These results provided clear examples of how subtle differences in sequence fine-tune EFCaBPs to interact with their specific targets. The structural approach stands at a critical crossroad, shifting in emphasis from descriptive structural biochemistry to integrated biology and medicine. We present our dual-molecular-switch model for Ca(2+) regulation of gating functions of voltage-gated sodium channels in which both CaM and an intrinsic EF-hand domain serve as coupled Ca(2+) sensors. A second example involves novel EFCaBP extracellular function, that is, the role of S100A8/S100A9 heterodimer in the innate immune response to bacterial pathogens. A mechanism for the antimicrobial activity of S100A8/S100A9 was discovered. We describe interactions of S100A8/S100A9 and S100B with the cell surface receptor for advanced glycation end products. Biochemical and structural studies are now uncovering the mechanisms by which EFCaBPs work and are helping to define their biological activities, while simultaneously expanding knowledge of the roles of these proteins in normal cellular physiology and the pathology of disease.

Project description:Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr2+ interference with Ca2+ binding to proteins of the EF-hand family, we studied Sr2+/Ca2+ interaction with a canonical EF-hand protein, α-parvalbumin (α-PA). Evaluation of the equilibrium metal association constants for the active Ca2+ binding sites of recombinant human α-PA ('CD' and 'EF' sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 109 M-1 and 4 × 109 M-1 for Ca2+, and 2 × 107 M-1 and 2 × 106 M-1 for Sr2+. Inactivation of the EF site by homologous substitution of the Ca2+-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca2+/Sr2+ affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca2+/Sr2+ affinity. These results suggest that Sr2+ and Ca2+ bind to CD/EF sites of α-PA and the Ca2+/Sr2+ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr2+ titration of the Ca2+-loaded α-PA revealed presence of secondary Sr2+ binding site(s) with an apparent equilibrium association constant of 4 × 105 M-1. Fourier-transform infrared spectroscopy data evidence that Ca2+/Sr2+-loaded forms of α-PA exhibit similar states of their COO- groups. Near-UV circular dichroism (CD) data show that Ca2+/Sr2+ binding to α-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca2+/Sr2+ binding are accompanied by nearly identical changes in secondary structure of α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr2+-induced increase in stability of tertiary structure of α-PA, compared to the Ca2+-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca2+-binding sites of α-PA are well protected against exchange of Ca2+ for Sr2+ regardless of coordination number of Sr2+, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca2+/Sr2+ selectivity. Overall, despite lowered affinity of α-PA to Sr2+, the latter competes with Ca2+ for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr2+ binding site(s) could be a factor contributing to Sr2+ impact on the functional activity of proteins.

Dataset Information

Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

Publications

Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets