Selectivity mechanism of magnesium and calcium in cation-binding pocket structures of phosphotyrosine

Magnesium $\left(\mathrm{M}{\mathrm{g}}^{2+}\right)$ and calcium $\left(\mathrm{C}{\mathrm{a}}^{2+}\right)$ are of essential importance in biological activity, but the molecular understanding of their selectivity is still lacking. Here, based on density functional theory calculations and ab initio molecular dynamics simulations, we show that $\mathrm{M}{\mathrm{g}}^{2+}$ binds more tightly to phosphotyrosine (pTyr) and stabilizes the conformation of pTyr, while $\mathrm{C}{\mathrm{a}}^{2+}$ binds more flexibly to pTyr with less structural stability. The key for the selectivity is attributed to the cation-π interactions between the hydrated cations and the aromatic ring together with the synergic interaction between the cations and the side groups in pTyr to form a cation-binding pocket structure, which we refer as side-group-synergetic hydrated cation-π interaction. The existence and relative strength of the cation-π interactions in the pocket structures as well as their structural stability have been demonstrated experimentally with ultraviolet (UV) absorption spectra and ${}^1\mathrm{H}$ NMR spectra. The findings offer insight into understanding the selectivity of $\mathrm{M}{\mathrm{g}}^{2+}$ and $\mathrm{C}{\mathrm{a}}^{2+}$ in a variety of biochemical and physiological essential processes.

Figure 1

Cation-binding pocket of phosphotyrosine (pTyr) and side-group-synergetic hydrated cation-π interactions. Representative AIMD trajectories of the conformations in the pocket structures of pTyr for $\mathrm{M}{\mathrm{g}}^{2+}$ (a) and $\mathrm{C}{\mathrm{a}}^{2+}$ (b), respectively. The numbers indicate the time of snapshots. The structures at $t=0\mathrm{ps}$ are achieved by geometry optimization with DFT calculations. The aromatic ring in pTyr are shown as big bonded spheres, the side groups as bonded sticks, and $\mathrm{M}{\mathrm{g}}^{2+}/\mathrm{C}{\mathrm{a}}^{2+}$ as a sphere (C: cyan; N: blue; O: red; H: white; Mg: purple; and Ca: yellow), and the surfaces of side groups, i.e., the carboxyl, amino and phosphate groups, are shown in pale red. The first-shell waters, whose oxygens do not exceed 2.8 Å from the cation, are shown as bonded spheres with their surfaces shown in pale pink, and the waters that are hydrogen-bonded with the phosphate group and the first-shell water are shown as small bonded spheres, (O: pink; H: grey), and the other waters are omitted for a clear view. The cations and their first-shell water and the key atoms in the side groups are linked by blue dashed lines, and the hydrogen bonds by pink dashed lines. The transparent six-face cones (purple, yellow) comprising the cation ($\mathrm{M}{\mathrm{g}}^{2+}$, $\mathrm{C}{\mathrm{a}}^{2+}$) and six aromatic-carbon atoms are indicative of the cation-π interaction. In (b), the wholly green molecules represent the intruding $\left(i\right)$, exchanging $\left(ex\right)$, or escaping $\left(es\right)$ water molecules within the hydration water around the $\mathrm{C}{\mathrm{a}}^{2+}$ ion, with the blue dashed arrows indicating the $i$, $ex$, and $es$ directions.

Figure 2

Structural variables of side-group-synergetic hydrated cation-π interactions. (a) and (b) Time evolution of the distances and deflection angles between the cation and the aromatic ring for both representative trajectories shown in Fig. 1. Three vertical lines indicate the times 72.0, 82.0, and 126.6 ps in (b) and also in (e). (c) Definition diagram of structural variables. D is the distance of the link line between the cation and the center of aromatic ring. $\varphi$ is the polar angle between the link line and the normal direction of aromatic ring plane (${\stackrel{\rightharpoonup }{n}}_{\pi }$). The red small sphere marks the position of the center of the aromatic ring, and atom representations and color settings are as in Fig. 1. (d) Probability distributions of $D_{\mathrm{cation}\text{−}\pi }$ and ${\varphi }_{\mathrm{cation}\text{−}\pi }$. The data within the whole 250 ps of the trajectory with $\mathrm{M}{\mathrm{g}}^{2+}$ and the data within [0,72] ps and [82,126.6] ps of the trajectory with $\mathrm{C}{\mathrm{a}}^{2+}$ are adopted to obtain the distributions for $\mathrm{M}{\mathrm{g}}^{2+}\text{−}\pi$, $\mathrm{C}{\mathrm{a}}^{2+}\text{−}\pi$ (I) and $\mathrm{C}{\mathrm{a}}^{2+}\text{−}\pi$ (II) interactions, respectively. The most probable values are indicated by relevant vertical lines and numbers. (e) Root-mean-square deviation (RMSD) of all atoms except for hydrogen atoms in the pocket structures of pTyr in both representative trajectories. RMSDs are calculated relative to the structures at $t=0\mathrm{ps}$. (f) Average distances with $\mathrm{M}{\mathrm{g}}^{2+}\text{−}\pi$, $\mathrm{C}{\mathrm{a}}^{2+}\text{−}\pi$ (I) and $\mathrm{C}{\mathrm{a}}^{2+}\text{−}\pi$ (II) interactions. $D_{\mathrm{OC}}$, $D_{\mathrm{OH}}$, $D_{\mathrm{N}}$, $D_{\mathrm{OP}}$, and $D_{\mathrm{OW}}$ correspond to the distances between cations and the key atoms in side groups, i.e., carbonyl oxygen, hydroxyl oxygen, amino nitrogen, phosphate oxygen near cations, and the oxygen of the first-shell water around cations, respectively.

Figure 3

Cation-binding structures of Tyr for $\mathrm{M}{\mathrm{g}}^{2+}/\mathrm{C}{\mathrm{a}}^{2+}$. Representative AIMD trajectories of the conformations of Tyr with $\mathrm{M}{\mathrm{g}}^{2+}$ (a) and $\mathrm{C}{\mathrm{a}}^{2+}$ (b), respectively. The numbers indicate the times of snapshots. Atom representations and color settings are as in Fig. 1.

Figure 4

Cation-π distances and deflection angles. (a) and (b) Time evolution of cation-π distances (D, black) and deflection angles ($\varphi$, red) for both representative trajectories shown in Fig. 3. (c) and (d) Probability distributions of D and $\varphi$ for both cation-π interactions. The data within the first 12.0 and the 7.6 ps are adopted to obtain the distributions for $\mathrm{M}{\mathrm{g}}^{2+}\text{−}\pi$ and $\mathrm{C}{\mathrm{a}}^{2+}\text{−}\pi$ interactions, respectively.

Figure 5

Experimental evidence of side-group-synergetic hydrated cation-π interactions. (a) Dependence of UV absorbance difference at $\sim 230\mathrm{nm}$ on cation concentrations. UV absorbance difference spectrum is determined with the UV absorbance spectrum of pure pTyr solution $plus$ the spectrum of pure $\mathrm{CaC}{\mathrm{l}}_2/\mathrm{MgC}{\mathrm{l}}_2$ solution $minus$ the spectrum of the pTyr solution with addition of the $\mathrm{CaC}{\mathrm{l}}_2/\mathrm{MgC}{\mathrm{l}}_2$ solution. Error bars indicate the standard deviation from three parallel experiments. (b) and (c) Chemical shifts $\left({\delta }_{\mathrm{H}}\right)$ as well as chemical shift variations ($\mathrm{\Delta }{\delta }_{\mathrm{H}}$) of the hydrogen atoms on the aromatic ring of pTyr in ${}^1\mathrm{H}$ NMR spectra, induced by addition of $\mathrm{CaC}{\mathrm{l}}_2/\mathrm{MgC}{\mathrm{l}}_2$ with various concentrations. In (b), two groups of split bimodal peaks $\left({\delta }_{\mathrm{H}}\right)$ in ${}^1\mathrm{H}$ NMR spectra correspond to four hydrogen atoms on the para-disubstituted aromatic ring of pTyr, and the numbers correspond to cation concentrations. $\mathrm{\Delta }{\delta }_{\mathrm{H}}$ in (c) is estimated as the peak ${\delta }_{\mathrm{H}}$ marked by dashed lines $minus$ the ${\delta }_{\mathrm{H}}$ at zero cation concentration in (b).