A prochelator peptide designed to use heterometallic cooperativity to enhance metal ion affinity

Abstract

A peptide has been designed so that its chelating affinity for one type of metal ion regulates its affinity for a second, different type of metal ion. The prochelator peptide (PCP), which is a fusion of motifs evocative of calcium loops and zinc fingers, forms a 1 : 2 Zn : peptide complex at pH 7.4 that increases its affinity for Zn²⁺ ∼3-fold in the presence of Tb³⁺ (log β₂ from 13.8 to 14.3), while the 1 : 1 luminescent complex with Tb³⁺ is brighter, longer lived, and 20-fold tighter in the presence of Zn²⁺ (log K from 6.2 to 7.5). This unique example of cooperative, heterometallic allostery in a biologically compatible construct suggests the possibility of designing conditionally active metal-binding agents that could respond to dynamic changes in cellular metal status.

Fig. 1. Top: structure of the parent Tb–LBT complex (PDB 1TJB). For clarity, only the Tb-binding ligands and flanking Tyr₁ and Leu₁₃ residues are shown, with their proximity emphasized by the purple line. Bottom: amino acid sequences of LBT, the ProChelator Peptide (PCP), and several controls. Putative Zn-binding residues are highlighted in yellow, with known Tb-binding residues of the LBT highlighted in cyan. Monodentate carboxylates from Asp1 and Asp5, bidentate carboxylates from Glu9 and Glu12, carboxamide O from Asn3, and backbone carbonyl O of Trp7 provide an 8-coordinate binding site for Tb³⁺.

Fig. 2. Titrations of PCP with TbCl₃ monitored by sensitized Tb emission at 545 nm in the absence (blue, circles) or presence (red, squares) of ZnSO₄ that is either (a) present initially or (b) added subsequently to Tb. Insets show full emission spectra from 300–650 nm. Conditions: [PCP] = 0.5 μM; [Tb] = 0–5 μM; [Zn] = 0 or 0.5 μM; [HEPES] = 5 mM with 50 μM DTT, pH 7.4; λ_ex = 280 nm.

Fig. 3. Luminescence decay rates for PCP–Tb in the presence (red squares) or absence (blue circles) of Zn at several H₂O/D₂O ratios. Solid lines are the linear fit. Conditions: [PCP] = 2 μM; [Tb] = 5 μM; [Zn] = 0 or 2 μM; [HEPES] = 5 mM pH 7.4; [TCEP] = 50 μM.

Fig. 4. Tb competition between PCP and ΔY_LBT in the presence (red squares) or absence of Zn (blue circles); solid lines represent the best fit. Conditions: [PCP] = 15 μM; [Tb] = 12 μM; [Zn] = 0 or 15 μM; [HEPES] = 5 mM pH 7.4; [TCEP] = 100 μM.

Fig. 5. UV-vis spectra of Zn(PAR)₂ (blue trace) followed by addition of PCP (a) or CysHis_PCP (b) (long arrow to green trace), followed by addition of Tb (orange trace). Inset shows excess Tb (orange to red) produces a Tb–PAR complex. Conditions: [PAR] = 20 μM; [Zn] = 3 μM; [PCP] = 0–5 μM; [Tb] = 0–50 μM; [CysHis_PCP] = 0–30 μM; [HEPES] = 5 mM pH 7.4; [TCEP] = 100 μM.

Fig. 6. Calculated concentration (logarithmic scale) of unbound Tb (left panel) or Zn (right panel) in the presence or absence of the second metal. Free metal calculated at 2 μM PCP.

Scheme 1. Proposed model of cooperative metal binding of two different metal ions (Tb³⁺ green, Zn²⁺ red) to prochelator peptide PCP (flexible black cylinder). The higher Tb emission observed in the presence of Zn is emphasized by a glow around Tb.