Lanthanide binding and IgG affinity construct: potential applications in solution NMR, MRI, and luminescence microscopy

Abstract

Paramagnetic lanthanide ions when bound to proteins offer great potential for structural investigations that utilize solution nuclear magnetic resonance spectroscopy, magnetic resonance imaging, or optical microscopy. However, many proteins do not have native metal ion binding sites and engineering a chimeric protein to bind an ion while retaining affinity for a protein of interest represents a significant challenge. Here we report the characterization of an immunoglobulin G-binding protein redesigned to include a lanthanide binding motif in place of a loop between two helices (Z-L2LBT). It was shown to bind Tb³⁺ with 130 nM affinity. Ions such as Dy³⁺, Yb³⁺, and Ce³⁺ produce paramagnetic effects on NMR spectra and the utility of these effects is illustrated by their use in determining a structural model of the metal-complexed Z-L2LBT protein and a preliminary characterization of the dynamic distribution of IgG Fc glycan positions. Furthermore, this designed protein is demonstrated to be a novel IgG-binding reagent for magnetic resonance imaging (Z-L2LBT:Gd³⁺ complex) and luminescence microscopy (Z-L2LBT: Tb³⁺ complex).

Figure 1

The chimeric Z-L2LBT protein (A) is built upon a IgG-binding Z-domain scaffold and contains a lanthanide binding motif in place of the loop between helices 2 and 3. (B) Luminescence intensity changes with the Tb³⁺coordination state. The shift of the Tb³⁺:Z-L2LBT profile to the UV region likely reflects sensitization of the Tb³⁺ ion through Tyr residues in the protein. A sharp instrument artifact observed at ∼272 nm in all samples including a water blank was removed from these plots. (C) Luminescence intensity saturates at higher concentrations of Tb³⁺ when titrated into a 0.85 μM solution of Z-L2LBT. These data are fit with Eq. (1).

Figure 2

¹⁵N heteronuclear single quantum coherence spectrum of Z-L2LBT bound to a diamagnetic Lu³⁺ ion (black spectrum) or a paramagnetic Dy³⁺ ion (grey spectrum). The PCS and PRE effects of the Dy³⁺ ion appear as frequency shifts (diagonal lines), broadened lines and reduced intensity (inset), respectively, of ¹H-¹⁵N cross peaks. The numbers indicate residue numbers in the amino acid sequence. For protein preparation and purity see also Supporting Information Figure S1.

Figure 3

Models of Z-L2LBT calculated with restraints from paramagnetic ion perturbations. Overlay of the backbone is shown for the ensemble of 10 models (A, and as rotated 90°, B). A ribbon diagram of the lowest energy model shows the structural elements (C and as rotated 90°, D). (E) A model of the (Z-L2LBT:Ln³⁺)₂–Fc complex. This figure was generated using PyMol.

Figure 4

The ¹⁵N relaxation rates R₁ (A) and R₂ (B) of Z-L2LBT show the Z domain and LBT regions have different dynamic characteristics.

Figure 5

Fc N-glycan resonances are perturbed by lanthanides bound to Z-L2LBT. (A) A portion of the ¹³C-HSQC spectrum at 14.1 T shows Gal resonances of Fc with a Gal-terminated N-glycan. (B) A slice through the C2-H2 crosspeak in (A) shows the H2 lineshape. (C) A portion of the ¹³C HSQC spectrum showing Gal resonances of (Gd³⁺:Z-L2LBT)₂:(¹³C_U-Gal Fc) complex; (D) a slice through the C2-H2 crosspeak in (C). The intense peak at 3.5 ppm in (C) is folded from another part of the spectrum and does not represent a new peak in the viewing window.

Figure 6

A gradient profile at 14.1 T of IgG2a-coated sepharose® beads sandwiched between two layers of unconjugated Sepharose® beads. This solution contains Gd³⁺:Z-L2LBT. Reduced ¹H T₁ times resulting from the concentration of Gd³⁺ by the IgG2a-coated beads give rise to greater intensity in the gradient profile which corresponds to the location of IgG2a-coated beads in a 3 mm NMR tube. A photograph of the phantom is shown.

Figure 7

Z-L2LBT bound to Tb³⁺ is a reagent for luminescence microscopy. Images of control Sepharose® beads (A) or mouse IgG2a-coated Sepharose® beads were obtained in the presence of Tb³⁺:Z-L2LBT. The beads are 45 to 165 μm Sepharose® 4B resin (Sigma). The average signal above background for ∼50 beads from each trial is shown ± standard error (C).