A Structural Basis for 129 Xe Hyper-CEST Signal in TEM-1 β-Lactamase

Abstract

Genetically encoded (GE) contrast agents detectable by magnetic resonance imaging (MRI) enable non-invasive visualization of gene expression and cell proliferation at virtually unlimited penetration depths. Using hyperpolarized ¹²⁹ Xe in combination with chemical exchange saturation transfer, an MR contrast approach known as hyper-CEST, enables ultrasensitive protein detection and biomolecular imaging. GE MRI contrast agents developed to date include nanoscale proteinaceous gas vesicles as well as the monomeric bacterial proteins TEM-1 β-lactamase (bla) and maltose binding protein (MBP). To improve understanding of hyper-CEST NMR with proteins, structural and computational studies were performed to further characterize the Xe-bla interaction. X-ray crystallography validated the location of a high-occupancy Xe binding site predicted by MD simulations, and mutagenesis experiments confirmed this Xe site as the origin of the observed CEST contrast. Structural studies and MD simulations with representative bla mutants offered additional insight regarding the relationship between local protein structure and CEST contrast.

Keywords: CEST; Xenon; contrast; hyperpolarized; magnetic resonance.

Figure 1.

Chain A of the bla-Xe complex (PDB ID 5HW1), with Xe shown as red spheres (van der Waals radii enlarged for clarity). (Inset) Xe1 binding site, with surrounding protein side chains shown as sticks (Xe-derivative in gray, native bla in cyan). Gray dashes indicate protein-Xe contacts within 4.5 Å. Isomorphous difference Fourier map for Xe1 shown as blue mesh and contoured at 10 σ.

Figure 2.

¹²⁹Xe hyper-CEST z-spectra of 80 μM bla in pH 7.2 PBS at 300 K. Unliganded bla (red) compared to bla in the presence of 1 mM tazobactam (green). Z-spectrum of PBS only (gray) shown for reference. Pulse length, τ_pulse = 3.80 ms; field strength, B_1,max = 77 μT.

Figure 3.

Xe occupancy map calculated from a 1 μs trajectory (only the last 400 ns included in the map). Blue shading is the density at an arbitrary isovalue. Red spheres are Xe atoms from a representative frame of the MD trajectory. Yellow spheres are Xe atoms found in the Xe-derivative crystal structure of WT bla.

Figure 4.

(a) Xe1 shown as a red sphere, with nearby side chains shown as sticks (Xe-derivative of WT bla in gray, “open” bla bound to allosteric inhibitor in magenta). (b) Number of Xe atoms in the Xe1 binding site correlated to the minimum distance between I263 and I279 side chains (minimum among all possible pairwise atom-atom distances). (c) Number of Xe atoms in the major binding site anti-correlated to the number of atom-atom contacts between I263 and I279 side chains.

Figure 5.

Xe1 cavity of the Xe-derivative of WT bla, with mutated side chains highlighted in green. Closest side chain contacts to Xe1 measured in Å.

Figure 6.

¹²⁹Xe hyper-CEST z-spectra of 80 μM bla in pH 7.2 PBS at 300 K. Bla mutants (various colors) compared to WT bla (red). Z-spectrum of PBS only (gray) shown for reference. Pulse length, τ_pulse = 3.80 ms; field strength, B_1,max = 77 μT.

Figure 7.

Xe1 and Xe5 (red spheres) bound to bla mutants I263L (a), I263N (b), and I263A (c), with distances shown in units Å. Isomorphous difference maps corresponding to Xe shown as blue mesh and contoured at 5 σ. Simulated annealing (SA) omit maps corresponding to mutated I263L and I263N side chains shown as green mesh and contoured at 3 σ. The I263 side chain of WT bla (yellow sticks) and Xe bound to WT bla (yellow sphere) shown for reference.

Figure 8.

Combined Xe1 entry and exit pathways from 1 μs MD simulations for WT bla (top-left). The thickness of each trace correlates to the number of Xe atoms following that trajectory. Blue blobs indicate average Xe density. Plots show the correlation between Xe binding and inter-side chain distances of residues 263 and 279 from MD Xe flooding simulations of bla mutants following the same parameters used for WT bla (Figs. 4b, 4c).

Scheme 1.

During hyper-CEST, exchangeable solute-bound Xe atoms that resonate at a frequency different from solution-phase hyperpolarized Xe (green) are selectively spin-flipped using RF irradiation. Rapid exchange leads to the depolarization of bulk solution-phase Xe (gray), thereby generating MR contrast.