Analysis of O 2 -binding Sites in Proteins Using Gas-Pressure NMR Spectroscopy: Outer Surface Protein A

Abstract

Internal cavities in proteins produce conformational fluctuations and enable the binding of small ligands. Here, we report a NMR analysis of O-binding sites by O-induced paramagnetic relaxation enhancements (PREs) on amide groups of proteins in solution. Outer surface protein A contains a nonglobular single-layer β-sheet that connects the N- and C-terminal globular domains. Several cavities have been observed in both domains of the crystallized protein structure. The receptor-binding sites are occluded and line the largest cavity of the C-terminal domain. We observed significant O-induced PREs for amide protons located around the largest cavity and at the central β-sheet. We suggested three potential O-accessible sites in the protein based on the 1/r distance dependence of the PRE. Two sites were in or close to the largest cavity and the third site was in the surface crevice of the central β-sheet. These results provide, to our knowledge, the first evidence of ligand binding to the surface crevice and cavity of the protein in solution. Because O generally binds more specifically to hydrophobic rather than hydrophilic cavities within a protein, the results also indicated that the receptor-binding sites lining the largest cavity were in the hydrophobic environment in the ground-state conformation. Molecular dynamics simulations permitted the visualization of the rotational and translational motions of O within the largest cavity, egress of O from the cavity, and ingress of O in the surface crevice of the β-sheet. These molecular dynamics simulation results qualitatively explained the O-induced changes in NMR observations. Exploring cavities that are sufficiently dynamic to enable access by small molecules can be a useful strategy for the design of stable proteins and their ligands.

Nuclear magnetic resonance‐based determination of dioxygen binding sites in protein cavities

“…These results indicate that O 2 molecules penetrate the hydrophobic interior of the protein, thereby causing the observed changes. Indeed, similar O 2 ‐induced chemical shifts and peak broadening were observed in the cavity enlarged mutant L99A of the protein and OspA, both of which contain interior hydrophobic cavities.…”

“…All nuclei are located around cavity 4, and thus their chemical shift changes would be sensitive to O 2 occupancy. Similar O 2 ‐induced chemical shift changes were observed for both the L99A mutant of the protein and OspA . As is likely to be the situation for both cases, the O 2 ‐induced chemical shift changes for the WT* were considered to originate from the paramagnetic shift, particularly the contact shift.…”

“…PREs originate from unpaired electrons of the paramagnetic triplet O 2 . We previously proposed a method to analyze O 2 ‐accessible sites in proteins based on the 1/ r 6 distance‐dependent PRE . 1 H longitudinal relaxation enhancements, Δ R 1 , was expressed by the 1/ r 6 distance‐dependent PRE contributions from each O 2 ‐binding site.…”

“…NMR provides a high‐resolution approach to investigate the structure and dynamics of proteins in solution. Several studies have shown that NMR spectroscopy in combination with dioxygen (O 2 ) gas‐pressure is a powerful tool for investigating the location of internal protein cavities and pockets in solution and their accessibility to ligands, owing to the paramagnetic properties of O 2 . In previous studies, methods to analyze O 2 ‐binding sites in proteins in solution were reported, using the O 2 ‐induced enhancement of backbone amide proton spin‐lattice relaxation .…”

“…Several studies have shown that NMR spectroscopy in combination with dioxygen (O 2 ) gaspressure is a powerful tool for investigating the location of internal protein cavities and pockets in solution and their accessibility to ligands, owing to the paramagnetic properties of O 2 . [12][13][14][15][16][17][18] In previous studies, methods to analyze O 2 -binding sites in proteins in solution were reported, using the O 2 -induced enhancement of backbone amide proton spin-lattice relaxation. 12,15,16 We found that O 2 bound selectively to the hydrophobic, rather than hydrophilic (water-containing) cavities in the cavity-enlarged T4 lysozyme mutant L99A.…”