Combined ligand-observe 19F and protein-observe 15N,1H-HSQC NMR suggests phenylalanine as the key Δ-somatostatin residue recognized by human protein disulfide isomerase

Abstract

Human protein disulphide isomerase (hPDI) is an endoplasmic reticulum (ER) based isomerase and folding chaperone. Molecular detail of ligand recognition and specificity of hPDI are poorly understood despite the importance of the hPDI for folding secreted proteins and its implication in diseases including cancer and lateral sclerosis. We report a detailed study of specificity, interaction and dissociation constants (Kd) of the peptide-ligand Δ-somatostatin (AGSKNFFWKTFTSS) binding to hPDI using 19F ligand-observe and 15N,1H-HSQC protein-observe NMR methods. Phe residues in Δ-somatostatin are hypothesised as important for recognition by hPDI therefore, step-wise peptide Phe-to-Ala changes were progressively introduced and shown to raise the Kd from 103 + 47 μM until the point where binding was abolished when all Phe residues were modified to Ala. The largest step-changes in Kd involved the F11A peptide modification which implies the C-terminus of Δ-somatostatin is a prime recognition region. Furthermore, this study also validated the combined use of 19F ligand-observe and complimentary 15N,1H-HSQC titrations to monitor interactions from the protein’s perspective. 19F ligand-observe NMR was ratified as mirroring 15N protein-observe but highlighted the advantage that 19F offers improved Kd precision due to higher spectrum resolution and greater chemical environment sensitivity.

19F NMR as a tool in chemical biology

“…Indeed, the high resolution observed in the fluorine spectra enabled the direct calculation of most of the ligand binding constants. The superior capacity of 19 F NMR to monitor binding events was also noted by Richards et al, when studying the interaction of human protein disulphide isomerase (hPDI) to Δ-somastatin [ 35 ]. In this work there was improved precision in the analysis of the dissociation constants due to the higher spectrum resolution and greater chemical environment sensitivity of the 19 F nuclei when compared to that offered by the 15 N.…”

A cell is more than the sum of its (dilute) parts: A brief history of quinary structure

“…128 Although 19 F NMR does not offer the breadth of information afforded by heteronuclear multidimensional in-cell NMR experiments, it is a simple approach for obtaining general information about proteins in cells, including larger proteins that may not afford two-dimensional in-cell spectra. Furthermore, 19 F NMR can be used to study protein-ligand interactions 132,133 with biomedical applications. [134][135][136][137] Quantitative assessments of quinary structure…”

Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

“…Therefore, a different NMR method is needed to study ligand and inhibitor binding to R67 DHFR. 19 F NMR spectroscopy is sensitive enough to characterize protein‐ligand interactions, and for drug discovery 9–13 . 19 F NMR can potentially overcome the problems of lost symmetry as it offers the advantage of being able to label select residues, which helps minimize the complexity of the spectra and the possibility of structural perturbations 14,15 .…”