Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR)

Abstract

We describe a high-throughput in-cell nuclear magnetic resonance (NMR)-based method for mapping the structural changes that accompany protein-protein interactions (STINT-NMR). The method entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring the protein interactions using in-cell NMR spectroscopy. The resulting spectra provide a complete titration of the interaction and define structural details of the interacting surfaces at atomic resolution.

Figure 1

Overview of STINT-NMR methodology. (a) In-cell NMR of interacting proteins. Target protein was over-expressed in uniformly labeled [U-¹⁵N] medium and a sample containing no interactor protein was prepared for in-cell NMR (top). Cells were then washed and resuspended in label-free medium. Samples were taken as the interacting protein was overexpressed (bottom). Changes in the target protein structure are measured as the concentration of the interactor is increased. (b) SDS-PAGE of ubiquitin and STAM2 sequential expression. Rosetta(DE3) cells were induced with l-arabinose and then with IPTG for the indicated amounts of time to overexpress ubiquitin and STAM2, respectively. Lane 1, uninduced Rosetta(DE3) cells. Note that the ubiquitin level remains essentially constant as STAM2 overexpression increases.

Figure 2

NMR spectra and interaction maps of ubiquitin-ligand complexes. (a) Overlay of ¹H{¹⁵N}HSQC-spectra of E. coli after 3-h overexpression of [¹⁵N]ubiquitin and 0-h (black), 2-h (red) and 3-h (blue) overexpression of AUIM. Individual peaks exhibiting large chemical shifts are labeled with corresponding assignments. Progression of colors in the ¹H{¹⁵N}HSQC spectrum was chosen for ease of viewing. (b) Overlay of ¹H{¹⁵N}HSQC-spectra of free [¹⁵N]ubiquitin (black), [¹⁵N]ubiquitin-AUIM complex at a molar ratio of 1:1 (red) and at a molar ratio of 1:2 (blue). (c) ¹H{¹⁵N}HSQC-spectrum of E. coli after 3-h overexpression of [¹⁵N]ubiquitin and 3-h overexpression of STAM2. Resonance peaks exhibiting extreme broadening are indicated by crosses. Insets , one-dimensional traces of selected peaks exhibiting differential broadening after 3-h overexpression of [¹⁵N]ubiquitin and 0-h (black), 2-h (red) and 3-h (green) overexpression of STAM2. (d) Interaction interface of ubiquitin-AUIM mapped on the three-dimensional structure of ubiquitin (PDB code 1D3Z) based on the chemical shift changes from the in-cell titration experiments. (e) Interaction interface of ubiquitin-AUIM complex based on the chemical shift changes from the in vitro titration experiment. Ubiquitin residues exhibiting either chemical shift change above 0.1 p.p.m. or extreme broadening are colored in red. (f) Interaction interface of ubiquitin-STAM2 based on the differentially broadened peaks from the in-cell titration experiment. The HSQC spectra were acquired as described in Supplementary Methods.