In-Cell Biochemistry Using NMR Spectroscopy

Abstract

Biochemistry and structural biology are undergoing a dramatic revolution. Until now, mostly in vitro techniques have been used to study subtle and complex biological processes under conditions usually remote from those existing in the cell. We developed a novel in-cell methodology to post-translationally modify interactor proteins and identify the amino acids that comprise the interaction surface of a target protein when bound to the post-translationally modified interactors. Modifying the interactor proteins causes structural changes that manifest themselves on the interacting surface of the target protein and these changes are monitored using in-cell NMR. We show how Ubiquitin interacts with phosphorylated and non-phosphorylated components of the receptor tyrosine kinase (RTK) endocytic sorting machinery: STAM2 (Signal-transducing adaptor molecule), Hrs (Hepatocyte growth factor regulated substrate) and the STAM2-Hrs heterodimer. Ubiquitin binding mediates the processivity of a large network of interactions required for proper functioning of the RTK sorting machinery. The results are consistent with a weakening of the network of interactions when the interactor proteins are phosphorylated. The methodology can be applied to any stable target molecule and may be extended to include other post-translational modifications such as ubiquitination or sumoylation, thus providing a long-awaited leap to high resolution in cell biochemistry.

The role of spartin and its novel ubiquitin binding region in DALIS occurrence

“…This patch is located in the immediate vicinity of the ubiquitin-binding surface and may explain the tighter binding of the spartin UBR to ubiquitin compared with the UBDs of other proteins (Burz and Shekhtman, 2008). The HSQC spectrum of free [ U- 15 N]spartin UBR revealed backbone amide proton and nitrogen peaks between 7.5 and 8.5 ppm (Figure 4A).…”

PTP1B Targets the Endosomal Sorting Machinery

“…A recent study suggested another potential function of STAM2 phosphorylation: NMR analysis of proteins in intact bacterial cells indicated that phosphorylation at Tyr 291 , Tyr 371 , and Tyr 374 may regulate the affinity of STAM2 for ubiquitin (31). To confirm these results using an alternative method, we tested the ability of recombinant GST-tagged ubiquitin, either a single ubiquitin group or diubiquitin (2Ub; supplemental Fig.…”

“…Plasmids-Mammalian expression plasmids encoding GSTtagged human PTP1B, TCPTP, and PTP-PEST (pEBG, WT, and DA mutant) (29) and myc-tagged WT human STAM2 (pEF1) (30) as well as the bacterial expression plasmids encoding Fyn (pDB1) (31) and GST-ubiquitin (mono-and diubiquitin, pGEX) (32) were described previously. STAM2 YF mutants and siRNA-resistant constructs containing four silent mutations within the STAM2 siRNA target sequence were prepared from pEF1-STAM2 WT by site-directed mutagenesis (QuikChange kit; Stratagene).…”

“…His-tagged recombinant STAM2 was purified from E. coli co-transformed with an inducible expression construct encoding Fyn (a protein-tyrosine kinase that phosphorylates STAM2) (31). For details, see supplemental Experimental Procedures.…”

CHAPTER 4. In‐Cell NMR Spectroscopy to Study Protein–Drug Interactions

“…Each resulting conformation has the potential to interact with drug-like molecules differently to produce different outcomes. Burz and Shekhtman 52 developed an in-cell methodology to introduce PTMs onto interactor proteins in bacterial cells and identify the changes in the interaction surface of a target protein when bound to the biochemically modified interactors. Modifying the interactor protein causes structural changes that manifest on the interacting surface of the target protein and these changes are monitored by using STINT-NMR.…”