In-cell biochemistry using NMR spectroscopy.
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.
Burz
David S
DS
Department of Chemistry, State University of New York at Albany, Albany, New York, United States of America.
Shekhtman
Alexander
A
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D003584:Cytological Techniques; D019281:Dimerization; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D015233:Models, Statistical; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D056827:Endosomal Sorting Complexes Required for Transport; D010750:Phosphoproteins; C491647:STAM2 protein, human; D025801:Ubiquitin; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D020794:Receptor Protein-Tyrosine Kinases