The sterile alpha motif (SAM) for protein-protein interactions is encountered in over 200 proteins, but the structural bases for its interactions is just becoming clear. Here we solved the structure of the EphA2-SHIP2 SAM:SAM heterodimeric complex by use of NMR restraints from chemical shift perturbations, NOE and RDC experiments. Specific contacts between the protein surfaces differ significantly from a previous model and from other SAM:SAM complexes. Molecular dynamics and docking simulations indicate fluctuations in the complex towards alternate, higher energy conformations. The interface suggests that EphA family members bind to SHIP2 SAM whereas EphB members may not; correspondingly we demonstrate binding of EphA1 but not of EphB2 to SHIP2 SAM. A variant of EphB2 SAM was designed that binds SHIP2. Functional characterization of a mutant EphA2 compromised in SHIP2 binding reveals two previously unrecognized functions of SHIP2 in suppressing ligand-induced activation of EphA2 and in promoting chemotactic cell migration in coordination with the receptor.

Protein unfolding, disassembly, and aggregation underlie many diseases, but detailed study of these processes in intact cells has been limited. Cysteine Shotgun labeling utilizes cell-permeable fluorescent dyes to label exposed cysteine residues. We applied it to study protein structure changes in response to mechanical stress on red blood cell ghosts in live mice and in whole-cell lysates in native versus urea-denaturing conditions. Labeling rate constants are calculated for any given Cys site by normalizing the protein labeling kinetics to the rapid labeling under denaturing conditions. Proteins can be identified and further analyzed by mass spectrometry to pinpoint specific, susceptible domains involved. A number of proteins contain cys with a wide variety of rate constants. This study focuses on human and mouse spectrin, Filamin A and B, Talin, and pyruvate kinase. These various proteins contain many cysteine-rich domains and have been amenable to studying by this in-cell technique. Results are confirmed by studies of purified protein.