Visualizing protein breathing motions associated with aromatic ring flipping.

Aromatic residues cluster in the core of folded proteins, where they stabilize the structure through multiple interactions. Nuclear magnetic resonance (NMR) studies in the 1970s showed that aromatic side chains can undergo ring flips-that is, 180° rotations-despite their role in maintaining the protein fold1-3. It was suggested that large-scale 'breathing' motions of the surrounding protein environment would be necessary to accommodate these ring flipping events1. However, the structural details of these motions have remained unclear. Here we uncover the structural rearrangements that accompany ring flipping of a buried tyrosine residue in an SH3 domain. Using NMR, we show that the tyrosine side chain flips to a low-populated, minor state and, through a proteome-wide sequence analysis, we design mutants that stabilize this state, which allows us to capture its high-resolution structure by X-ray crystallography. A void volume is generated around the tyrosine ring during the structural transition between the major and minor state, and this allows fast flipping to take place. Our results provide structural insights into the protein breathing motions that are associated with ring flipping. More generally, our study has implications for protein design and structure prediction by showing how the local protein environment influences amino acid side chain conformations and vice versa.

Structural basis of homodimerization of the JNK scaffold protein JIP2 and its heterodimerization with JIP1.

The scaffold proteins JIP1 and JIP2 intervene in the c-Jun N-terminal kinase (JNK) pathway to mediate signaling specificity by coordinating the simultaneous assembly of multiple kinases. Using NMR, we demonstrate that JIP1 and JIP2 heterodimerize via their SH3 domains with the affinity of heterodimerization being comparable to homodimerization. We present the high-resolution crystal structure of the JIP2-SH3 homodimer and the JIP1-JIP2-SH3 heterodimeric complex. The JIP2-SH3 structure reveals how charge differences in residues at its dimer interface lead to formation of compensatory hydrogen bonds and salt bridges, distinguishing it from JIP1-SH3. In the JIP1-JIP2-SH3 complex, structural features of each homodimer are employed to stabilize the heterodimer. Building on these insights, we identify key residues crucial for stabilizing the dimer of both JIP1 and JIP2. Through targeted mutations in cellulo, we demonstrate a functional role for the dimerization of the JIP1 and JIP2 scaffold proteins in activation of the JNK signaling pathway.