Abstract
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.
Highlights
We capture ring flipping events of a buried tyrosine residue in the SH3 domain of the JNK-interacting protein 1 ( JIP1)
We show how a substantial void volume is generated around the tyrosine ring during the structural transition from the major to the minor state, which can be associated with the breathing motions that allow fast-timescale ring flipping events to take place
These results show that the relaxation dispersion that affects around 40% of the residues in the SH3 domain arises from a single exchange process, with Y526 being the origin of the observed exchange
Summary
524–529) in the wild-type protein (left) and in the H493A (middle) and V517A (right) variants. d, e, Two examples of linear correlations between the chemical shifts of wild-type JIP1-SH3 (blue spectrum) and the two variants H493A (grey spectrum) and V517A (red spectrum) as observed in 1H–15N HSQC spectra acquired at 35 °C (d, residue E522; e, residue D524). f, Energy landscapes illustrating the effect of single point mutations on the exchange rate constants and fractional populations of the major (eclipsed) and minor (staggered) conformations as determined by relaxation dispersion experiments acquired at 15 °C. During the structural transition between the major and the minor state (Supplementary Video 1), a void volume is created around the ring of Y526 that corresponds to a pocket expansion of 65 Å3; this is mainly due to the structural reorganization of the side chain of Q520 (Fig. 4e, f) This cavity expansion is in agreement with previous studies that have reported activation volumes between 40 and 85 Å3 for ring flipping events of aromatic residues in other proteins[12,14,21,40,41]. The β-bulge to β-sheet transition is completed and the aromatic ring becomes trapped in a staggered conformation that is stabilized by CH–π interactions with L519—a process that gives rise to the observed relaxation dispersion. These events are rare and occur on a slow timescale All mutants—including A541L, which stabilizes Y526 in a staggered conformation by a different mechanism—share the same initial structural trajectory and report on identical breathing motions
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