Edited by Wolfgang Peti Forkhead box protein O1 (FOXO1) is a transcription factor involved in various cellular processes such as glucose metabolism, development, stress resistance, and tumor suppression. FOXO1's transcriptional activity is controlled by different environmental cues through a myriad of posttranslational modifications. In response to growth factors, the serine/threonine kinase AKT phosphorylates Thr 24 and Ser 256 in FOXO1 to stimulate binding of 14-3-3 proteins, causing FOXO1 inactivation. In contrast, low nutrient and energy levels induce FOXO1 activity. AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, partly mediates this effect through phosphorylation of Ser 383 and Thr 649 in FOXO1. In this study, we identified Ser 22 as an additional AMPK phosphorylation site in FOXO1's N terminus, with Ser 22 phosphorylation preventing binding of 14-3-3 proteins. The crystal structure of a FOXO1 peptide in complex with 14-3-3 at 2.3 Å resolution revealed that this is a consequence of both steric hindrance and electrostatic repulsion. Furthermore, we found that AMPK-mediated Ser 22 phosphorylation impairs Thr 24 phosphorylation by AKT in a hierarchical manner. Thus, numerous mechanisms maintain FOXO1 activity via AMPK signaling. AMPK-mediated Ser 22 phosphorylation directly and indirectly averts binding of 14-3-3 proteins, whereas phosphorylation of Ser 383 and Thr 649 complementarily stimulates FOXO1 activity. Our results shed light on a mechanism that integrates inputs from both AMPK and AKT signaling pathways in a small motif to fine-tune FOXO1 transcriptional activity.

Interactions between a protein and a peptide motif of its protein partner are prevalent in nature. Often, a protein also has multiple interaction partners. X-ray protein crystallography is commonly used to examine these interactions in terms of bond distances and angles as well as to describe hotspots within protein complexes. However, the crystallization process presents a significant bottleneck in structure determination since it often requires notably time-consuming screening procedures, which involve testing a broad range of crystallization conditions via a trial-and-error approach. This difficulty is also increased as each protein–peptide complex does not necessarily crystallize under the same conditions. Here, a new co-crystallization/peptide-soaking method is presented which circumvents the need to return to the initial lengthy crystal screening and optimization processes for each consequent new complex. The 14-3-3σ protein, which has multiple interacting partners with specific peptidic motifs, was used as a case study. It was found that co-crystals of 14-3-3σ and a low-affinity peptide from one of its partners, c-Jun, could easily be soaked with another interacting peptide to quickly and easily generate new structures at high resolution. Not only does this significantly reduce the production time, but new 14-3-3–peptide structures that were previously not accessible with the 14-3-3σ isoform, despite screening hundreds of other different conditions, were now also able to be resolved. The findings achieved in this study may be considered as a supporting and practical guide to potentially enable the acceleration of the crystallization process of any protein–peptide system.

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