A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation

The IκB Kinase (IKK) complex, containing catalytic IKK2 and noncatalytic NEMO subunits, plays essential roles in the induction of transcription factors of the NF-κB family. Catalytic activation of IKK2 via phosphorylation of its activation loop is promoted upon noncovalent association of linear or K63-linked polyubiquitin chains to NEMO within the IKK complex. The mechanisms of this activation remain speculative. To investigate interaction dynamics within the IKK complex during activation of IKK2, we conducted hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS) on NEMO and IKK2 proteins in their free and complex-bound states. Altered proton exchange profiles were observed in both IKK2 and NEMO upon complex formation, and changes were consistent with the involvement of distinct regions throughout the entire length of both proteins, including previously uncharacterized segments, in direct or allosteric interactions. Association with linear tetraubiquitin (Ub4) affected multiple regions of the IKK2:NEMO complex, in addition to previously identified interaction sites on NEMO. Intriguingly, observed enhanced solvent accessibility of the IKK2 activation loop within the IKK2:NEMO:Ub4 complex, coupled with contrasting protection of surrounding segments of the catalytic subunit, suggests an allosteric role for NEMO:Ub4 in priming IKK2 for phosphorylation-dependent catalytic activation.

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Protein Language Model Identifies Disordered, Conserved Motifs Driving Phase Separation

Zhang,

Zheng,

Zhang

2024

Preprint

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Intrinsically disordered regions (IDRs) play a critical role in phase separation and are essential for the formation of membraneless organelles (MLOs). Mutations within IDRs can disrupt their multivalent interaction networks, altering phase behavior and contributing to various diseases. Therefore, examining the evolutionary fitness of IDRs provides valuable insights into the relationship between protein sequences and phase separation. In this study, we utilized the ESM2 protein language model to map the fitness landscape of IDRs. Our findings reveal that IDRs, particularly those actively participating in phase separation, contain conserved amino acids. This conservation is evident through mutational constraints predicted by ESM2 and supported by direct analyses of multiple sequence alignments. These conserved, disordered amino acids include residues traditionally identified as "stickers" as well as "spacers" and frequently form continuous sequence motifs. The strong conservation, combined with their critical role in phase separation, suggests that these motifs act as functional units under evolutionary selection to support stable MLO formation. Our findings underscore the insights into phase separation's molecular grammar made possible through evolutionary analysis enabled by protein language models.

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A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation

Cited by 3 publications

References 67 publications

Control of nuclear localization of the nucleocapsid protein of SARS-CoV-2

Control of nuclear localization of the nucleocapsid protein of SARS-CoV-2

HDX-MS Analysis of Catalytic Activation of IKK2 in the IκB Kinase Complex

Protein Language Model Identifies Disordered, Conserved Motifs Driving Phase Separation

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