A structural basis for IκB kinase 2 activation via oligomerization-dependent trans auto-phosphorylation.

Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase

Mechanism of Filamentation-Induced Allosteric Activation of the SgrAI Endonuclease

Filament formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the cryoelectron microscopy (cryo-EM) structure of the filament of SgrAI in its activated configuration. The structural data illuminate the mechanistic origin of hyperaccelerated DNA cleavage activity and suggests how indirect DNA sequence readout within filamentous SgrAI may enable recognition of substantially more nucleotide sequences than its low-activity form, thereby altering and partially relaxing its DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAI's catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.United States Department of Health & Human Services, National Institutes of Health (NIH) - USA [P41 GM103311]; National Science Foundation (NSF) [MCB-1410355]; United States Department of Health & Human Services, National Institutes of Health (NIH) - USA [DP5 OD021396]; DBT Wellcome Trust India Alliance [IA/I/15/1/501852]12 month embargo; published online: 1 October 2019This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Regulatory subunit NEMO promotes polyubiquitin-dependent induction of NF-κB through a targetable second interaction with upstream activator IKK2

Canonical NF-κB signaling through the inhibitor of κB kinase (IKK) complex requires induction of IKK2/IKKβ subunit catalytic activity via specific phosphorylation within its activation loop. This process is known to be dependent upon the accessory ubiquitin (Ub)-binding subunit NF-κB essential modulator (NEMO)/IKKγ as well as poly-Ub chains. However, the mechanism through which poly-Ub binding serves to promote IKK catalytic activity is unclear. Here, we show that binding of NEMO/IKKγ to linear poly-Ub promotes a second interaction between NEMO/IKKγ and IKK2/IKKβ, distinct from the well-characterized interaction of the NEMO/IKKγ N terminus to the "NEMO-binding domain" at the C terminus of IKK2/IKKβ. We mapped the location of this second interaction to a stretch of roughly six amino acids immediately N-terminal to the zinc finger domain in human NEMO/IKKγ. We also showed that amino acid residues within this region of NEMO/IKKγ are necessary for binding to IKK2/IKKβ through this secondary interaction in vitro and for full activation of IKK2/IKKβ in cultured cells. Furthermore, we identified a docking site for this segment of NEMO/IKKγ on IKK2/IKKβ within its scaffold-dimerization domain proximal to the kinase domain-Ub-like domain. Finally, we showed that a peptide derived from this region of NEMO/IKKγ is capable of interfering specifically with canonical NF-κB signaling in transfected cells. These in vitro biochemical and cell culture-based experiments suggest that, as a consequence of its association with linear poly-Ub, NEMO/IKKγ plays a direct role in priming IKK2/IKKβ for phosphorylation and that this process can be inhibited to specifically disrupt canonical NF-κB signaling

Structural Basis for the Activation of IKK1/α.

Distinct signaling pathways activate the NF-κB family of transcription factors. The canonical NF-κB-signaling pathway is mediated by IκB kinase 2/β (IKK2/β), while the non-canonical pathway depends on IKK1/α. The structural and biochemical bases for distinct signaling by these otherwise highly similar IKKs are unclear. We report single-particle cryoelectron microscopy (cryo-EM) and X-ray crystal structures of human IKK1 in dimeric (∼150 kDa) and hexameric (∼450 kDa) forms. The hexamer, which is the representative form in the crystal but comprises only ∼2% of the particles in solution by cryo-EM, is a trimer of IKK1 dimers. While IKK1 hexamers are not detectable in cells, the surface that supports hexamer formation is critical for IKK1-dependent cellular processing of p100 to p52, the hallmark of non-canonical NF-κB signaling. Comparison of this surface to that in IKK2 indicates significant divergence, and it suggests a fundamental role for this surface in signaling by these kinases through distinct pathways

A Structural Basis for IκB Kinase 2 Activation Via Oligomerization-Dependent <i>Trans</i> Auto-Phosphorylation

<div><p>Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of <i>Xenopus</i> IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote <i>trans</i> auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via <i>trans</i> auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent <i>trans</i> auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.</p></div

Oligomerization of hIKK2 dimers.

<p>(A) Ribbon diagram of the interaction between two neighboring hIKK2 dimers in the crystal. Their asymmetric association gives rise to two unique intersubunit interfaces. (B) Close-up view of residues that interact between the KDs at the V-shaped interface. (C) Additional residues that mediate V-shaped interface interactions between the ULD an SDD. (D) Close-up view of interacting residues within the anti-parallel interface. (E) In vitro kinase assay reveals that catalytic activity of hIKK2 with mutations that disrupt the V-shaped interface (lanes 3–5) is drastically reduced compared to wild-type protein (WT-lane 2). (F) In vitro kinase assays with the same WT mutant proteins in which activation loop serines are mutated to glutamate. (G) Immunoblotting with anti-phospho-Ser177,181 antibody reveals that the decrease in catalytic activity observed in the V-shaped interface mutants correlates with activation loop phosphorylation status. (H) In vitro kinase assays reveal the modest effects on hIKK2 catalytic activity of mutation at the antiparallel interface.</p

IKK2 oligomerization activation model.

<p>(A) The hIKK2 X-ray crystal structure in space filling representation viewed from three different angles. The four surfaces that mediate oligomerization in the X-ray crystal structure are colored purple (dimer interface), blue (antiparallel interface), orange (V-shaped interface), and green (KD–KD interface). (B) A structure-based model for IKK2 activation via <i>trans</i> auto-phosphorylation. IKK2 interconverts between its open and closed dimeric forms. The open dimer can further associate to form transient homooligomers, such as observed in the hIKK2 X-ray crystal structure. Phosphorylation of one IKK2 subunit by an upstream kinase activates the kinase activity of that subunit and, as a consequence of its propensity to assemble into higher order oligomers through it V-shaped and KD-KD interfaces, is rapidly amplified via <i>trans</i> auto-phosphorylation.</p