In vitro detection of NEMO-ubiquitin binding using DELFIA and microscale thermophoresis assays.

Canonical NF-κB signaling in response to various stimuli converges at the level of the IκB kinase (IKK) complex to ultimately activate NF-κB. To achieve this, the IKK complex uses one of its regulatory subunit (IKKγ/NEMO) to sense ubiquitin chains formed by upstream complexes. Various studies have shown that different Ubiquitin chains are involved in the binding of NEMO and thereby the activation of NF-κB. We have utilized two distinct biochemical methods, i.e., Dissociation-Enhanced Lanthanide Fluorescence Immunoassay (DELFIA) and Microscale Thermophoresis (MST), to detect the interaction of NEMO to linear and K63-linked Ubiquitin chains, respectively. Here, we describe the brief basis of the methods and a detailed underlying protocol.

The human IKKbeta subunit kinase domain displays CK2-like phosphorylation specificity.

NF-kappaB activation in response to pro-inflammatory stimuli relies upon phosphorylation of IkappaB alpha at serines 32 and 36 by the beta subunit of the IkappaB kinase complex (IKK). In this study, we build upon the observation that highly purified human IKKbeta subunit preparations retain this specificity in vitro. We show that IKKbeta constructs that lack their carboxy-terminus beginning at the leucine zipper motif fail to phosphorylate IkappaB alpha at Ser-32 and Ser-36. Rather, these constructs, which contain the entire IKKbeta subunit kinase domain, phosphorylate serine and threonine residues contained within the IkappaB alpha carboxy-terminal PEST region. Furthermore, removal of the leucine zipper and helix-loop-helix regions converts IKKbeta to monomer. We propose that the helix-loop-helix of the human IKKbeta subunit is necessary for restricting substrate specificity toward Ser-32 and Ser-36 in IkappaB alpha and that in the absence of its carboxy-terminal protein structural motifs the human IKKbeta subunit kinase domain exhibits a CK2-like phosphorylation specificity.

Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samples.

Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific (15)N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific (15)N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The (15)N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the (15)N-amino acid prevents the scrambling of the (15)N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" (15)N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples.

Structure of a NEMO/IKK-associating domain reveals architecture of the interaction site.

The phosphorylation of IkappaB by the IKK complex targets it for degradation and releases NF-kappaB for translocation into the nucleus to initiate the inflammatory response, cell proliferation, or cell differentiation. The IKK complex is composed of the catalytic IKKalpha/beta kinases and a regulatory protein, NF-kappaB essential modulator (NEMO; IKKgamma). NEMO associates with the unphosphorylated IKK kinase C termini and activates the IKK complex's catalytic activity. However, detailed structural information about the NEMO/IKK interaction is lacking. In this study, we have identified the minimal requirements for NEMO and IKK kinase association using a variety of biophysical techniques and have solved two crystal structures of the minimal NEMO/IKK kinase associating domains. We demonstrate that the NEMO core domain is a dimer that binds two IKK fragments and identify energetic hot spots that can be exploited to inhibit IKK complex formation with a therapeutic agent.