Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-κB Essential Modulator/IKKβ Protein–Protein Interface

Abstract

We report a comprehensive analysis of binding energy hot spots at the protein-protein interaction (PPI) interface between NF-κB Essential Modulator (NEMO) and IκB kinase subunit β (IKKβ), an interaction that is critical for NF-κB pathway signaling, using experimental alanine scanning mutagenesis and also the FTMap method for computational fragment screening. The experimental results confirm that the previously identified NBD region of IKKβ contains the highest concentration of hot spot residues, the strongest of which are W739, W741 and L742 (ΔΔG = 4.3, 3.5 and 3.2 kcal/mol, respectively). The region occupied by these residues defines a potentially druggable binding site on NEMO that extends for ~16 Å to additionally include the regions that bind IKKβ L737 and F734. NBD residues D738 and S740 are also important for binding but do not make direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues. We additionally found two previously unknown hot spot regions centered on IKKβ residues L708/V709 and L719/I723. The computational approach successfully identified all three hot spot regions on IKKβ. Moreover, the method was able to accurately quantify the energetic importance of all hot spots residues involving direct contact with NEMO. Our results provide new information to guide the discovery of small molecule inhibitors that target the NEMO/IKKβ interaction. They additionally clarify the structural and energetic complementarity between “pocket-forming” and “pocket occupying” hot spot residues, and further validate computational fragment mapping as a method for identifying hot spots at PPI interfaces.

Ligand deconstruction: Why some fragment binding positions are conserved and others are not

“…First, experimental or computational mapping of protein binding sites using small fragment probes shows that individual probe structures are rather indiscriminate, such that binding subsites occupied by ligands are characterized by their ability to bind a variety of different probe structures. Indeed, the FTMap algorithm works by identifying such "consensus sites," and it is well validated that these consensus sites coincide with the experimentally observed binding sites for drugs and other ligands (31,34), and with the locations of binding energy hot spots at protein-protein interfaces identified by alanine-scanning mutagenesis (32,33). Moreover, the locations of FTMap consensus sites are robust to conformational changes in the protein, and the same sites can be reliably identified using experimental X-ray crystal structures that show the protein in any of a variety of bound or unbound conformational states (34).…”

“…Although the experimental determination of hot spots can be technically demanding to carry out (24), it has been shown that hot spots can also be reliably determined computationally using the solvent mapping algorithm FTMap, a close computational analog of the X-ray-and NMR-based approaches (26)(27)(28)(29)(30)(31). We have further shown that, for protein-protein interface sites, the small molecule hot spots identified by FTMap correspond closely to the protein-protein interaction hot spots identified by alanine scanning mutagenesis (32,33).…”

The identification of affinity peptide ligands specific to the variable region ofhuman antibodies

“…We used an alanine-scanning approach (12,19,34) to find a specific motif involved in binding to scFv. Impressively, Li et al reported compatible results with an experimental alanine-scanning study while using an in silico kinesin-microtubule binding model (19).…”

A Synthetic Loop Replacement Peptide That Blocks Canonical NF‐κB Signaling

“…[8] When D738 and S740 are substituted with allylglycine residues, the lowest free energy conformer is observed to have backbone dihedral angles at W739 that are dissimilar to those in the native sequence, suggesting these modifications increase the local flexibility of the modified NBD peptide (Figure 2, panel B). [7,14] However, MD simulations of a peptide in which a C-C bond replaces the loop hydrogen bond reveal that the key dihedral angles are analogous to those in the wild-type NBD peptide (Figure 2, panel C), demonstrating that the constrained version of the NBD sequence can adopt the key dihedral angles needed to interact with NEMO.…”

“…(Supplemental Figure 2). [7] Significantly, the modification does not alter the ability of the hydrophobic hot spot residues to engage NEMO in the context of cellular lysates. As shown in Figure 3b, photoactive variants of NBD WT (bNBD WT ) and NBD2 (bNBD2) capture NEMO in a pulldown experiment in HEK293T cell lysates.…”