Enhancing the Binding Affinity of SxIP Ligands to the EB1 Protein through Rational Peptide Design

Core Concepts

The binding of SxIP-containing ligands to the EB1 protein involves a two-step "dock-and-lock" mechanism, where the initial docking through the SxIP motif provides specificity, and the subsequent folding of the EB1 C-terminus defines the full binding affinity.

Abstract

The content discusses the binding mechanism between the EB1 protein and its SxIP-containing ligands. EB1 is a key component of the signaling networks located at the plus ends of microtubules and interacts with various proteins through its C-terminal EBH domain. The authors show that the SxIP motif alone binds to EBH with low affinity, and the full interaction requires contributions from the post-SxIP residues. They propose a two-step "dock-and-lock" model, where the SxIP motif initially binds to a partially-formed EBH pocket, which then induces folding of the unstructured C-terminus and transition to the stable complex. The authors use NMR structural and dynamics analysis to dissect the contributions from different interactions. They demonstrate that the post-SxIP region enhances affinity by two orders of magnitude through hydrophobic interactions with the EBH C-terminus. By rationally designing MACF peptide mutations in the post-SxIP region, they are able to increase the affinity to the nanomolar range. The authors also show that the high-affinity mutant peptides can be recruited more efficiently to the dynamic plus ends of microtubules in live cell experiments, supporting the functional importance of the post-SxIP region. The proposed model explains EB1's interaction with SxIP-containing ligands and can be used to design small molecule inhibitors targeting this interaction.

Stats

The dissociation constant (Kd) for the EB1 interaction with different MACF peptides: 4MACF (SKIP): Kd ~10 mM 6MACF (SKIPTP): Kd ~2 mM 11MACF (KPSKIPTPQRK): Kd 3.5 μM 11MACF-LLL: Kd 0.35 μM 11MACF-VLL: Kd 0.12 μM 11MACF-VLLRK: Kd 16 nM The dissociation rate (koff) for the EB1/11MACF interaction is ~130 s^-1.

Quotes

"The folding of the EBH C-terminus after the peptide binding brings the highly hydrophobic region 253FVIP256 of EBH into the contact with a relatively hydrophilic 5481TPQ5483 region of the peptide." "The dock-and-lock mechanism of the EBH binding has a number of functional benefits. The open, partly formed binding pocket is easily accessible, leading to the fast on-rate." "The additional residues that follow the SxIP motif contribute in two different ways. First, they enhance the affinity through the interaction with the folded EBH coiled-coil. The second effect of the post-SxIP residues is to induce the folding of the EBH C-terminus, which locks the peptide into the fully folded state."

Key Insights Distilled From

Dock-and-lock binding of SxIP ligands is required for stable and selective EB1 interactions

by Almeida,T., ... at www.biorxiv.org 02-29-2024

https://www.biorxiv.org/content/10.1101/2024.02.27.581919v1

Deeper Inquiries

How could the dock-and-lock binding mechanism of EB1 be exploited for the development of small molecule inhibitors targeting EB1 interactions

The dock-and-lock binding mechanism of EB1 can be leveraged for the development of small molecule inhibitors targeting EB1 interactions by focusing on disrupting either the initial docking step or the subsequent locking step of the interaction. For the initial docking step, small molecules can be designed to target the SxIP motif recognition pocket on EB1, inhibiting the specific recognition of the ligand. This can be achieved by developing compounds that competitively bind to the pocket, preventing the SxIP motif from interacting with EB1. On the other hand, for the locking step, molecules can be designed to interfere with the folding of the EBH C-terminus induced by the post-SxIP region of the ligand. By disrupting this step, the stable complex formation between EB1 and its ligands can be prevented, leading to the inhibition of the interaction. Through structure-based drug design approaches and high-throughput screening, small molecule inhibitors targeting different stages of the dock-and-lock mechanism can be identified and further optimized for therapeutic applications.

What other types of intrinsically disordered protein interactions may utilize a similar dock-and-lock binding mechanism, and how could the insights from this study be applied to understand those systems

The dock-and-lock binding mechanism observed in the EB1 interactions with SxIP-containing ligands may be applicable to other intrinsically disordered protein (IDP) interactions that involve a similar two-step recognition process. IDPs often engage in transient and dynamic interactions with their binding partners, where the initial recognition is followed by induced folding or conformational changes that stabilize the complex. Proteins with short linear motifs (SLiMs) that undergo induced fit upon binding to their partners could potentially utilize a dock-and-lock mechanism similar to EB1. By studying the post-binding conformational changes and the role of specific residues in stabilizing the complex, insights from the EB1 study can be applied to understand and potentially target other IDP interactions. This knowledge can aid in the development of therapeutics targeting a wide range of protein-protein interactions involving IDPs, providing new avenues for drug discovery and intervention strategies.

The authors mention the importance of EB1 interactions in diseases like cancer. What are some potential therapeutic applications of modulating EB1-ligand interactions, beyond just small molecule inhibitors

Modulating EB1-ligand interactions can have significant therapeutic applications beyond small molecule inhibitors in the treatment of diseases like cancer. One potential application is the development of peptide-based therapeutics that mimic or disrupt the interactions between EB1 and its ligands. By designing peptides that specifically target the SxIP motif recognition pocket or interfere with the folding of the EBH C-terminus, it is possible to modulate EB1 function in cancer cells. Additionally, targeting EB1 interactions can be explored for the development of antibody-based therapies that selectively block the binding of EB1 to its ligands, leading to the disruption of critical signaling pathways involved in cancer progression. Furthermore, understanding and manipulating EB1 interactions can also have implications in other diseases where MT dynamics and cytoskeletal organization play a role, opening up new avenues for precision medicine and targeted therapies.