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

Reviewer #1 (Public Review):

Summary:

In this article, Almeida and colleagues use a combination of NMR and ITC to study the interaction of the EBH domain of microtubule end-binding protein 1 (EB1) with SxIP peptides derived from the MACF plus-end tracking protein. EBH forms a dimer and in isolation has previously been shown to have a disordered C-terminal tail. Here, the authors use NMR to determine a solution structure of the EBH dimer bound to 11-mer SxIP peptides derived from MACF, and observe that the disordered C-terminal of EBH is recruited by residues C-terminal to the SxIP motif to fold into the final complex. By comparison of binding in different length peptides, and of EBH lacking the C-terminal tail, they show that these additional contacts increase binding affinity by an order of magnitude, greatly stabilising the interaction, in a binding mode they term 'dock-and-lock'.

The authors also use their new structural knowledge to design peptides with higher affinities and show in a cell model that these can be weakly recruited to microtubule ends - although a dimeric construct is necessary for efficient recruitment. Ultimately, by demonstrating the feasibility of targeting these proteins, this work points towards the possibility of designing small-molecules to block the interactions.

Strengths:

The authors determine an NMR structure of the dimeric complex, and additional report nuclear spin relaxation measurements to explore conformational dynamics within the complex via S2 order parameters and exchange contributions to relaxation (Rex terms).

A variety of appropriate experimental techniques are applied to probe the thermodynamics and kinetics of peptide binding: ITC, 2D NMR lineshape analysis, and chemical exchange saturation transfer (CEST) NMR. These yield consistent results, and a thoughtful analysis is described, based on the non-observation of exchange broadening in 2D titration and CEST measurements, in order to conclude that the proposed locking step, in which the C-terminal tail of EBH folds against the bound peptide, must occur on a rapid (sub-ms) timescale.

The use of 2D NMR lineshape analysis enables authors to extract the fullest information from their titration data, permitting an analysis of binding kinetics in addition to affinities. They also mention briefly that this enables them to account for the fact that binding occurs to two symmetric sites on the EBH dimer.

The authors use a range of peptide lengths, and mutations of EBH, to explore the contribution of different parts of the sequence to the overall binding affinity. They also use their structural observations to design a new peptide that binds with sub-micromolar affinity. They develop a simple but effective fluorescence assay to test the interaction of these peptides with microtubule ends within cells and show that their designed peptide can compete with native ligands for EBH.

Weaknesses:

There is no direct experimental evidence for independent dock and lock steps. The model is certainly plausible given their structural data, but all titration and CEST measurements are fully consistent with a simple one-step binding mechanism. Indeed, it is acknowledged that the results for the VLL peptide are not consistent with the predictions of this model, as affinity and dissociation rates do not co-vary. The model may still be a helpful way to interpret and discuss their results, and may indeed be the correct mechanism, but this has not yet been proven.

There is little discussion of the fact that binding occurs to EBH dimers - either in terms of the functional significance of this or in the acquisition and analysis of their data. There is no discussion of cooperation in binding (or its absence), either in the analysis of NMR titrations or in ITC measurements. Complete ITC fit results have not been reported so it is not possible to evaluate this for oneself.

Three peptides are used to examine the role of C-terminal residues in SxIP motifs: 4-MACF (SKIP), 6-MACF (SKIPTP), and 11-MACF (KPSKIPTPQRK). The 11-mer demonstrates the strongest binding, but this has added residues to the N-terminal as well. It has also introduced charges at both termini, further complicating the interpretation of changes in binding affinities. Given this, I do not believe the authors can reasonably attribute increased affinities solely to post-SxIP residues.

Experimental uncertainties are, with exceptions, not reported.

Reviewer #2 (Public Review):

Barsukov and his colleagues investigate the interaction mechanism between the EB1 C-terminal domain (EBH) and its binding motif, "SxIP," from MACF. From the crystal structure of the C-terminus of EB1 and SxIP, it has been postulated that complex formation is a simple protein-peptide interaction, achieved by only four residues. The authors demonstrate that the post-SxIP region is involved in EBH interactions using NMR and ITC, and propose that a more complex system exists - a two-step "dock-and-lock" model. The CEST data clearly show that EBH possesses two structural conformations and that the C-terminal EBH conformation undergoes a change upon binding to 11MACF. The authors then mutate the 11MACF peptide sequence and identify peptides with much higher affinities for EBH. These findings may contribute to the development of peptide drugs targeting EB1/microtubules.

This work provides a novel structural insight into EB1 and its binding proteins, and the authors present solid experimental evidence to support the idea. One thing the authors should do is, I think, to use the longer EB1 construct. As the authors describe in the Introduction, each domain of EB1 has a distinct function. The C-terminal tail of EB1, which is adjacent to EBH and is not analyzed in this study, is highly acidic and plays an important role in protein interactions. If the authors discuss the C-terminus of EB1, they should analyze the whole C-terminus of EB1, which would strengthen the conclusion they have made.