Autoinhibited kinesin-1 adopts a hierarchical folding pattern

Reviewer #1 (Public Review):

Using a combination of structural biology methods, this report aims at describing the auto-inhibited architecture of kinesin 1 either as homodimers or hetero-tetramers. Hence, the multiple contacts between the protein domains and their folding pattern is addressed using cross-linking mass spectrometry (XL-MS), negative stain electron microscopy and Alpha Fold based structure prediction. Based on the existing literature, the key domains and amino acids responsible for kinesin 1 inhibited state were not clearly deciphered. The synergetic use of different methods now seems to describe in detail the molecular cues which could induce kinesin-1 refolding and opening. Multiple interactions between the different domains seem to induce the folded conformation.

The combination of methodologies is an efficient way to unravel details that could not be addressed previously. The paper is well written. However, the methodology is sometimes not sufficiently detailed and the paper would benefit from additional explanations and demonstrations. The methods for generating the electron microscopy data and its relevance and quality, for instance, are barely described. In addition, the conclusions drawn would be more convincing if similar investigations would be carried out similarly for all isoforms (KIF5B and FIF5C) in parallel.

This article raises the potential strength and power of deep learning structure prediction methods combined simultaneously with other structural biology methods to answer specific questions. In the present context, this study will certainly be helpful to reveal and understand the activation mechanism of kinesin motor proteins.

Reviewer #2 (Public Review):

The authors sought to define the molecular structure of autoinhibited Kinesin-1, which is the major kinesin providing plus-end directed transport on microtubules. The paper reports a structural model of full-length kinesin-1 which builds on the known folded conformation of kinesin-1 and describes its autoinhibitory mechanism using cryo-EM, alphafold structural predictions, cross-linking and mass spectrometry. The authors study the conformation of dimeric Kinesin Heavy Chain (KHC) and tetrameric KHC bound to the Kinesin Light Chains (KLCs), where KLC stabilize the autoinhibited conformation. The combination of these various approaches leads to an integrated molecular model of autoinhibited Kinesin-1. Until now, there was some debate over the role of the small coiled coil 3 (a and b) and where the hinge region of Kinesin-1. The authors resolve this question and present data indicating the hinge is between cc3a and cc3b.

In some places the absence of crosslinks is reported as a lack of interaction, however it could also be that there are no residues that can be crosslinked in this region. The distance is also not reported in the figures so we do not know how valid these model are. For example for TRAP binding to KHC, there are not many crosslinks but it is not clear if there was an issue with the complex assembly or crosslinking reaction-as there is no EM data of this complex. There is also a structural model of KHC and KLC (Fig 4) where the domains are too far apart for the crosslinks to be allowed, raising a question about whether that model is correct or not. The structural data are supported by single molecule motility assays with various mutants of Kinesin-1, which greatly help characterising the domains functionally.

Overall there are some interesting novel data on the autoinhibitory mechanism of Kinesin-1, with well performed and analyzed data with KLC and TRAP. The topic and paper will be of interest to many.