Vilela, F. | Search bioRxiv

bioRxiv2022

Insights into the activation of Kinesin1 from the molecular characterisation of JIP3/4 binding to Kif5b

Whereas our understanding of kinesin auto-inhibition mechanisms is improving faster, important insights into kinesin activation mechanisms such as those controlled by cargo-motor adaptors are still missing. JIP3 and JIP4 are versatile motor-cargo adaptors for kinesin1 and dynein-dynactin motors enabling bi-directional transport on microtubules. JIP3 activates kinesin1 heavy chains, independently of kinesin1 light chains. In this report, we characterize the molecular details of the binding of the kinesin1 heavy chain, Kif5b to the motor-cargo adaptors, JIP3 and JIP4, using biophysical approaches. The definition of the exact binding site of Kif5b, as well as the specificity of interaction between JIP3 and JIP4 provide new insights into kinesin1 activation.

biophysics↗

bioRxiv2022

Characterization of heterogeneity in nanodisc samples using Feret signatures

Nanodiscs have become a popular tool in structure determination of membrane proteins using cryogenic electron microscopy and single particle analysis. However, the structure determination of small membrane proteins remains challenging. When the embedded protein is in the same size range as the nanodisc, the nanodisc can significantly contribute to the alignment and classification during the structure determination process. In those cases, it is crucial to minimize the heterogeneity in the nanodisc preparations to assure maximum accuracy in the classification and alignment steps of single particle analysis. Here, we introduce a new in-silico method for the characterization of nanodisc samples that is based on analyzing the Feret diameter distribution of their particle projection as imaged in the electron microscope. We validated the method with comprehensive simulation studies and show that Feret signatures can detect subtle differences in nanodisc morphologies and composition that might otherwise go unnoticed. We used the method to identify a specific biochemical nanodisc preparation with low size variations, allowing us to obtain a structure of the 23-kDa single-span membrane protein Bcl-xL while embedded in a nanodisc. Feret signature analysis can steer experimental data collection strategies, allowing more efficient use of high-end data collection hardware, as well as image analysis investments in studies where nanodiscs significantly contribute to the total volume of the full molecular species. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/501900v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@19ea37aorg.highwire.dtl.DTLVardef@1fed1aeorg.highwire.dtl.DTLVardef@781962org.highwire.dtl.DTLVardef@ca7859_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LINew methodology to characterize nanodiscs based on Feret signatures C_LIO_LIFeret signatures distinguish nanodisc morphologies and compositions C_LIO_LIAnalysis is highly sensitive to sample quality C_LIO_LIMethod selected condition to solve structure of small membrane protein Bcl-xL C_LI

molecular biology↗