A multiscale approach reveals the molecular architecture of the autoinhibited kinesin KIF5A.

Kinesin-1 is a microtubule motor that transports cellular cargo along microtubules. KIF5A is one of three kinesin-1 isoforms in humans, all of which are autoinhibited by an interaction between the motor and an IAK motif in the proximal region of the C-terminal tail. The C-terminal tail of KIF5A is ∼80 residues longer than the other two kinesin-1 isoforms (KIF5B and KIF5C) and it is unclear if it contributes to autoinhibition. Mutations in KIF5A cause neuronal diseases and could affect autoinhibition, as reported for a mutation that skips exon 27, altering its C-terminal sequence. Here, we combined negative-stain electron microscopy, crosslinking mass spectrometry (XL-MS) and AlphaFold2 structure prediction to determine the molecular architecture of the full-length autoinhibited KIF5A homodimer, in the absence of light chains. We show that KIF5A forms a compact, bent conformation, through a bend between coiled-coils 2 and 3, around P687. XL-MS of WT KIF5A revealed extensive interactions between residues in the motor, between coiled-coil 1 and the motor, between coiled-coils 1 and 2, with coiled-coils 3 and 4, and the proximal region of the C-terminal tail and the motor in the autoinhibited state, but not between the distal C-terminal region and the rest of the molecule. While negative-stain electron microscopy of exon-27 KIF5A splice mutant showed the presence of autoinhibited molecules, XL-MS analysis suggested that its autoinhibited state is more labile. Our model offers a conceptual framework for understanding how mutations within the motor and stalk domain may affect motor activity.

Effects of specific disease mutations in non-muscle myosin 2A on its structure and function.

Non-muscle myosin 2A (NM2A), a widely expressed class 2 myosin, is important for organizing actin filaments in cells. It cycles between a compact inactive 10S state in which its regulatory light chain (RLC) is dephosphorylated and a filamentous state in which the myosin heads interact with actin, and the RLC is phosphorylated. Over 170 missense mutations in MYH9, the gene that encodes the NM2A heavy chain, have been described. These cause MYH9 disease, an autosomal-dominant disorder that leads to bleeding disorders, kidney disease, cataracts, and deafness. Approximately two-thirds of these mutations occur in the coiled-coil tail. These mutations could destabilize the 10S state and/or disrupt filament formation or both. To test this, we determined the effects of six specific mutations using multiple approaches, including circular dichroism to detect changes in secondary structure, negative stain electron microscopy to analyze 10S and filament formation in vitro, and imaging of GFP-NM2A in fixed and live cells to determine filament assembly and dynamics. Two mutations in D1424 (D1424G and D1424N) and V1516M strongly decrease 10S stability and have limited effects on filament formation in vitro. In contrast, mutations in D1447 and E1841K, decrease 10S stability less strongly but increase filament lengths in vitro. The dynamic behavior of all mutants was altered in cells. Thus, the positions of mutated residues and their roles in filament formation and 10S stabilization are key to understanding their contributions to NM2A in disease.

Structure of the shutdown state of myosin-2.

Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain promotes an inactive, 'shutdown' state with the filament-forming tail folded onto the two heads1, which prevents filament formation and inactivates the motors2. The mechanism by which this happens is unclear. Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin with a resolution of 6 Å in the head region. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. The N-terminal extension of one regulatory light chain interacts with the tail, and the other with the partner head, revealing how the regulatory light chains stabilize the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and the light chains stabilize the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all isoforms of myosin-2 and provides a framework for understanding their disease-causing mutations.

Optimization of Membrane Protein Production Using Titratable Strains of E. coli.

The heterologous expression of membrane proteins driven by T7 RNA polymerase in E. coli is often limited by a mismatch between the transcriptional and translational rates resulting in saturation of the Sec translocon and non-insertion of the membrane protein. In order to optimize the levels of folded, functional inserted protein, it is important to correct this mismatch. In this protocol, we describe the use of titratable strains of E. coli where two small-molecule inducers are used in a bi-variate analysis to optimize the expression levels by fine tuning the transcriptional and translational rates of an eGFP-tagged membrane protein.