Acetylation of K40 in α-tubulin is the sole posttranslational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule stabilization is correlative or causative. We have obtained high-resolution cryo-electron microscopy (cryo-EM) reconstructions of pure samples of αTAT1-acetylated and SIRT2-deacetylated microtubules to visualize the structural consequences of this modification and reveal its potential for influencing the larger assembly properties of microtubules. We modeled the conformational ensembles of the unmodified and acetylated states by using the experimental cryo-EM density as a structural restraint in molecular dynamics simulations. We found that acetylation alters the conformational landscape of the flexible loop that contains αK40. Modification of αK40 reduces the disorder of the loop and restricts the states that it samples. We propose that the change in conformational sampling that we describe, at a location very close to the lateral contacts site, is likely to affect microtubule stability and function.
Microtubule (MT)–associated protein 7 (MAP7) is a required cofactor for kinesin-1–driven transport of intracellular cargoes. Using cryo–electron microscopy and single–molecule imaging, we investigated how MAP7 binds MTs and facilitates kinesin-1 motility. The MT-binding domain (MTBD) of MAP7 bound MTs as an extended α helix between the protofilament ridge and the site of lateral contact. Unexpectedly, the MTBD partially overlapped with the binding site of kinesin-1 and inhibited its motility. However, by tethering kinesin-1 to the MT, the projection domain of MAP7 prevented dissociation of the motor and facilitated its binding to available neighboring sites. The inhibitory effect of the MTBD dominated as MTs became saturated with MAP7. Our results reveal biphasic regulation of kinesin-1 by MAP7 in the context of their competitive binding to MTs.
Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma in humans and afflicts more than 58 million people worldwide. The HCV envelope E1 and E2 glycoproteins are essential for viral entry and comprise the primary antigenic target for neutralizing antibody responses. The molecular mechanisms of E1E2 assembly, as well as how the E1E2 heterodimer binds broadly neutralizing antibodies, remain elusive. Here, we present the cryo–electron microscopy structure of the membrane-extracted full-length E1E2 heterodimer in complex with three broadly neutralizing antibodies—AR4A, AT1209, and IGH505—at ~3.5-angstrom resolution. We resolve the interface between the E1 and E2 ectodomains and deliver a blueprint for the rational design of vaccine immunogens and antiviral drugs.
28Acetylation of K40 in α-tubulin is the sole post-translational modification to mark the 29 luminal surface of microtubules. It is still controversial whether its relationship with 30 microtubule stabilization is correlative or causative. We have obtained high-resolution 31 cryo-electron microscopy reconstructions of pure samples of αTAT1-acetylated and 32 SIRT2-deacetylated microtubules to visualize the structural consequences of this 33 modification and reveal its potential for influencing the larger assembly properties of 34 microtubules. We modeled the conformational ensembles of the unmodified and 35 acetylated states by using the experimental cryo-EM density as the structural restraint in 36 molecular dynamics simulations. We found that acetylation alters the conformational 37 landscape of the flexible loop that contains αK40. Modification of αK40 reduces the 38 disorder of the loop and restricts the states that it samples. We propose that the change 39 in conformational sampling that we describe, at a location very close to the lateral contacts 40 site, is likely to affect microtubule stability and function. 41 42 ABBREVIATIONS 43 MT, microtubule; PF, protofilament; PTM, post-translational modification; MAP, 44 microtubule-associated protein; αTAT1, acetyltransferase TAT1 for α-tubulin; SIRT2 45 deacetylase SIRT2. 46 47 131 cryo-EM samples as previously described 2,26 of Ac 96 and Ac 0 MTs in the presence of end-132 binding protein 3 (EB3). EB3 served as a fiducial marker of the dimer that facilitated 133 alignment of MT segments during image processing 27 . Table S1 summarizes the data 134
Microtubule (MT)-associated proteins (MAPs) regulate intracellular transport by selectively recruiting or excluding kinesin and dynein motors from MTs. We used single-molecule and cryo-electron imaging to determine the mechanism of MAP-motor interactions in vitro. Unexpectedly, we found that the regulatory role of a MAP cannot be predicted based on whether it overlaps with the motor binding site or forms liquid condensates on the MT. Although the MT binding domain (MTBD) of MAP7 overlaps with the kinesin-1 binding site, tethering of kinesin-1 by the MAP7 projection domain supersedes this inhibition and results in biphasic regulation of kinesin-1 motility. Conversely, the MTBD of tau inhibits dynein motility without overlapping with the dynein binding site or by forming tau islands on the MT. Our results indicate that MAPs sort intracellular cargos moving in both directions, as neither dynein nor kinesin can walk on a MAP-coated MT without favorably interacting with that MAP.
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