Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms

Long-distance transport in cells is driven by kinesin and dynein motors that move along microtubule tracks. These motors must be tightly regulated to ensure the spatial and temporal fidelity of their transport events. Transport motors of the kinesin-1 and kinesin-3 families are regulated by autoinhibition, but little is known about the mechanisms that regulate kinesin-2 motors. We show that the homodimeric kinesin-2 motor KIF17 is kept in an inactive state in the absence of cargo. Autoinhibition is caused by a folded conformation that enables nonmotor regions to directly contact and inhibit the enzymatic activity of the motor domain. We define two molecular mechanisms that contribute to autoinhibition of KIF17. First, the C-terminal tail interferes with microtubule binding; and second, a coiled-coil segment blocks processive motility. The latter is a new mechanism for regulation of kinesin motors. This work supports the model that autoinhibition is a general mechanism for regulation of kinesin motors involved in intracellular trafficking events.     

Role of the kinesin-2 family protein, KIF3, during mitosis

During mitosis, kinesin and dynein motor proteins play critical roles in the equal segregation of chromosomes between two daughter cells. Kinesin-2 is composed of two microtubule-based motor subunits, KIF3A/3B, and a kinesin-associated protein known as KAP3, which links KIF3A/3B to cargo that is carried to cellular organelles along microtubules in interphase cells. We have shown here that the kinesin-2 complex is localized with components of the mitotic apparatus such as spindle microtubules and centrosomes. Furthermore, we found that expression of a mutant KIF3B, which is able to associate with KIF3A but not KAP3 in NIH3T3 cells, caused chromosomal aneuploidy and abnormal spindle formation. Our data suggested that the kinesin-2 complex plays an important role not only in interphase but also in mitosis.     

Rod cyclic nucleotide-gated channels have a stoichiometry of three CNGA1 subunits and one CNGB1 subunit

Phototransduction relies on the precise balance of speed and sensitivity to achieve optimal performance. The cyclic nucleotide-gated (CNG) ion channels, with their Ca(2+) permeability, high sensitivity to changes in cytosolic cGMP, rapid gating kinetics, and Ca(2+)-calmodulin modulation, are beautifully optimized for their role in light detection. Many of these specializations come about from the heteromeric composition of the native channel, comprised of CNGA1 and CNGB1 subunits. However, the stoichiometry and arrangement of these subunits is unknown. Here we have used an approach based on fluorescence resonance energy transfer (FRET) to determine the composition of the intact functional channel in the surface membrane. We find, surprisingly, that the channel contains three CNGA1 subunits and only one CNGB1 subunit. These results have implications for CNG channel function in particular and assembly of membrane proteins in general.     

Pathogenic mutations in the kinesin-3 motor KIF1A diminish force generation and movement through allosteric mechanisms

The kinesin-3 motor KIF1A functions in neurons, where its fast and superprocessive motility facilitates long-distance transport, but little is known about its force-generating properties. Using optical tweezers, we demonstrate that KIF1A stalls at an opposing load of ~3 pN but more frequently detaches at lower forces. KIF1A rapidly reattaches to the microtubule to resume motion due to its class-specific K-loop, resulting in a unique clustering of force generation events. To test the importance of neck linker docking in KIF1A force generation, we introduced mutations linked to human neurodevelopmental disorders. Molecular dynamics simulations predict that V8M and Y89D mutations impair neck linker docking. Indeed, both mutations dramatically reduce the force generation of KIF1A but not the motor’s ability to rapidly reattach to the microtubule. Although both mutations relieve autoinhibition of the full-length motor, the mutant motors display decreased velocities, run lengths, and landing rates and delayed cargo transport in cells. These results advance our understanding of how mutations in KIF1A can manifest in disease.     

The microtubule-binding protein Cep170 promotes the targeting of the kinesin-13 depolymerase Kif2b to the mitotic spindle

Microtubule dynamics are essential throughout mitosis to ensure correct chromosome segregation. Microtubule depolymerization is controlled in part by microtubule depolymerases, including the kinesin-13 family of proteins. In humans, there are three closely related kinesin-13 isoforms (Kif2a, Kif2b, and Kif2c/MCAK), which are highly conserved in their primary sequences but display distinct localization and nonoverlapping functions. Here we demonstrate that the N-terminus is a primary determinant of kinesin-13 localization. However, we also find that differences in the C-terminus alter the properties of kinesin-13, in part by facilitating unique protein–protein interactions. We identify the spindle-localized proteins Cep170 and Cep170R (KIAA0284) as specifically associating with Kif2b. Cep170 binds to microtubules in vitro and provides Kif2b with a second microtubule-binding site to target it to the spindle. Thus the intrinsic properties of kinesin-13s and extrinsic factors such as their associated proteins result in the diversity and specificity within the kinesin-13 depolymerase family.     

Plk1 regulates the kinesin-13 protein Kif2b to promote faithful chromosome segregation

Solid tumors are frequently aneuploid, and many display high rates of ongoing chromosome missegregation in a phenomenon called chromosomal instability (CIN). The most common cause of CIN is the persistence of aberrant kinetochore-microtubule (k-MT) attachments, which manifest as lagging chromosomes in anaphase. k-MT attachment errors form during prometaphase due to stochastic interactions between kinetochores and microtubules. The kinesin-13 protein Kif2b promotes the correction of k-MT attachment errors in prometaphase, but the mechanism restricting this activity to prometaphase remains unknown. Using mass spectrometry, we identified multiple phosphorylation sites on Kif2b, some of which are acutely sensitive to inhibition of Polo-like kinase 1 (Plk1). We show that Plk1 directly phosphorylates Kif2b at threonine 125 (T125) and serine 204 (S204), and that these two sites differentially regulate Kif2b function. Phosphorylation of S204 is required for the kinetochore localization and activity of Kif2b in prometaphase, and phosphorylation of T125 is required for Kif2b activity in the correction of k-MT attachment errors. These data demonstrate that Plk1 regulates both the localization and activity of Kif2b during mitosis to promote the correction of k-MT attachment errors to ensure mitotic fidelity.     

LDL receptor related protein 1 requires the I3 domain of discs-large homolog 1/DLG1 for interaction with the kinesin motor protein KIF13B

KIF13B, a kinesin-3 family motor, was originally identified as GAKIN due to its biochemical interaction with human homolog of Drosophila discs-large tumor suppressor (hDLG1). Unlike its homolog KIF13A, KIF13B contains a carboxyl-terminal CAP-Gly domain. To investigate the function of the CAP-Gly domain, we developed a mouse model that expresses a truncated form of KIF13B protein lacking its CAP-Gly domain (KIF13BΔCG), whereas a second mouse model lacks the full-length KIF13A. Here we show that the KIF13BΔCG mice exhibit relatively higher serum cholesterol consistent with the reduced uptake of [³H]CO-LDL in KIF13BΔCG mouse embryo fibroblasts. The plasma level of factor VIII was not significantly elevated in the KIF13BΔCG mice, suggesting that the CAP-Gly domain region of KIF13B selectively regulates LRP1-mediated lipoprotein endocytosis. No elevation of either serum cholesterol or plasma factor VIII was observed in the full length KIF13A null mouse model. The deletion of the CAP-Gly domain region caused subcellular mislocalization of truncated KIF13B concomitant with the mislocalization of LRP1. Mechanistically, the cytoplasmic domain of LRP1 interacts specifically with the alternatively spliced I₃ domain of DLG1, which complexes with KIF13B via their GUK-MBS domains, respectively. Importantly, double mutant mice generated by crossing KIF13A null and KIF13BΔCG mice suffer from perinatal lethality showing potential craniofacial defects. Together, this study provides first evidence that the carboxyl-terminal region of KIF13B containing the CAP-Gly domain is important for the LRP1-DLG1-KIF13B complex formation with implications in the regulation of metabolism, cell polarity, and development.     

Loss of CNGB1 protein leads to olfactory dysfunction and subciliary cyclic nucleotide-gated channel trapping

Olfactory receptor neurons (ORNs) employ a cyclic nucleotide-gated (CNG) channel to generate a receptor current in response to an odorant-induced rise in cAMP. This channel contains three types of subunits, the principal CNGA2 subunit and two modulatory subunits (CNGA4 and CNGB1b). Here, we have analyzed the functional relevance of CNGB1 for olfaction by gene targeting in mice. Electro-olfactogram responses of CNGB1-deficient (CNGB1-/-) mice displayed a reduced maximal amplitude and decelerated onset and recovery kinetics compared with wild-type mice. In a behavioral test, CNGB1-/- mice exhibited a profoundly decreased olfactory performance. Electrophysiological recordings revealed that ORNs of CNGB1-/- mice weakly expressed a CNG current with decreased cAMP sensitivity, very rapid flicker-gating behavior and no fast modulation by Ca2+-calmodulin. Co-immunoprecipitation confirmed the presence of a CNGA2/CNGA4 channel in the olfactory epithelium of CNGB1-/- mice. This CNGA2/CNGA4 channel was targeted to the plasma membrane of olfactory knobs, but failed to be trafficked into olfactory cilia. Interestingly, we observed a similar trafficking defect in mice deficient for the CNGA4 subunit. In conclusion, these results demonstrate that CNGB1 has a dual function in vivo. First, it endows the olfactory CNG channel with a variety of biophysical properties tailored to the specific requirements of olfactory transduction. Second, together with the CNGA4 subunit, CNGB1 is needed for ciliary targeting of the olfactory CNG channel.     

Association of the kinesin motor KIF1A with the multimodular protein liprin-alpha

Liprin-alpha/SYD-2 is a multimodular scaffolding protein important for presynaptic differentiation and postsynaptic targeting of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid glutamate receptors. However, the molecular mechanisms underlying these functions remain largely unknown. Here we report that liprin-alpha interacts with the neuron-specific kinesin motor KIF1A. KIF1A colocalizes with liprin-alpha in various subcellular regions of neurons. KIF1A coaccumulates with liprin-alpha in ligated sciatic nerves. KIF1A cofractionates and coimmunopreciptates with liprin-alpha and various liprin-alpha-associated membrane, signaling, and scaffolding proteins including alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors, GRIP/ABP, RIM, GIT1, and beta PIX. These results suggest that liprin-alpha functions as a KIF1A receptor, linking KIF1A to various liprin-alpha-associated proteins for their transport in neurons.     

Monomeric and dimeric states exhibited by the kinesin-related motor protein KIF1A.

KIF1A, a kinesin-related motor protein that transports pre-synaptic vesicles in neurons, was originally presumed to translocate along microtubules (MT) as a monomer. Protein structure predictions from its amino acid sequence failed to identify the long coiled-coil domains typical of kinesins, which led researchers to believe it does not oligomerize into the canonical kinesin dimer. However, mounting evidence using recombinant chimeric protein indicates that KIF1A, like conventional kinesin, requires dimerization for fast, unidirectional processive movement along MTs. Because these studies are somewhat indirect, we wished to test the oligomerization state of native KIF1A, and to compare that to full-length recombinant protein. We have performed hydrodynamic analyses to determine the molecular weights of the respective complexes. Our results indicate that most native KIF1A is soluble and indeed monomeric, but recombinant KIF1A is a dimer. MT-binding studies also showed that native KIF1A did not bind to MTs in either the presence of AMP-PNP, apyrase, or adenosine triphosphate (ATP), but recombinant KIF1A bound to MTs most stably in the presence of ATP, indicating very different motor functional states. To further characterize KIF1A's dimerization potential, we prepared peptides corresponding to the neck domains of MmKIF1A and CeUnc104, and by circular dichroism spectroscopy compared these peptides for their ability to form coiled-coils. Interestingly, both MmKIF1A and CeUnc104 neck peptides formed homodimeric coiled-coils, with the MmKIF1A neck coiled-coil exhibiting the greater stability. Collectively, from our data and from previous studies, we predict that native KIF1A can exist as both an inactive monomer and an active homodimer formed in part through its neck coiled-coil domain.     

The motor protein kinesin-1 links neurofibromin and merlin in a common cellular pathway of neurofibromatosis

Mutations in either of the two tumor suppressor genes NF1 (neurofibromin) and NF2 (merlin) result in Neurofibromatosis, a condition predisposing individuals to developing a variety of benign and malignant tumors of the central and peripheral nervous systems. Here we report the identification of two distinct NF1-containing complexes, one in the soluble and the other in the particulate fraction of HeLa extract. We show that the soluble NF1 complex delineates a large holo-NF1 complex (2 MDa) encompassing the components of a smaller particulate core-NF1 complex (400 kDa). Purification of the core-NF1 complex followed by mass spectrometric analysis revealed the motor protein, kinesin-1 heavy chain (HsuKHC/KIF5B), as a catalytic subunit of both NF-1-containing complexes. Importantly, although NF1 and NF2 are not in a stable association, NF2 is also a component of a distinct kinesin-1-containing complex. These results point to kinesin-1 as a common denominator between NF1 and NF2.     

Modulation of myosin phosphatase targeting subunit and protein phosphatase 1 in the heart

Myosin light chain 2 (LC2) phosphorylation is of both physiological and pathological importance to myocardial function. The phosphatase that directly dephosphorylates LC2 is a type 1 protein phosphatase (PP1) that contains a catalytic subunit that complexes with a myosin-binding phosphatase targeting subunit (MYPT). The goal of the present study was to examine the role of MYPT in the regulation of PP1 in ventricular myocytes. In the first part of the study, regional distribution of MYPT expression and phosphorylation were determined in unstimulated hearts. The pattern of MYPT phosphorylation was inversely related to the LC2 phosphorylation spatial gradient as described by Epstein and colleagues (Davis JS, Hassanzadeh S, Winitsky S, Lin H, Satorius C, Vemuri R, Aletras AH, Wen H, and Epstein ND. Cell 107: 631-641, 2001). In the second part of the study, adult rat isolated ventricular myocytes were exposed to an alpha-adrenergic receptor agonist, and properties of MYPT, PP1, and LC2 were studied. We found MYPT associates with cardiac myofilaments, and this association increases upon alpha-adrenergic receptor stimulation. Activation of alpha-adrenergic receptors also led to a decrease in the PP1-myofilament association. Furthermore, alpha-adrenergic receptor stimulation results in phosphorylation of MYPT and LC2 and an increase in myocyte Ca(2+) sensitivity of tension that all depend on Rho kinase activation. These data support the hypothesis that alpha-adrenergic receptor activation works through Rho kinase to phosphorylate MYPT, and phosphorylated MYPT dissociates from PP1 so that PP1 is no longer physically associated with LC2. Hence, we propose a pathway for the dynamic modulation of LC2 phosphorylation through receptor-dependent phosphorylation of MYPT, and a spatial gradient of LC2 phosphorylation under basal conditions that occurs due to varied levels of phosphorylation of MYPT in ventricles.     

Positive charge in the K-loop of the kinesin-3 motor KIF1A regulates superprocessivity by enhancing microtubule affinity in the one-head–bound state

KIF1A is an essential neuronal transport motor protein in the kinesin-3 family, known for its superprocessive motility. However, structural features underlying this function are unclear. Here, we determined that superprocessivity of KIF1A dimers originates from a unique structural domain, the lysine-rich insertion in loop-12 termed the ‘K-loop’, which enhances electrostatic interactions between the motor and the microtubule. In 80 mM PIPES buffer, replacing the native KIF1A loop-12 with that of kinesin-1 resulted in a 6-fold decrease in run length, whereas adding additional positive charge to loop-12 enhanced the run length. Interestingly, swapping the KIF1A loop-12 into kinesin-1 did not enhance its run length, consistent with the two motor families using different mechanochemical tuning to achieve persistent transport. To investigate the mechanism by which the KIF1A K-loop enhances processivity, we used microtubule pelleting and single-molecule dwell time assays in ATP and ADP. First, the microtubule affinity was similar in ATP and in ADP, consistent with the motor spending the majority of its cycle in a weakly bound state. Second, the microtubule affinity and single-molecule dwell time in ADP were 6-fold lower in the loop-swap mutant than WT. Thus, the positive charge in loop-12 of KIF1A enhances the run length by stabilizing binding of the motor in its vulnerable one-head–bound state. Finally, through a series of mutants with varying positive charge in the K-loop, we found that KIF1A processivity is linearly dependent on the charge of loop-12, further highlighting how loop-12 contributes to the function of this key motor protein.     

A variant-proof SARS-CoV-2 vaccine targeting HR1 domain in S2 subunit of spike protein

The emerging SARS-CoV-2 variants, commonly with many mutations in S1 subunit of spike (S) protein are weakening the efficacy of the current vaccines and antibody therapeutics. This calls for the variant-proof SARS-CoV-2 vaccines targeting the more conserved regions in S protein. Here, we designed a recombinant subunit vaccine, HR121, targeting the conserved HR1 domain in S2 subunit of S protein. HR121 consisting of HR1–linker1–HR2–linker2–HR1, is conformationally and functionally analogous to the HR1 domain present in the fusion intermediate conformation of S2 subunit. Immunization with HR121 in rabbits and rhesus macaques elicited highly potent cross-neutralizing antibodies against SARS-CoV-2 and its variants, particularly Omicron sublineages. Vaccination with HR121 achieved near-full protections against prototype SARS-CoV-2 infection in hACE2 transgenic mice, Syrian golden hamsters and rhesus macaques, and effective protection against Omicron BA.2 infection in Syrian golden hamsters. This study demonstrates that HR121 is a promising candidate of variant-proof SARS-CoV-2 vaccine with a novel conserved target in the S2 subunit for application against current and future SARS-CoV-2 variants.Subject terms: Mechanisms of disease, Molecular modelling     

15 A resolution model of the monomeric kinesin motor, KIF1A

A two-headed structure has been widely believed to be essential for the kinesin molecular motor to move processively on the track, microtubules. However, we have recently demonstrated that a monomeric motor domain construct of KIF1A (C351), a kinesin superfamily protein, moves processively, taking about 700 steps before being detached from microtubules. To elucidate the mechanism of its single-headed processivity, we examined the C351 -MT interaction by mutant analysis and high-resolution cryo-EM. Mutant analysis indicated the importance of a highly positively charged loop, the "K loop," for such processivity. A 15 A resolution structure unambiguously docked with the available atomic models revealed "K loop" as an extra microtubule-binding domain specific to KIF1A, and bound to the C terminus of tubulin. The site-specific cross-linking further confirmed this model.     

Regulated targeting of the monotopic hairpin membrane protein Erg1 requires the GET pathway

The guided entry of tail-anchored proteins (GET) pathway targets C-terminally anchored transmembrane proteins and protects cells from lipotoxicity. Here, we reveal perturbed ergosterol production in ∆get3 cells and demonstrate the sensitivity of GET pathway mutants to the sterol synthesis inhibiting drug terbinafine. Our data uncover a key enzyme of sterol synthesis, the hairpin membrane protein squalene monooxygenase (Erg1), as a non-canonical GET pathway client, thus rationalizing the lipotoxicity phenotypes of GET pathway mutants. Get3 recognizes the hairpin targeting element of Erg1 via its classical client-binding pocket. Intriguingly, we find that the GET pathway is especially important for the acute upregulation of Erg1 induced by low sterol conditions. We further identify several other proteins anchored to the endoplasmic reticulum (ER) membrane exclusively via a hairpin as putative clients of the GET pathway. Our findings emphasize the necessity of dedicated targeting pathways for high-efficiency targeting of particular clients during dynamic cellular adaptation and highlight hairpin proteins as a potential novel class of GET clients.     

Planar cell polarization requires Widerborst,a B' regulatory subunit of protein phosphatase 2A

We have identified widerborst (wdb), a B' regulatory subunit of PP2A, as a conserved component of planar cell polarization mechanisms in both Drosophila and in zebrafish. In Drosophila, wdb acts at two steps during planar polarization of wing epithelial cells. It is required to organize tissue polarity proteins into proximal and distal cortical domains, thus determining wing hair orientation. It is also needed to generate the polarized membrane outgrowth that becomes the wing hair. Widerborst activates the catalytic subunit of PP2A and localizes to the distal side of a planar microtubule web that lies at the level of apical cell junctions. This suggests that polarized PP2A activation along the planar microtubule web is important for planar polarization. In zebrafish, two wdb homologs are required for convergent extension during gastrulation, supporting the conjecture that Drosophila planar cell polarization and vertebrate gastrulation movements are regulated by similar mechanisms.