Pathogenic Mutations in the Kinesin-3 Motor KIF1A Diminish Force Generation and Movement Through Allosteric Mechanisms

Kaur

et al. 2020

Preprint

Self Cite

KIF1A Associated Neurological Disorder (KAND) encompasses a recently identified group of rare neurodegenerative conditions caused by variants in KIF1A, a member of the kinesin-3 family of microtubule (MT) motor proteins. Here we characterize the natural history of KAND in 117 individuals using a combination of caregiver or self-reported medical history, a standardized measure of adaptive behavior, clinical records, and neuropathology. We developed a heuristic severity score using a weighted sum of common symptoms to assess disease severity. Focusing on 100 individuals, we compared the average clinical severity score for each variant with in silico predictions of deleteriousness and location in the protein. We found increased severity is strongly associated with variants occurring in regions involved with ATP and MT-binding: the P-loop, switch I, and switch II. For a subset of identified variants, we generated recombinant mutant proteins which we used to assess transport in vivo by assessing neurite tip accumulation, and to assess MT binding, motor velocity, and processivity using total internal reflection fluorescence microscopy. We find all patient variants result in defects in transport, and describe three classes of protein dysfunction: reduced MT binding, reduced velocity and processivity, and increased non-motile rigor MT binding. The molecular rigor phenotype is consistently associated with the most severe clinical phenotype, while reduced binding is associated with milder clinical phenotypes. Our findings suggest the clinical phenotypic heterogeneity in KAND likely reflects and parallels diverse molecular phenotypes. We propose a new way to describe KAND subtypes to better capture the breadth of disease severity.

Section: Resultsmentioning

confidence: 97%

Genotype and defects in microtubule-based motility correlate with clinical severity in KIF1A Associated Neurological Disorder

Boyle

Kaur

et al. 2020

Preprint

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“…Our recent work has shown that the velocity and force-generation properties of KIF1A expressed in E. coli bacteria and mammalian cells are statistically indistinguishable. 55 A similar construct has also been used in other studies. 49 , 56 , 57 …”

Section: Resultsmentioning

confidence: 99%

Genotype and defects in microtubule-based motility correlate with clinical severity in KIF1A-associated neurological disorder

Boyle

Human Genetics and Genomics Advances

Kaur

et al. 2021

Self Cite

Summary KIF1A-associated neurological disorder (KAND) encompasses a group of rare neurodegenerative conditions caused by variants in KIF1A ,a gene that encodes an anterograde neuronal microtubule (MT) motor protein. Here we characterize the natural history of KAND in 117 individuals using a combination of caregiver or self-reported medical history, a standardized measure of adaptive behavior, clinical records, and neuropathology. We developed a heuristic severity score using a weighted sum of common symptoms to assess disease severity. Focusing on 100 individuals, we compared the average clinical severity score for each variant with in silico predictions of deleteriousness and location in the protein. We found increased severity is strongly associated with variants occurring in protein regions involved with ATP and MT binding: the P loop, switch I, and switch II. For a subset of variants, we generated recombinant proteins, which we used to assess transport in vivo by assessing neurite tip accumulation and to assess MT binding, motor velocity, and processivity using total internal reflection fluorescence microscopy. We find all modeled variants result in defects in protein transport, and we describe three classes of protein dysfunction: reduced MT binding, reduced velocity and processivity, and increased non-motile rigor MT binding. The rigor phenotype is consistently associated with the most severe clinical phenotype, while reduced MT binding is associated with milder clinical phenotypes. Our findings suggest the clinical phenotypic heterogeneity in KAND likely reflects and parallels diverse molecular phenotypes. We propose a different way to describe KAND subtypes to better capture the breadth of disease severity.

“…We have previously shown that introduction of the human KIF1A gene into unc-104(e1265) background results in restoration of movement to near WT levels (14), indicating that the human gene can largely compensate for unc-104's cellular functions in worms. Indeed, single-molecule studies with recombinant proteins show that UNC104 and KIF1A have similar motile and force-generation properties (30).…”

Section: A Genetic Model Reveals Kif1a P305l Behaves As a Null Mutatimentioning

confidence: 99%

“…To assess any effects on force generation, we performed single-molecule optical tweezers assays (7,39,40) with our human KIF1A proteins. In a parallel study (30), we have shown that full-length rat KIF1A and tail-truncated rat KIF1A (1-393)-LZ both detach at an average load of ~2.7 pN and then rapidly reattach to the MT to resume motion, resulting in a sawtooth-like force-generation pattern in which forcegeneration events cluster tightly together (30). We find that human KIF1A (1-393-LZ), which differs from a similar rat KIF1A construct by a single amino acid (valine instead of an isoleucine at residue 359), also exhibits a sawtooth-like force-generation pattern (Fig.…”

Section: The P305l Mutation Impairs Kif1a's Ability To Generate Forcementioning

confidence: 99%

A Highly Conserved 3₁₀-Helix Within the Kinesin Motor Domain is Critical for Kinesin Function and Human Health

Lam

Anazawa

et al. 2020

Preprint

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KIF1A, a kinesin-3 family member, plays critical roles as a long-distance cargo-transporter within neurons. Over 100 known KIF1A mutations in humans result in KIF1A Associated Neurological Disease (KAND), developmental and degenerative neurological conditions for which there is no cure. A de novo missense mutation, P305L, was recently identified in several children diagnosed with KAND, but the underlying molecular basis for the disease phenotype is unknown. Interestingly, this residue is highly conserved in kinesin-family proteins, and together with adjacent conserved residues also implicated in KAND, forms an unusual 310-helical element immediately C-terminal to loop-12 (L12, also known as the K-loop in KIF1A) in the conserved kinesin motor core. In KIF1A, the disordered K-loop contains a highly charged insertion of lysines that is thought to endow the motor with a high microtubule-association rate. Here, we characterize the molecular defects of the P305L mutation in KIF1A using genetic, biochemical, and single-molecule approaches. We find the mutation negatively impacts the velocity, run-length, and force generation of the motor. However, a much more dramatic effect is observed on the microtubule-association rate of the motor, revealing that the presence of an intact K-loop is not sufficient for its function. We hypothesize that an elusive K-loop conformation, mediated by formation of a highly conserved adjacent 310-helix that is modulated via P305, is critically important for the kinesin-microtubule interaction. Importantly, we find that the function of this proline is conserved in the canonical kinesin, KIF5, revealing a fundamental principle of the kinesin motor mechanism.