Subunit Interactions and Cooperativity in the Microtubule-severing AAA ATPase Spastin

Eckert, Thomas; Link, Susanne; Le, Doan Tuong-Van; Sobczak-Thépot, Joëlle; Gieseke, Anja; Richter, Klaus; Woehlke, Günther

doi:10.1074/jbc.m111.291898

Cited by 37 publications

(58 citation statements)

References 45 publications

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“…N-terminal GFP fusions (GFP-Δ227 HsSpastin) were generated from the Δ227 human spastin GST expression vector used previously [8]. The protein was expressed in E. coli BL21(RIL), and purified by GSH-sepharose affinity, ion exchange, and gel filtration chromatography in buffers 1 and 2, as described (Eckert & al., Roll-Mecak & Vale, 2008).…”

Section: Methodsmentioning

confidence: 99%

“…Evidently, one of the crucial steps of the severing process is the interaction with the microtubule. As other AAA ATPases, severing enzymes have been reported to be active in the form of homo-hexameric rings [5]–[8]. It has been hypothesized that the C-termini of α or β tubulin are threaded through the central pores of hexameric katanin or spastin rings [6], [7].…”

Section: Introductionmentioning

confidence: 99%

“…Spastin's microtubule binding activity has been mapped to a region of 59 amino acid residues N-terminal of the catalytic AAA domain, and has been termed microtubule-binding domain (MTBD; Figure 1; [6]). The situation is convoluted, though, because the human and the Drosophila ortholog differ greatly in their non-AAA domains (Figure 1; [8]). Drosophila spastin requires a combination of parts from two domains for microtubule binding [7].…”

Section: Introductionmentioning

confidence: 99%

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Spastin's Microtubule-Binding Properties and Comparison to Katanin

Eckert¹,

Le²,

Link³

et al. 2012

PLoS ONE

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Spastin and katanin are ring-shaped hexameric AAA ATPases that sever microtubules, and thus crucially depend on a physical interaction with microtubules. For the first time, we report here the microtubule binding properties of spastin at the single-molecule level, and compare them to katanin. Microscopic fluorescence assays showed that human spastin bound to microtubules by ionic interactions, and diffused along microtubules with a diffusion coefficient comparable to katanin. The microscopic measurement of landing and dissociation rates demonstrated the ionic character of the interaction, which could be mapped to a patch of three lysine residues outside of the catalytic domain of human spastin. This motif is not conserved in Drosophila spastin or katanin, which also bound by non-catalytic parts of the protein. The binding affinities of spastin and katanin were nucleotide-sensitive, with the lowest affinities under ADP,, the highest under ATP-γS conditions. These changes correlated with the formation of higher oligomeric states, as shown in biochemical experiments and electron microscopic images. Vice versa, the artificial dimerization of human spastin by addition of a coiled coil led to a constitutively active enzyme. These observations suggest that dimer formation is a crucial step in the formation of the active complex, and thus the severing process by spastin.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Spastin's Microtubule-Binding Properties and Comparison to Katanin

Eckert¹,

Le²,

Link³

et al. 2012

PLoS ONE

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“…However, some subunits, such as Mcm4, Mcm5, and Mcm7, appear to have a greater contribution toward the overall MCM2-7 ATP hydrolysis rate than others, as mutations in their WB motifs result in stronger ATP hydrolysis defects (Bochman et al 2008;Bochman and Schwacha 2010;Bell and Botchan 2013). Many other ring-shaped hexameric motors show high cooperativity between active sites and sequential order of ATP hydrolysis (ATP hydrolysis in one subunit at the time following the order in the ring) (Abrahams et al 1994;Liao et al 2005;Adelman et al 2006;Thomsen and Berger 2008;Eckert et al 2012) but not all of them. For instance, studies on the hexameric protein unfoldase ClpX revealed that ATP hydrolysis in the hexamer is probabilistic.…”

Section: Mcm2-7 Atp Hydrolysis Activities Of the Occm Dh And Cmgmentioning

confidence: 99%

From structure to mechanism—understanding initiation of DNA replication

et al. 2017

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DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability.

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“…Spastin is a ring-shaped hexameric AAA ATPase that physically interacts with microtubules by ionic interactions [10] and severs them by enzymatic activity [11] at the expense of ATP hydrolysis energy [12]. …”

mentioning

confidence: 99%

Cytoskeletal Protection: Acting on Notch to Prevent Neuronal Dysfunction

et al. 2013

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Functional and structural plasticity is a fundamental property of the brain involving chemical, electrical, molecular and cellular responses and leading to reorganization of connections within a brain region and/or between brain regions. The Notch pathway has been recognized as one of the main contributors in regulating neural development and has been proposed as a key mediator in neuroplasticity. We supported this concept, demonstrating that Notch plays a role in determining the only possible ‘cell fate' decisions in post-mitotic mature neurons: synaptic remodelling or neurite extension/retraction. We demonstrated that Notch pathway activation causes a decrease in neurite branching and a loss of varicosities, with consequent reduction in the release of neurotransmitters. Furthermore, in dysfunctional neurons that present Notch pathway hyper-activation, neuronal morphology was reverted by Notch-inhibiting agents. Potentially, a better understanding of the molecular events participating in neuroplasticity may provide relevant information for innovative therapeutic approaches in a variety of neurological disorders. Hence, we propose a Notch-signalling fine-tuned manipulation as a novel approach to modulate neuronal cytoskeleton plasticity in order to prevent dysfunctional structural plasticity in neurodegenerative diseases.

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Subunit Interactions and Cooperativity in the Microtubule-severing AAA ATPase Spastin

Cited by 37 publications

References 45 publications

Spastin's Microtubule-Binding Properties and Comparison to Katanin

Spastin's Microtubule-Binding Properties and Comparison to Katanin

From structure to mechanism—understanding initiation of DNA replication

Cytoskeletal Protection: Acting on Notch to Prevent Neuronal Dysfunction

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