Dataset Information

Solution structures and dynamics of ADF/cofilins UNC-60A and UNC-60B from Caenorhabditis elegans.

ABSTRACT: The nematode Caenorhabditis elegans has two ADF (actin-depolymerizing factor)/cofilin isoforms, UNC-60A and UNC-60B, which are expressed by the unc60 gene by alternative splicing. UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A. To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B proteins and characterized their backbone dynamics. Both UNC-60A and UNC-60B show a conserved ADF/cofilin fold. The G-actin (globular actin)-binding regions of the two proteins are structurally and dynamically conserved. Accordingly, UNC-60A and UNC-60B individually bind to rabbit muscle ADP-G-actin with high affinities, with Kd values of 32.25 nM and 8.62 nM respectively. The primary differences between these strong and weak severing proteins were observed in the orientation and dynamics of the F-actin (filamentous actin)-binding loop (F-loop). In the strong severing activity isoform UNC-60B, the orientation of the F-loop was towards the recently identified F-loop-binding region on F-actin, and the F-loop was relatively more flexible with 14 residues showing motions on a nanosecond-picosecond timescale. In contrast, in the weak severing protein isoform UNC-60A, the orientation of the F-loop was away from the F-loop-binding region and inclined towards its own C-terminal and strand ?6. It was also relatively less flexible with only five residues showing motions on a nanosecond-picosecond timescale. These differences in structure and dynamics seem to directly correlate with the differential F-actin site-binding and severing properties of UNC-60A and UNC-60B, and other related ADF/cofilin proteins.

SUBMITTER: Shukla VK

PROVIDER: S-EPMC4325984 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Solution structures and dynamics of ADF/cofilins UNC-60A and UNC-60B from Caenorhabditis elegans.

Shukla Vaibhav Kumar VK Kabra Ashish A Maheshwari Diva D Yadav Rahul R Jain Anupam A Tripathi Sarita S Ono Shoichiro S Kumar Dinesh D Arora Ashish A

The Biochemical journal 20150101 1

The nematode Caenorhabditis elegans has two ADF (actin-depolymerizing factor)/cofilin isoforms, UNC-60A and UNC-60B, which are expressed by the unc60 gene by alternative splicing. UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A. To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B pr ...[more]

PMID: 25279657

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Project description:Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1.SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.

Dataset Information

Solution structures and dynamics of ADF/cofilins UNC-60A and UNC-60B from Caenorhabditis elegans.

Publications

Solution structures and dynamics of ADF/cofilins UNC-60A and UNC-60B from Caenorhabditis elegans.

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