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Published February 27, 2015 | public
Journal Article Open

Filament Assembly by Spire: Key Residues and Concerted Actin Binding


The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers.

Additional Information

© 2014 Elsevier Ltd. Received 7 April 2014, Revised 28 July 2014, Accepted 4 September 2014, Available online 16 September 2014. The authors would like to thank Dr. Edward J. Miracco for helpful discussions and for performing the homology modeling and structural comparisons. This work was supported by the National Institutes of Health (R01-GM096133) and the Burroughs-Wellcome Fund (Career Award in the Biomedical Sciences) grants to M.E.Q and funding from a Human Frontier Science Program Cross-Disciplinary Fellowship to J.S.B.

Attached Files

Supplemental Material - 1-s2.0-S0022283614004720-mmc1.pdf

Accepted Version - nihms631419.pdf


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August 22, 2023
August 22, 2023