Abstract
L-plastin is a calcium-regulated actin-bundling protein that is expressed in cells of hematopoietic origin and in most metastatic cancer cells. These cell types are mobile and require the constant remodeling of their actin cytoskeleton, where L-plastin bundles filamentous actin. The calcium-dependent regulation of the actin-bundling activity of L-plastin is not well understood. We have used NMR spectroscopy to determine the solution structure of the EF-hand calcium-sensor headpiece domain. Unexpectedly, this domain does not bind directly to the four CH-domains of L-plastin. A novel switch helix is present immediately after the calcium-binding region and it binds tightly to the EF-hand motifs in the presence of calcium. We demonstrate that this switch helix plays a major role during actin-bundling. Moreover a peptide that competitively inhibits the association between the EF-hand motifs and the switch helix was shown to deregulate the actin-bundling activity of L-plastin. Overall, these findings may help to develop new drugs that target the L-plastin headpiece and interfere in the metastatic activity of cancer cells.
Highlights
L-plastin (LPL) is a 70 kDa Ca2+- and actin-binding protein that plays an important role in both the adaptive and innate immune system[1,2,3,4]
The backbone amide resonances of Ca2+-free EF-H5 were completed except for Arg 91 and Tyr 48, which could not be identified due to extensive signal overlap in the middle of the spectrum. This overlap is characteristic of the presence of some unstructured elements that are present in part of the apo-protein
By determining the NMR solution structures of the EF-hand domains of L-plastin protein (LPL), we have discovered that an extra ‘switch-helix’ can be formed between the EF construct and the actin-binding domains (ABDs) which may act as the Ca2+-sensor
Summary
L-plastin (LPL) is a 70 kDa Ca2+- and actin-binding protein that plays an important role in both the adaptive and innate immune system[1,2,3,4]. A calcium signal is induced when cancer cells bind to proteins of the basal lamina[23]. This calcium-mediated signal suggests that LPL plays a crucial role in invasion and cellular adhesion of cancer cells. Studies of the formation of invadopodia and filopodia in cancer cells showed that the two actin-bundling proteins L-plastin and fascin played distinct and complementary roles. Both proteins were required to create the actin-based cellular protrusions that give rise to the migration, invasion, and the metastatic properties of cancer cells[25]. Very recently it has been shown that S-glutathionylation may contribute to the regulation of LPL function[44]
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