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A molecular trajectory of ?-actinin activation.

ABSTRACT: The mechanisms by which living cells respond to mechanical stimuli are not yet fully understood. It has been suggested that mechanosensing proteins play an important role in mechanotransduction because their binding affinities are directly affected by the external stress. ?-Actinin is an actin cross-linker and may act as a mechanosensor in adhesion sites. Its interaction with vinculin is suggested to be mechanically regulated. In this study, the free energy of activation is explored using the umbrella sampling method. An activation trajectory is generated in which ?-actinin's vinculin-binding site swings out of the rod domain, leading to approximately an 8 kcal/mol free energy release. The activation trajectory reveals several local and global conformational changes along the activation pathway accompanied by the breakage of a number of key interactions stabilizing the inhibited structure. These results may shed light on the role of ?-actinin in cellular mechanotransduction and focal adhesion formation.

SUBMITTER: Shams H

PROVIDER: S-EPMC3512038 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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A molecular trajectory of α-actinin activation.

Shams Hengameh H Golji Javad J Mofrad Mohammad R K MR

Biophysical journal 20121120 10

The mechanisms by which living cells respond to mechanical stimuli are not yet fully understood. It has been suggested that mechanosensing proteins play an important role in mechanotransduction because their binding affinities are directly affected by the external stress. α-Actinin is an actin cross-linker and may act as a mechanosensor in adhesion sites. Its interaction with vinculin is suggested to be mechanically regulated. In this study, the free energy of activation is explored using the um ...[more]

PMID: 23200039

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Project description:BackgroundAnnexins are calcium dependent phospholipid binding proteins that are expressed in a wide variety of tissues and implicated in various extra- and intracellular processes. In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal. Conflicting reports from transgenic mice overexpressing annexin A6 or null mice lacking annexin A6 showed imbalances in intracellular calcium turnover and disturbed cardiac contractility. However, few studies have focussed on the signalling module of annexin A6 in the heart either in normal or in pathological state.ResultsTo identify the putative binding partners of annexin A6 in the heart, ventricular extracts were subjected to glutathione S-transferase (GST)- annexin A6 pull down assay and the GST- annexin A6 bound proteins were identified by mass spectrometry. The pull down fractions of ventricular extracts with GST-full length annexin A6 as well as GST-C terminus deleted annexin A6 when immunoblotted with anti sarcomeric alpha (α)-actinin antibody showed the presence of α-actinin in the immunoblot which was absent when GST-N terminus deleted annexin A6 was used for pull down. Overexpression of green fluorescent protein (GFP) tagged full length annexin A6 showed z-line like appearance in cardiomyocytes whereas GFP-N termimus deleted annexin A6 was mostly localized to the nucleus. Overexpression of GFP-C terminus deleted annexin A6 in cardiomyocytes showed aggregate like appearance in the cytoplasm. Double immunofluorescent staining of cardiomyocytes with anti annexin A6 and anti sarcomeric α-actinin antibodies showed perfect co-localization of these two proteins with annexin A6 appearing like a component of sarcomere. Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells.ConclusionsIn overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.

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A molecular trajectory of ?-actinin activation.

Publications

A molecular trajectory of α-actinin activation.

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