Abstract
Intrinsically disordered proteins (IDPs) play a major role in various cellular functions ranging from transcription to cell migration. Mutations/modifications in such IDPs are shown to be associated with various diseases. Current strategies to study the mode of action and regulatory mechanisms of disordered proteins at the structural level are time consuming and challenging. Therefore, using simple and swift strategies for identifying functionally important regions in unstructured segments and understanding their underlying mechanisms is critical for many applications. Here we propose a simple strategy that employs dissection of human paxillin (residues 1–313) that comprises intrinsically disordered regions, followed by its interaction study using FAT (Focal adhesion targeting domain of focal adhesion kinase) as its binding partner to retrace structural behavior. Our findings show that the paxillin interaction with FAT exhibits a masking and unmasking effect by a putative intra-molecular regulatory region. This phenomenon suggests how cancer associated mutations in paxillin affect its interactions with Focal Adhesion Kinase (FAK). The strategy could be used to decipher the mode of regulations and identify functionally relevant constructs for other studies.
Highlights
Genomic data suggests that a large proportion of eukaryotic proteins appear to adopt disordered structures in physiological conditions [1, 2]
Focal Adhesion Kinase (FAK) has to bind to both LD2 and LD4, failing which phosphorylation during signalling is reduced [8], which is observed in case of cancer [3], resulting in abnormal functioning of paxillin
Paxillin mutations associated with lung cancer were observed in the unstructured segments, the regulatory region of LD2 and masking region of LD4 [3]
Summary
Genomic data suggests that a large proportion of eukaryotic proteins appear to adopt disordered structures in physiological conditions [1, 2]. Mode of Action in Disordered Regions of Paxillin deciphering mode of action in IDPs has been challenging given that unstructured segments render poor chemical shift dispersions and electron density in major techniques like NMR and X-ray, respectively [4]. It took almost 10 years to decipher the mode of action of Sic, a disordered protein involved in inhibition of a cyclin-dependent kinase [5]. We present a simple high throughput (HTP) screening strategy (Fig 1a), doi:10.1371/journal.pone.0150153.g001
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