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Pan1 is an intrinsically disordered protein with homotypic interactions.

ABSTRACT: The yeast scaffold protein Pan1 contains two EH domains at its N-terminus, a predicted coiled-coil central region, and a C-terminal proline-rich domain. Pan1 is also predicted to contain regions of intrinsic disorder, characteristic of proteins that have many binding partners. In vitro biochemical data suggest that Pan1 exists as a dimer, and we have identified amino acids 705 to 848 as critical for this homotypic interaction. Tryptophan fluorescence was used to further characterize Pan1 conformational states. Pan1 contains four endogenous tryptophans, each in a distinct region of the protein: Trp(312) and Trp(642) are each in an EH domain, Trp(957) is in the central region, and Trp(1280) is a critical residue in the Arp2/3 activation domain. To examine the local environment of each of these tryptophans, three of the four tryptophans were mutagenized to phenylalanine to create four proteins, each with only one tryptophan residue. When quenched with acrylamide, these single tryptophan mutants appeared to undergo collisional quenching exclusively and were moderately accessible to the acrylamide molecule. Quenching with iodide or cesium, however, revealed different Stern-Volmer constants due to unique electrostatic environments of the tryptophan residues. Time-resolved fluorescence anisotropy data confirmed structural and disorder predictions of Pan1. Further experimentation to fully develop a model of Pan1 conformational dynamics will assist in a deeper understanding of the mechanisms of endocytosis.

SUBMITTER: Pierce BD

PROVIDER: S-EPMC3800252 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Pan1 is an intrinsically disordered protein with homotypic interactions.

Pierce B D BD Toptygin Dmitri D Wendland Beverly B

Proteins 20130819 11

The yeast scaffold protein Pan1 contains two EH domains at its N-terminus, a predicted coiled-coil central region, and a C-terminal proline-rich domain. Pan1 is also predicted to contain regions of intrinsic disorder, characteristic of proteins that have many binding partners. In vitro biochemical data suggest that Pan1 exists as a dimer, and we have identified amino acids 705 to 848 as critical for this homotypic interaction. Tryptophan fluorescence was used to further characterize Pan1 conform ...[more]

PMID: 23801378

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Project description:CK2, a serine/threonine (Ser/Thr) kinase present in eukaryotic cells, is known to have a vast number of substrates. We have recently shown that it localizes to nuclei and at pores between hyphal compartments in Magnaporthe oryzae We performed a pulldown proteomics analysis of M. oryzae CK2 catalytic subunit MoCKa to detect interacting proteins. The MoCKa pulldown was enriched for septum and nucleolus proteins and intrinsically disordered proteins (IDPs) containing a CK2 phosphorylation motif that is proposed to destabilize and unfold α-helices. This points to a function for CK2 phosphorylation and corresponding phosphatase dephosphorylation in the formation of functional protein-protein aggregates and protein-RNA/DNA binding. To test this as widely as possible, we used secondary data downloaded from databases from a large range of M. oryzae experiments, as well as data for a relatively closely related plant-pathogenic fungus, Fusarium graminearum We found that CKa expression was strongly positively correlated with Ser/Thr phosphatases, as well as with disaggregases (HSP104, YDJ1, and SSA1) and an autophagy-indicating protein (ATG8). The latter points to increased protein aggregate formation at high levels of CKa expression. Our results suggest a general role for CK2 in chaperoning aggregation and disaggregation of IDPs and their binding to proteins, DNA, and RNA.IMPORTANCE CK2 is a eukaryotic conserved kinase enzyme complex that phosphorylates proteins. CK2 is known to phosphorylate a large number of proteins and is constitutively active, and thus a "normal" role for a kinase in a signaling cascade might not be the case for CK2. Previous results on localization and indications from the literature point to a function for CK2 phosphorylation in shaping and folding of proteins, especially intrinsically disordered proteins, which constitute about 30% of eukaryotic proteins. We used pulldown of interacting proteins and data downloaded from a large range of transcriptomic experiments in M. oryzae and complemented these with data downloaded from a large range of transcriptomic experiments in Fusarium graminearum We found support for a general role for CK2 in aggregation and disaggregation of IDPs and their binding to proteins, DNA, and RNA-interactions that could explain the importance of CK2 in eukaryotic cell function and disease.

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Pan1 is an intrinsically disordered protein with homotypic interactions.

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Pan1 is an intrinsically disordered protein with homotypic interactions.

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