Structural features of FAP174, a MYCBP-1 orthologue from: Chlamydomonas reinhardtii, revealed by computational and experimental analyses

M. Yogesha, Venkatramanan G. Rao, Elvis A.F. Martis, Evans C. Coutinho, Holger Gohlke, Santhosh Chidangil, Prabhakar M. Dongre, Jacinta S. D'Souza

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Abstract

The dimers of regulatory sub-units of protein kinase A (RIα, RIIα, RIβ, and RIIβ isoforms) and DPY-30 interact with their respective A-Kinase Anchoring Proteins (AKAPs) via their Dimerization and Docking domains (D/D). The mammalian MYCBP-1 protein, which stimulates the E-box-dependent transcriptional activity of the c-Myc protein, also interacts with AKAPs present in various sub-cellular locales. We have earlier shown that the Flagellar Associated Protein 174 (FAP174) from Chlamydomonas reinhardtii is an orthologue of human MYCBP-1 and binds to AKAP240. Here, we set out to identify the D/D domain of FAP174 and the amino acid residues involved in oligomerization. By means of electrophoresis of recombinant proteins with an increasing concentration of a reducing agent (DTT), MALDI-TOF, FT-IR, and micro-Raman spectroscopy, we show that the D/D domain resides in and around the C46 residue. Using in silico methods, we infer that the neighboring amino acids of C46, viz. A39, L40, Y42, and L47, might also be involved in dimerization. The secondary structure of FAP174 was investigated using computer-assisted prediction, CD, micro-Raman spectroscopy, and FT-IR. Accordingly, FAP174 is made of four α-helices. Helix B contains two VLV stretches at positions 21-23 and 25-27. Mutating these VLV stretches to alanines followed by native gel electrophoresis of the respective recombinant proteins indicated their role in oligomerization. The results imply that FAP174, unlike most of the other AKAP-interacting proteins, harbors two different domains, one for dimerization and the other for oligomerization.

Original languageEnglish
Pages (from-to)51391-51402
Number of pages12
JournalRSC Advances
Volume7
Issue number81
DOIs
Publication statusPublished - 01-01-2017

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All Science Journal Classification (ASJC) codes

  • Chemistry(all)
  • Chemical Engineering(all)

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