Recognition of a structural domain (RWDBD) in Gcn1 proteins that interacts with the RWD domain containing proteins

Abstract

The protein Gcn1 (General control non-derepressible 1) is found in virtually all eukaryotes, and is a key component of the general amino acid control signal transduction pathway. This pathway is best known for its importance for cells to sense and overcome amino acid starvation. Gcn1 directly binds to the RWD (RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases) domain of the protein kinase Gcn2, and this is essential for delivering the starvation signal to Gcn2. Gcn2, and thus the GAAC (General Amino Acid Control) pathway, then becomes activated enabling the cell to cope and overcome the starvation condition. Using sensitive homology detection and fold recognition methods a conserved structural domain in Gcn1, RWD Binding Domain (RWDBD), has been recognized that encompasses the region experimentally shown previously to be involved in Gcn2 binding. Further, the structural fold for this domain has been recognized as the ARM (Armadillo) domain, and residues likely to be involved in the binding of Gcn2 RWD domain have been identified within this structural domain. Thus, the current analysis provides a structural basis of Gcn1-Gcn2 association.ReviewersThis article was reviewed by Dr Oliviero Carugo and Dr Michael Gromiha.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-017-0184-3) contains supplementary material, which is available to authorized users.

Yeast as a Model to Understand Actin-Mediated Cellular Functions in Mammals—Illustrated with Four Actin Cytoskeleton Proteins

“…Fewer studies have addressed Gcn2 activation in mammals but they suggest that the mechanisms are highly conserved throughout the eukaryotic kingdom. For example, mammalian Gcn1 is required for mammalian Gcn2 activation [197] and the amino acid residue in yeast Gcn1 (Arg2259) that is crucial for direct Gcn1-Gcn2 interaction is also found in mammalian Gcn1 [201,206,220]. Overexpression of a fragment of yeast Gcn1p that is sufficient for Gcn2 binding impairs Gcn1-Gcn2 interaction and Gcn2 activation in yeast [206].…”

A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation

“…We have shown previously that in Gcn1, Arg-2259 is critical for Gcn2 binding [ 5 ]. To predict additional amino acids relevant for Gcn2 binding, we interrogated our computationally modelled structure of the RWDBD [ 20 ], to identify surface exposed amino acids that are in close proximity to Arg-2259. Since Arg-2259 is a charged amino acid, and is located within a patch of similarly charged amino acids [ 20 ], we reasoned that these amino acids may also be involved in Gcn2 binding.…”

“…Phe-2291 is buried inside the RWDBD, and appears to be involved in maintaining the RWDBD three-dimensional structure [ 20 ]. If that is the case, then substitution of Phe with a charged amino acid, Glu, should affect the proper folding of the RWDBD, rendering it unable to disrupt Gcn1-Gcn2 interaction.…”

“…The 2052–2428 Gcn1 fragment co-precipitates in vitro with the Gcn2 N-terminal 598 amino acids containing the RWD domain (a domain found in R ING finger-containing proteins, W D-repeat-containing proteins, and yeast D EAD (DEXD)-like helicases) [ 5 ]. Therefore, for simplicity this Gcn1 fragment was called the RWD binding domain (RWDBD) [ 20 ]. Though it shall be noted that the RWDBD does not necessarily constitute an autonomous structural domain.…”

“…The first helices of each repeat pack next to each other in parallel to form one surface-side of the protein, while the second helices of each repeat also pack next to each other in parallel to form the second layer of helices to form the other surface-side of the protein. According to our RWDBD structure model, Arg-2259 is surface exposed and situated at the end of one helix [ 5 , 20 ]. Arg-2259 is located amongst similarly charged amino acids, which are surrounded by an uncharged region, suggesting that the charged amino acids are contributing to Gcn2 binding.…”