The role of helix stabilizing residues in GCN4 basic region folding and DNA binding

Abstract

Basic region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a coiled-coil leucine zipper dimerization domain and a highly charged basic region that directly contacts DNA. The basic region is largely unfolded in the absence of DNA, but adopts a helical conformation upon DNA binding. Although a coil → helix transition is entropically unfavorable, this conformational change positions the DNA-binding residues appropriately for sequence-specific interactions with DNA. The N-terminal residues of the GCN4 DNA-binding domain, DPAAL, make no DNA contacts and are not part of the conserved basic region, but are nonetheless important for DNA binding. Asp and Pro are often found at the N-termini of ␣-helices, and such N-capping motifs can stabilize ␣-helical structure. In the present study, we investigate whether these two residues serve to stabilize a helical conformation in the GCN4 basic region, lowering the energetic cost for DNA binding. Our results suggest that the presence of these residues contributes significantly to helical structure and to the DNA-binding ability of the basic region in the absence of the leucine zipper. Similar helix-capping motifs are found in approximately half of all bZip domains, and the implications of these findings for in vivo protein function are discussed.

Multifarious Roles of Intrinsic Disorder in Proteins Illustrate Its Broad Impact on Plant Biology

“…The basic regions can be completely structured (e.g., ATF4; Podust et al, 2001), contain a certain amount of helical secondary structure (e.g., HY5; Yoon et al, 2006), or be completely disordered (e.g., Opaque-2; Moreau et al, 2004). Experimental evidence has suggested that, in complex with DNA, the basic regions uniformly form a-helical conformations (Hollenbeck et al, 2002). A study of conformational ensembles for 15 different bZIPs proposed that the basic regions of the bZIPs have quantifiable preferences for a-helical conformations in their unbound monomeric forms and this helicity varies from one basic region to another despite significant sequence similarity of the bZIP domains.…”

N-Terminal Segments Modulate the α-Helical Propensities of the Intrinsically Disordered Basic Regions of bZIP Proteins

“…Conservation Consensus 10 20 The first column provides the name for each bZIP-bR, 10,18,22 and the numbers in parentheses represent the net charge per residue 23 calculated based on the expected protonation states of free amino acids at neutral pH. Residues 9-18 make up the DBM.…”

“…Furthermore, stopped-flow fluorescence experiments show that bZIP-bRs bind their cognate half-sites independently as monomers, 11,16,17 and available spectroscopic evidence suggests that, in complex with DNA, basic regions form α-helical conformations irrespective of the presence or the absence of the leucine zipper region. 18 It has been proposed that the high fraction of basic residues within basic regions destabilizes α-helical conformations in their unbound monomeric forms, thus biasing them towards heterogeneous randomcoil-like ensembles. 6,7,16,19 As a result, α-helicity within basic regions is believed to emerge as a consequence of DNA binding through an inducedfit mechanism 7,19,20 whereby disordered basic regions make sequence-specific contacts with cognate half-sites on the DNA and zipper up to form α-helices.…”

Lipid core peptide targeting the cathepsin D hemoglobinase ofSchistosoma mansonias a component of a schistosomiasis vaccine

“…The peptide alone (peptide 1), the peptide conjugated at both N-and C-termini to the yeast GCN4 coil promoting sequence 30 (peptide 2), and each of these two peptides linked to a lipid core to generate LCP-1 and LCP-2 respectively. CD analysis confirmed the helical nature of the two LCPs but not the corresponding unadjuvanted peptides, irrespective of the presence of GCN4 in LCP-2.…”