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Interleukin‐17 (IL‐17) is a family of pro‐inflammatory cytokines and has been involved in the pathogenesis of chronic inflammatory and autoimmune diseases. The IL‐17E, also known as IL‐25, is a distinct member of this family that binds to its unique receptor IL‐17Rb to induce the activation of nuclear factor kappa‐light‐chain enhancer of activated B cells. Here, we systematically examined the intermolecular recognition and association of IL‐25 with IL‐17Rb and demonstrated that the IL‐25 primarily adopts two discrete linear and cyclic epitopes to interact with IL‐17Rb. The two epitopes are separately located in the monomers 1 and 2 of IL‐25 homodimer and cover sequences 125DPRGNSELLYHN136 and 77ELDRDLNRLPQDLY90. They totally contribute 71.6% binding energy to the full‐length IL‐25. The linear epitope targets a site spanning over the extracellular fnIIID1 and fnIIID2 domains of IL‐17Rb, while the cyclic epitope primarily binds at the fnIIID1 domain. In addition, we also found that the linear and cyclic epitopes are natively folded into ordered single‐stranded and double‐stranded conformations in IL‐25 protein context, respectively, but would become largely disordered when splitting from the context to be free peptides, which, however, cannot bind effectively to IL‐17Rb as them in the native state. In this respect, we extended the cyclic epitope to cover the whole IL‐25 double‐stranded region and added a disulfide bridge across its two strands at three selected anchor residue pairs. It is revealed that the disulfide‐stapled peptides can be constrained into a native‐like conformation and thus exhibit an improved binding potency to IL‐17Rb as compared to their unstapled counterpart. [ABSTRACT FROM AUTHOR]

The cell growth, development, and regeneration of tissue and organ are associated with a large number of gene regulation events, which are mediated in part by transcription factors (TFs) binding to cis-regulatory elements involved in the genome. Predicting the binding affinity and inferring the binding specificity of TF-DNA interactions at the genomic level would be fundamentally helpful for our understanding of the molecular mechanism and biological implication underlying sequence-specific TF-DNA recognition. In this study, we report the development of a combination method to characterize the interaction behavior of a 11-mer oligonucleotide segment and its mutations with the Gcn4p protein, a homodimeric, basic leucine zipper TF, and to predict the binding affinity and specificity of potential Gcn4p binders in the genome-wide scale. In this procedure, a position-mutated energy matrix is created based on molecular modeling analysis of native and mutated Gcn4p-DNA complex structures to describe the position-independent interaction energy profile of Gcn4p with different nucleotide types at each position of the oligonucleotide, and the energy terms extracted from the matrix and their interactives are then correlated with experimentally measured affinities of 19 268 distinct oligonucleotides using statistical modeling methodology. Subsequently, the best one of built regression models is successfully applied to screen those of potential high-affinity Gcn4p binders from the complete genome. The findings arising from this study are briefly listed below: (i) The 11 positions of oligonucleotides are highly interactive and non-additive in contribution to Gcn4p-DNA binding affinity; (ii) Indirect conformational effects upon nucleotide mutations as well as associated subtle changes in interfacial atomic contacts, but not the direct nonbonded interactions, are primarily responsible for the sequence-specific recognition; (iii) The intrinsic synergistic effects among the sequence positions of oligonucleotides determine Gcn4p-DNA binding affinity and specificity; (iv) Linear regression models in conjunction with variable selection seem to perform fairly well in capturing the internal dependences hidden in the Gcn4p-DNA system, albeit ignoring nonlinear factors may lead the models to systematically underestimate and overestimate high- and low-affinity samples, respectively. [ABSTRACT FROM AUTHOR]