Mapping the recognition pathway of cyclobutane pyrimidine dimer in DNA by Rad4/XPC.

UV radiation-induced DNA damages have adverse effects on genome integrity and cellular function. The most prevalent UV-induced DNA lesion is the cyclobutane pyrimidine dimer (CPD), which can cause skin disorders and cancers in humans. Rad4/XPC is a damage sensing protein that recognizes and repairs CPD lesions with high fidelity. However, the molecular mechanism of how Rad4/XPC interrogates CPD lesions remains elusive. Emerging viewpoints indicate that the association of Rad4/XPC with DNA, the insertion of a lesion-sensing ß-hairpin of Rad4/XPC into the lesion site and the flipping of CPD's partner bases (5'-dA and 3'-dA) are essential for damage recognition. Characterizing these slow events is challenging due to their infrequent occurrence on molecular time scales. Herein, we have used enhanced sampling and molecular dynamics simulations to investigate the mechanism and energetics of lesion recognition by Rad4/XPC, considering multiple plausible pathways between the crystal structure of the Rad4-DNA complex and nine intermediate states. Our results shed light on the most likely sequence of events, their potential coupling and energetics. Upon association, Rad4 and DNA form an encounter complex in which CPD and its partner bases remain in the duplex and the BHD3 ß-hairpin is yet to be inserted into the lesion site. Subsequently, sequential base flipping occurs, with the flipping of the 5'-dA base preceding that of the 3'-dA base, followed by the insertion of the BHD3 ß-hairpin into the lesion site. The results presented here have significant implications for understanding the molecular basis of UV-related skin disorders and cancers and for paving the way for novel therapeutic strategies.

Response of Terahertz Protein Vibrations to Ligand Binding: Calmodulin-Peptide Complexes as a Case Study.

Terahertz vibrations are sensitive reporters of the structure and interactions of proteins. Ligand binding alters the nature and distribution of these collective vibrations. The ligand-induced changes in the terahertz protein vibrations contribute to the binding entropy and to the overall thermodynamic stability of the resultant protein-ligand complexes. Here, we have examined the response of the low-frequency (below 6 terahertz) collective vibrations of the calcium-loaded calmodulin (CaM) to binding to five different ligands, both in the presence and absence of water, using normal-mode analysis and molecular dynamics simulations. A comparison of the vibrational spectra of hydrated and dry systems reveals that protein-solvent interactions stiffen the terahertz protein vibrations and that these solvent-coupled collective vibrations contribute significantly to the hydration-sensitive variation in the vibrational entropy of CaM. In the absence of water, the low-frequency vibrations of CaM are stiffened by ligand binding. On the contrary, the number and the cumulative vibrational entropy of low-frequency vibrational modes (ω < 200 cm-1) of the hydrated CaM are increased noticeably after binding to the peptides, indicating binding-induced softening of collective vibrations of the protein. Although the calculated and experimental binding affinities of the chosen complexes correlated reasonably well, no systematic correlation was observed between the protein vibrational entropy and the binding affinity. The results underscored the importance of the interplay of protein-ligand and solvent interactions in modulating the low-frequency vibrations of proteins.

Allosteric Response of DNA Recognition Helices of Catabolite Activator Protein to cAMP and DNA Binding.

The homodimeric catabolite activator protein (CAP) regulates the transcription of several bacterial genes based on the cellular concentration of cyclic adenosine monophosphate (cAMP). The binding of cAMP to CAP triggers allosteric communication between the cAMP binding domains (CBD) and DNA binding domains (DBD) of CAP, which entails repositioning of DNA recognition helices (F-helices) in the DBD to dock favorably to the target DNA. Despite considerable progress, much remains to be understood about the mechanistic details of DNA recognition by CAP and about the map of allosteric pathways involved in CAP-mediated gene transcription. The present study uses molecular dynamics and umbrella sampling simulations to investigate the mechanism of cAMP- and DNA-induced changes in the conformation and energetics of F-helices observed during the allosteric regulation of CAP by cAMP and the subsequent binding to the DNA promoter region. Using novel collective variables, the free energy profiles associated with the orientation and dynamics of F-helices in the unliganded, cAMP-bound, and cAMP-DNA-bound states of CAP are calculated and compared. The binding-induced alterations in the resultant free energy profiles reveal important flexibility constraints imposed on DBD upon cAMP and DNA binding. A comprehensive analysis of residue-wise interaction maps reveals potential allosteric pathways between CBD and DBD that facilitate the allosteric transduction of regulatory signals in CAP. The revelation that the predicted allosteric pathways crisscross the intersubunit interface offers important clues on the microscopic origin of the intersubunit cooperativity and dimer stability of CAP.