Abstract

The pyrimidine-pyrimidone (6-4) photoproduct (6-4PP) is a UV-induced DNA lesion implicated in skin disorders and cancers. The repair protein XPC/RAD4 detects this lesion and initiates nucleotide excision repair to safeguard genomic integrity. While the X-ray crystallographic structure of the 6–4PP containing DNA-RAD4/XPC complex reveals DNA distortion and extrusion of the lesion and partner bases, the precise mechanism by which RAD4/XPC initiates lesion repair remains unclear. To investigate this, we employed molecular dynamics simulations, umbrella sampling, and the nudged elastic band method to map the minimum energy path (MEP) from RAD4’s initial encounter with damaged DNA to its bound state. Our results reveal that the initial interrogation phase involves partial opening of the DNA, marked by the partial extrusion of the lesion while its partner bases largely remain intrahelical, accompanied by significant DNA unwinding near the damage site. This partially opened state represents the rate-limiting distortion, with 5′ base flipping as the bottleneck for downstream events leading to the bound state. Upon overcoming the bottleneck, the DNA adopts a final untwisted conformation, with the lesion flipping out first, followed by the complete sequential flipping of the 5′ base and then the 3′ partner bases, while full β-hairpin insertion ultimately stabilizes the final bound DNA complex. The energetics and structural intermediates along the MEP provide key insights into conformational changes that drive lesion recognition, extrusion, and stable binding, advancing our understanding of nucleotide excision repair.

Abstract

The histone methyltransferase ASH1L, first discovered for its role in transcription, has been shown to accelerate the removal of ultraviolet (UV) light-induced cyclobutane pyrimidine dimers (CPDs) by nucleotide excision repair. Previous reports demonstrated that CPD excision is most efficient at transcriptional regulatory elements, including enhancers, relative to other genomic sites. Therefore, we analyzed DNA damage maps in ASH1L-proficient and ASH1L-deficient cells to understand how ASH1L controls enhancer stability. This comparison showed that ASH1L protects enhancer sequences against the induction of CPDs besides stimulating repair activity. ASH1L reduces CPD formation at C–containing but not at TT dinucleotides, and no protection occurs against pyrimidine-(6,4)-pyrimidone photoproducts or cisplatin crosslinks. The diminished CPD induction extends to gene promoters but excludes retrotransposons. This guardian role against CPDs in regulatory elements is associated with the presence of H3K4me3 and H3K27ac histone marks, which are known to interact with the PHD and BRD motifs of ASH1L, respectively. Molecular dynamics simulations identified a DNA-binding AT hook of ASH1L that alters the distance and dihedral angle between neighboring C nucleotides to disfavor dimerization. The loss of this protection results in a higher frequency of C–>T transitions at enhancers of skin cancers carrying ASH1L mutations compared to ASH1L-intact counterparts.

Abstract

Criticality, observed during second-order phase transitions, is an emergent phenomenon. The brain operates near criticality where complex systems exhibit high correlations. As a system approaches criticality, it develops “domain”-like regions with competing phases and increased spatio-temporal correlations that diverge. The dynamics of these domains depend on the system’s proximity to criticality. This study explores the differences in the proximity to criticality of Alzheimer’s-afflicted and cognitively normal brains through the use of a spin-lattice model derived from resting-state fMRI data and investigates the type of criticality found in the human brain - whether it is of the Ising class or something more complex. The autocorrelation relaxation time in both groups displays a stretched exponential nature, indicating closer alignment with the criticality of the spin-glass class rather than the Ising class. Longer relaxation times observed in cognitively normal subjects suggest increased proximity to the phase boundary. The weak distinction observed in the spatial characteristics related to proximity to criticality might once more point to a spin-glass scenario, necessitating nuanced order parameters to distinguish between phase-ordering in Alzheimer’s and cognitively normal brains.

Abstract

Criticality, observed during second-order phase transitions, is an emergent phenomenon. The brain operates near criticality, where complex systems exhibit high correlations. The critical brain hypothesis suggests that the brain becomes an efficient learning system in this state but poor in memory, while sub-criticality enhances memory but inhibits learning. As a system approaches criticality, it develops ”domain”-like regions with competing phases and increased spatiotemporal correlations that diverge. The dynamics of these domains depend on the system’s proximity to criticality. This study investigates the phase ordering properties of a spin-lattice model derived from Alzheimer’s and cognitively normal subjects, expecting significant differences in their proximity to criticality. However, our findings show no conclusive distinction in the distal properties from criticality, as reflected in the phase ordering behavior of the Alzheimer’s and cognitively normal brain.

Abstract

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 β-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 β- 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 β-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. INTRODUCTION DNA plays a central role in the

Abstract

G-protein coupled receptors (GPCRs) are the most prominent family of membrane proteins that serve as major targets for one-third of the drugs produced. A detailed understanding of the molecular mechanism of drug-induced activation and inhibition of GPCRs is crucial for the rational design of novel therapeutics. The binding of the neurotransmitter adrenaline to the β2-adrenergic receptor (β2AR) is known to induce a flight or fight cellular response, but much remains to be understood about binding-induced dynamical changes in β2AR and adrenaline. In this article, we examine the potential of mean force (PMF) for the unbinding of adrenaline from the orthosteric binding site of β2AR and the associated dynamics using umbrella sampling and molecular dynamics (MD) simulations. The calculated PMF reveals a global energy minimum, which corresponds to the crystal structure of β2AR−adrenaline complex, and a meta-stable state in which the adrenaline is moved slightly deeper into the binding pocket with a different orientation compared to that in the crystal structure. The orientational and conformational changes in adrenaline during the transition between these two states and the underlying driving forces of this transition are also explored. Based on the clustering of MD configurations and machine learning-based statistical analyses of time series of relevant collective variables, the structures and stabilizing interactions of these two states of the β2AR−adrenaline complex are also investigated.

Abstract

GPCRs are the most prominent family of membrane proteins that serve as major targets for one-third of the drugs produced. A detailed understanding of the molecular mechanism of drug-induced activation and inhibition of GPCRs is crucial for the rational design of novel therapeutics. The binding of the neurotransmitter adrenaline to the β2-adrenergic receptor (β2AR) is known to induce a flight or fight cellular response, but much remains to be understood about binding-induced dynamical changes in β2AR and adrenaline. In this article, we examine the potential of mean force (PMF) for the unbinding of adrenaline from the orthosteric binding site of β2AR and the associated dynamics using umbrella sampling and molecular dynamics (MD) simulations. The calculated PMF reveals a global energy minimum, which corresponds to the crystal structure of β2AR-adrenaline complex, and a metastable state in which the adrenaline is moved slightly deeper into the binding pocket with a different orientation compared to that in the crystal structure. The orientational and conformational changes in adrenaline during the transition between these two states and the underlying driving forces of this transition are also explored. Based on clustering of MD configurations and machine learning-based statistical analyses of time series of relevant collective variables, the structures and stabilizing interactions of these two states of the β2AR-adrenaline complex are also investigated.

Abstract

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

Abstract

Bacterial cellulase enzymes are potent candidates for the efficient production of bioethanol, a promising alternative to fossil fuels, from cellulosic biomass. These enzymes catalyze the breakdown of cellulose in plant biomass into simple sugars and then to bioethanol. In the absence of the enzyme, the cellulosic biomass is recalcitrant to decomposition due to fermentationresistant lignin and pectin coatings on the cellulose surface, which make them inaccessible for hydrolysis. Cellobiohydrolase CelS is a microbial enzyme that binds to cellulose fiber and efficiently cleaves it into a simple sugar (cellobiose) by a repeated processive chopping mechanism. The two contributing factors to the catalytic reaction rate and the yield of cellobiose are the efficient product expulsion from the product binding site of CelS and the movement of the substrate or cellulose chain into the active site. Despite progress in understanding product expulsion in other cellulases, much remains to be understood about the molecular mechanism of processive action of these enzymes. Here, nonequilibrium molecular dynamics simulations using suitable reaction coordinates are carried out to investigate the energetics and mechanism of the substrate dynamics and product expulsion in CelS. The calculated free energy barrier for the product expulsion is three times lower than that for the processive action indicating that product removal is relatively easier and faster than the sliding of the substrate to the catalytic active site. The water traffic near the active site in response to the product expulsion and the processive action is also explored.

Abstract

The configurational sampling is central to characterize the equilibrium properties of complex molecular systems, but it remains a significant computational challenge. The conventional molecular dynamics simulations of limited duration often result in inadequate sampling and thus inaccurate equilibrium estimates. Replica exchange with non-equilibrium switches (RENS) is a collective variable-free computational technique to achieve extensive sampling from a sequence of equilibrium and non-equilibrium MD simulations without modifying the underlying potential energy surface of the system. Unlike the conventional replica exchange molecular dynamics (REMD) simulation, which demands a significant number of replicas for better accuracy, RENS employs non-equilibrium heating (forward) and cooling (reverse) work simulations prior to configurational swaps to improve the acceptance probability for replica exchange. Here, we have implemented the RENS algorithm on four model systems and examined its performance against the conventional MD and REMD simulations. The desired equilibrium distributions were generated by RENS for all the model systems, whereas REMD and MD simulations could not do so due to inadequate sampling on the same timescales. The calculated work distributions from RENS obeyed the expected non-equilibrium fluctuation theorem. The results indicate that the switching time of the non-equilibrium simulations can be systematically altered to optimize the acceptance probability and the reduced work of switching. The modular implementation of RENS algorithm not only enables us to readily extend it to multiple replicas, but also paves the way for extension to larger molecular systems in the future