Enzymes effectively catalyze chemical reactions at their active sites. The reactions involve three microscopic events at the active sites: substrate binding, multi-step chemical reactions, and product release. These even...Enzymes effectively catalyze chemical reactions at their active sites. The reactions involve three microscopic events at the active sites: substrate binding, multi-step chemical reactions, and product release. These events are often coupled with enzyme conformational changes, making theoretical and computational analyses more challenging. Advanced molecular simulations, involving molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM), are now utilized to investigate the functions of enzymes such as tryptophan synthase and P-type ATPases. Here, we summarize recent multiscale molecular simulations that incorporate multiple microscopic events in enzyme functions. The coupling of enzyme conformational changes and chemical reactions can predict a proper direction in enzymatic reaction cycles, which requires accurate predictions of the free energy changes between different physiological states. Using machine learning (ML) methods, all the microscopic events in enzyme catalysis could be described with the same accuracy as quantum chemistry. We also discuss recent developments in ML/MM simulations for enzyme catalysis.
Proteolysis-targeting chimeras (PROTACs) achieve irreversible clearance of target proteins by hijacking the ubiquitin-proteasome system, breaking the design paradigm of traditional inhibitory drugs. The development of co...Proteolysis-targeting chimeras (PROTACs) achieve irreversible clearance of target proteins by hijacking the ubiquitin-proteasome system, breaking the design paradigm of traditional inhibitory drugs. The development of computational approaches has effectively promoted the rational design of PROTACs, yet existing methods mainly focus on predicting the static structure of PROTAC systems, with methodological gaps in analyzing their dynamic characteristics. Knowing that the dynamic behaviors can dramatically influence the stability and degradation efficacy of a PROTAC system, we systematically summarize the recent progresses of using structure-based and structure-artificial intelligence-hybrid methodologies for characterizing the dynamic behaviors of PROTAC systems, with a focus on elucidating the dynamic characteristics of target protein-PROTAC-E3 ligase ternary structures and prediction of their key properties.
Protein-ligand modeling is a cornerstone of modern drug discovery, facilitating the identification and optimization of bioactive compounds that modulate protein function. Computational approaches provide cost-effective a...Protein-ligand modeling is a cornerstone of modern drug discovery, facilitating the identification and optimization of bioactive compounds that modulate protein function. Computational approaches provide cost-effective and scalable strategies for exploring the growing chemical and biological spaces, accelerating early-stage drug development. Advances in both physics-based methods and data-driven machine learning techniques have expanded the range and accuracy of tools available for modeling protein-ligand interactions. This review provides a current and concise view of key methodologies in protein-ligand modeling, including binding site prediction and the generation and evaluation of target-bound ligand conformations. It also discusses state-of-the-art machine learning approaches that are reshaping how these tasks are performed and enhancing the accuracy of binding site, binding pose, and binding affinity predictions.
Our previously proposed Ras dimerization model is consistent with recent details observed by NMR in that Raf activation is centered on the Ras/Raf dimer, distinct from one in which Ras activates Raf as a monomer with the...Our previously proposed Ras dimerization model is consistent with recent details observed by NMR in that Raf activation is centered on the Ras/Raf dimer, distinct from one in which Ras activates Raf as a monomer with the Raf cysteine rich domain inserted in the membrane. We review mechanistic understanding of Raf activation within nanoclusters of Ras on the membrane, with a shift to dimers upon binding Raf. This sets the stage for a signaling platform composed of Ras/Raf and Galectin dimers that facilitates the release of Raf autoinhibition and folding of the Raf intrinsically disordered region between the Ras-binding domains and the kinase bound to 14-3-3 and MEK. This platform could provide synchronized units for signal amplification and is consistent with a Ras stationary phase observed in cells.
Curr Opin Struct Biol
· 2025 Oct · PMID 40884937
·
Full text
Drug residence time defines the duration the drug is bound to its protein target. It is a crucial determinant of drug action. Yet, a priori estimating it in the design could be the most challenging. The mechanisms of all...Drug residence time defines the duration the drug is bound to its protein target. It is a crucial determinant of drug action. Yet, a priori estimating it in the design could be the most challenging. The mechanisms of allosteric and orthosteric drugs differ in how they affect it. Binding at the active site, the residence time of orthosteric drugs is primarily affected by binding kinetics, which is not the case for allosteric drugs. Allosteric drugs determine the orthosteric drug residence time by the nature and extent of the population shift that they promote, which modulate the active site conformation. However, cooperative binding is bidirectional; orthosteric drug binding at the active site can increase (decrease) residence time at the allosteric site.
Curr Opin Struct Biol
· 2025 Oct · PMID 40850108
·
Full text
This perspective highlights recent applications and technological progress in dipolar electron paramagnetic resonance (EPR) spectroscopy, including double electron-electron resonance (DEER) spectroscopy. These methods pr...This perspective highlights recent applications and technological progress in dipolar electron paramagnetic resonance (EPR) spectroscopy, including double electron-electron resonance (DEER) spectroscopy. These methods provide nanoscale distance distributions between site-specific spin labels in biomacromolecules. The resulting data are particularly well suited for quantifying the structure and energetics of conformational ensembles of multi-state and flexible proteins. Recent applications span a wide range of systems and are accompanied by innovations in spin labeling, deuteration, in-cell measurements, integrative multi-technique approaches, and novel computational modeling methods combined with structure prediction tools.
Cryo-focused ion beam instruments to produce cellular thin sections for subsequent imaging by cryo-electron tomography have become an integral part of the methodologies for in situ structural biology, enabling high-resol...Cryo-focused ion beam instruments to produce cellular thin sections for subsequent imaging by cryo-electron tomography have become an integral part of the methodologies for in situ structural biology, enabling high-resolution imaging of biological structures in their native environment. The application of these instruments has opened windows into cells that allowed unprecedented insights into the ultrastructure of cells and more recently, small multicellular organisms and tissues. While great strides have been made in the characterization of cryo-FIB milling and the streamlining of workflows with these tools, many limitations and technical challenges remain to be resolved. Here, we discuss the technical and technological challenges ahead to continue the steep rise of biological discoveries by in-cell cryo-electron tomography to enable cellular structural biology in the multicellular context.
Curr Opin Struct Biol
· 2025 Oct · PMID 40840016
·
Full text
In mammalian cells, RNA species make up ∼10% of chromatin by mass and play a structural role in the nucleus by acting as scaffolds and influencing genome organisation. Although many proteins bind nuclear RNAs, these inte...In mammalian cells, RNA species make up ∼10% of chromatin by mass and play a structural role in the nucleus by acting as scaffolds and influencing genome organisation. Although many proteins bind nuclear RNAs, these interactions are often non-specific, making it challenging to define RNA's role in genome folding. Nonetheless, a clearer picture is emerging. Some RNAs, like NEAT1 and MALAT1, have high affinity for specific RNA-binding proteins and form the basis for nuclear bodies. In contrast, many nuclear proteins bind RNA weakly, resulting in numerous low-affinity interactions. We propose that these interactions generate a complex RNA-protein network with dynamic, gel-like properties that modulate chromatin folding and transcription factor mobility. This suggests an exciting feedback mechanism in which newly transcribed RNA contributes directly to shaping chromatin architecture.
RNA and RNA-protein (RNP) complexes are central to many cellular processes, but the determination of their structures remains challenging due to RNA flexibility and interaction diversity. This review highlights recent co...RNA and RNA-protein (RNP) complexes are central to many cellular processes, but the determination of their structures remains challenging due to RNA flexibility and interaction diversity. This review highlights recent computational advances, particularly from the past two years, in predicting and analyzing RNA and RNP structures. We discuss template-based modeling, docking, molecular simulations, and deep learning approaches, with an emphasis on emerging hybrid methods that integrate these strategies. Special attention is given to tools for modeling conformational heterogeneity, folding pathways, and dynamic binding. We also outline machine learning and simulation techniques for ensemble prediction and explore future directions including quantum-enhanced modeling. Together, these developments are enabling more accurate and scalable modeling of both the static and dynamic aspects of RNA and RNP complexes.
The intrinsic conformational flexibility of proteins creates structural heterogeneity, giving rise to conformational ensembles within the energy landscape. When conformational ensembles harbor distinct functional sub-sta...The intrinsic conformational flexibility of proteins creates structural heterogeneity, giving rise to conformational ensembles within the energy landscape. When conformational ensembles harbor distinct functional sub-states, mutations can reshape the conformational landscape, thereby altering the distribution of functional sub-states and driving the evolution of novel functions. In this review, we provide a conceptual framework that elucidates the importance of functional sub-states and how evolution can select them. We highlight key studies that have uncovered functional sub-states and discuss recent insights into the transitions of functional sub-states during evolutionary trajectories. Finally, we outline critical techniques for studying functional sub-states, address the challenges faced in analyzing these sub-states, and explore future advancements in the field of protein evolution.
Curr Opin Struct Biol
· 2025 Oct · PMID 40815918
·
Full text
RNA aptamers possess a remarkable ability to selectively target a diverse spectrum of biomolecules with exceptional affinity and specificity. Their distinctive physical and chemical attributes have driven extensive resea...RNA aptamers possess a remarkable ability to selectively target a diverse spectrum of biomolecules with exceptional affinity and specificity. Their distinctive physical and chemical attributes have driven extensive research into their therapeutic, diagnostic, and analytical applications. However, experimental approaches alone are insufficient to meet the growing demand. As a result, accurate and efficient computational methods are playing an increasingly vital role in RNA aptamer sequence design and structural modeling. Recent breakthroughs in biomolecular structure prediction, particularly through deep learning, have further spurred the development of innovative algorithms. In this review, we summarize current computational models for RNA aptamer structure prediction and design, highlighting recent advances in the field.
The advent of AlphaFold and consumer large language models have elicited unprecedented development of artificial intelligence (AI). AI has had substantial impact in every area of research, including in molecular biology....The advent of AlphaFold and consumer large language models have elicited unprecedented development of artificial intelligence (AI). AI has had substantial impact in every area of research, including in molecular biology. This is principally in thanks to contributions to the Protein Data Bank and various genome sequence databases, providing an astronomical amount of data for model training. These databases contain evolutionary information explicitly and implicitly, allowing accurate predictions and deep insights into biological questions. Here, we describe recent state-of-the-art applications of AI that exploit evolutionary relationships. This includes structure prediction and design, conformational ensemble generation, and functional site identification. We present a brief snapshot of AI usage in studying protein structure and dynamics, a field that is advancing at breakneck speed.
Enzymes are inherently dynamic entities, with their functions intricately governed by the interplay between conformational dynamics - ranging from local residue fluctuations to global motions - and biochemical activity....Enzymes are inherently dynamic entities, with their functions intricately governed by the interplay between conformational dynamics - ranging from local residue fluctuations to global motions - and biochemical activity. Deciphering how such dynamics coordinate higher-order cooperativity across multiple timescales to drive catalysis remains a fundamental challenge. This mini-review highlights the role of large-scale, collective motions involving domain-level displacements and hinge-based rearrangements, which not only facilitate substrate recognition, transformation, and release, but also emerge from and propagate through multidirectional allosteric interactions. Such dynamic mechanochemical coupling reflects evolutionary memory and provides a blueprint for enzyme design innovations.
Cryo-electron microscopy (cryo-EM) has emerged as a transformative tool in structural biology, enabling high-resolution visualization of macromolecules in their native states. Cryo-focused ion beam milling (cryo-FIB) and...Cryo-electron microscopy (cryo-EM) has emerged as a transformative tool in structural biology, enabling high-resolution visualization of macromolecules in their native states. Cryo-focused ion beam milling (cryo-FIB) and other advances in sample preparation have expanded the range of biological samples that can be studied with cryo-EM to include cells and tissues. While the dream of high-resolution structural analysis of proteins within their native, cellular context is now being realized, sample preparation, especially from tissues, is still labor-intensive and technically challenging. Here we review the latest innovations in cryo-EM sample preparation, including support fabrication and functionalization, cell micropatterning, and techniques for thinning frozen biological samples. Beyond streamlining and improving the repeatability of sample preparation, these advances are expanding the impact of cryo-EM by enabling unprecedented visualization of structures within cells and tissues in healthy and diseased states, as well as structural analysis of biological processes at well-controlled time points.
Curr Opin Struct Biol
· 2025 Oct · PMID 40782689
·
Full text
Membrane proteins have evolved to function as part of specialized biological membranes, and their structures and activities are highly susceptible to their local environment. Detergents and lipid mimetics replicate certa...Membrane proteins have evolved to function as part of specialized biological membranes, and their structures and activities are highly susceptible to their local environment. Detergents and lipid mimetics replicate certain aspects of biological membranes, and have been used to produce an exceptional body of structural data, but do not fully capture the complex, asymmetric properties of the native environment and can alter structure and function. Here, we review recent advances in nuclear magnetic resonance (NMR) that enable the examination of membrane protein structure and activity in situ, within native membranes. The development of optimized protein expression strategies, isotopic labeling schemes, powerful instrumentation and specialized pulse sequences offer new opportunities for exploring the new frontier of in situ structural biology. By outlining the framework for in situ NMR of membrane proteins from conceptualization to experiments we hope to inspire new research in this growing and important area.
Curr Opin Struct Biol
· 2025 Oct · PMID 40774150
·
Full text
The native cell membrane nanoparticles (NCMN) system utilizes membrane-active polymers specifically designed and optimized to extract and stabilize membrane proteins in the form of NCMN particlesfor biochemical and bioph...The native cell membrane nanoparticles (NCMN) system utilizes membrane-active polymers specifically designed and optimized to extract and stabilize membrane proteins in the form of NCMN particlesfor biochemical and biophysical characterization. The NCMN system is a genuine and advanced detergent-free approach inspired by the membrane activity of the styrene-maleic acid copolymers (SMA), distinguishing it from the nanodisc technology, Salipro technology, and Peptidisc technology. This review introduces the current advancements in the NCMN system, including the development of NCMN polymers, the application of the NCMN system for single-particle cryo-EM analysis, and the functional characterization of membrane proteins.
Curr Opin Struct Biol
· 2025 Oct · PMID 40769055
·
Full text
Optogenetically regulated enzymes offer unprecedented spatiotemporal control over protein activity, intermolecular interactions, and intracellular signaling. Many design strategies have been developed for their fabricati...Optogenetically regulated enzymes offer unprecedented spatiotemporal control over protein activity, intermolecular interactions, and intracellular signaling. Many design strategies have been developed for their fabrication based on the principles of intrinsic allostery, oligomerization or 'split' status, intracellular compartmentalization, and steric hindrance. In addition to employing photosensory domains as part of the traditional optogenetic toolset, the specificity of effector domains has also been leveraged for endogenous applications. Here, we discuss the dynamics of light activation while providing a bird's eye view of the crafting approaches, targets, and impact of optogenetic enzymes in orchestrating cellular functions, as well as the bottlenecks and an outlook into the future.
The stabilization of HIV-1 gp160 trimers (Env) within phospholipid bilayer nanodiscs has provided critical structural insights into the membrane-proximal external region (MPER) and the broader dynamics of gp160. Cryo-EM...The stabilization of HIV-1 gp160 trimers (Env) within phospholipid bilayer nanodiscs has provided critical structural insights into the membrane-proximal external region (MPER) and the broader dynamics of gp160. Cryo-EM and molecular simulations reveal that the membrane context preserves the MPER architecture and captures spontaneous trimer asymmetry, as well as ectodomain tilting. These dynamic properties expose vulnerable epitopes that are targeted by broadly neutralizing antibodies (bnAbs). Studies using nanodiscs have highlighted how interactions with the membrane affect the structure of gp160, the accessibility of epitopes, and the mechanisms of neutralization, providing important insights for immunogen design. Unlike soluble SOSIP and IDL constructs, full-length nanodisc-embedded gp160 maintains its native stability, flexibility, and the complete set of neutralization epitopes, suggesting that membrane-mimicking platforms are essential for the rational design of next-generation HIV vaccines targeting conserved regions, such as the MPER.
The controlled dissipation of chemical potentials is the fundamental way cells make a living. Enzyme-mediated catalysis allows the various transformations to proceed at biologically relevant rates with remarkable precisi...The controlled dissipation of chemical potentials is the fundamental way cells make a living. Enzyme-mediated catalysis allows the various transformations to proceed at biologically relevant rates with remarkable precision and efficiency. Theory, experiments, and computational studies coincide to show that local frustration is a useful concept to relate protein dynamics with catalytic power. Local frustration gives rise to the asperities of the energy landscapes that can harness the thermal fluctuations to guide the functional protein motions. We review here recent advances into these relationships from various fields of protein science. The biologically relevant dynamics is tuned by the evolution of protein sequences that modulate local frustration patterns to near-optimal values.