It is now widely accepted that allosteric signaling is beyond signal transmission to, or conformational change triggered at, a distal point within a structure; it also affects different cellular pathways and functions de...It is now widely accepted that allosteric signaling is beyond signal transmission to, or conformational change triggered at, a distal point within a structure; it also affects different cellular pathways and functions depending on the specific allosteric modulators. A family of signaling molecules that has attracted wide attention in recent years is the Class B1 G protein-coupled receptors (GPCRs). In the classic view of GPCR signaling, cyclic adenosine monophosphate (cAMP) production is accepted to be uniquely associated with signaling events at the plasma membrane. However, a growing number of studies over the past decade, especially for the parathyroid hormone type 1 receptor (PTHR), demonstrate that cAMP is also produced at the endosomes through a different pathway after receptor internalization. Recent advances in the structural and computational characterization of this family of allosteric receptors provide new insights into the mechanisms of their activation or inhibition, as well as determinants of differential allosteric signaling. We focus on PTHR as a prototypical member of the family and present our current understanding of the role of selected ligands in acting as positive or negative allosteric modulators and eliciting signaling location biases in the cell.
Though allostery is a well-established research field, its importance for biomedical applications, spanning from the allosteric effects of mutations in diagnostics to the design of innovative allosteric drugs, is yet to...Though allostery is a well-established research field, its importance for biomedical applications, spanning from the allosteric effects of mutations in diagnostics to the design of innovative allosteric drugs, is yet to be fully appreciated. The potential of allostery in resolving issues of current drug design and in targeting proteins considered difficult or undruggable is the main topic of this review. In particular, we discuss how the non-conservatism of allosteric sites enables selective targeting of individual members in conserved families, thereby minimizing off-target toxicity. The multiplicity of allosteric sites in any structure allows alleviation of challenges posed by emerging drug resistance driven by allosteric and orthosteric mutations. The modulatory action of allosteric drugs provides, at the same time, an important therapeutic advantage in gradual activation/inhibition of enzymatic function and signaling in receptors. We also discuss here an approach for rational design of allosteric drugs, illustrating its process and output in obtaining effectors with the above-mentioned advantages and characteristics. Reviewing recent advances in the development of allosteric effectors, we show that allostery is undoubtedly becoming an integral part of the drug discovery paradigm. We, therefore, anticipate that the pharmaceutical industry will be prompted to systematically incorporate allostery as an important complement to existing drug design strategies in the near future.
Curr Opin Struct Biol
· 2025 Dec · PMID 41130140
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Transcription factor (TF) paralogs provide unique insights into how DNA-binding specificity evolves and diversifies. While paralogous TFs share conserved, highly similar DNA-binding domains, they achieve distinct regulat...Transcription factor (TF) paralogs provide unique insights into how DNA-binding specificity evolves and diversifies. While paralogous TFs share conserved, highly similar DNA-binding domains, they achieve distinct regulatory functions through mechanisms that are now being elucidated. This review examines how sequence variations between paralogs translate into functional diversity, including how mutations distant from the DNA interface can allosterically modulate binding specificity. We focus on competitive binding dynamics when paralogs are co-expressed and discuss emerging evidence that TFs recognize extensive repertoires of lower-affinity binding sites. Differential preferences for lower-affinity binding sites create paralog-specific binding patterns that determine TF genomic occupancy. These insights have important implications for interpreting the impact of coding and noncoding variation on TF-DNA interactions and human disease.
Curr Opin Struct Biol
· 2025 Dec · PMID 41106034
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Protein kinases catalyze the transfer of phosphate groups from ATP to specific substrates, initiating, modulating, or terminating signaling cascades. Generally, the response of these enzymes to stimuli is characterized b...Protein kinases catalyze the transfer of phosphate groups from ATP to specific substrates, initiating, modulating, or terminating signaling cascades. Generally, the response of these enzymes to stimuli is characterized by ultrasensitive rather than graded responses and mediated by cooperative binding interactions. Here, we provide examples of positive and negative cooperativity processes regulating several protein kinases. We first examine the binding cooperativity between nucleotide and substrate in protein kinase A, showing how dysfunctional cooperativity may be linked to signalopathies. We then illustrate how certain drugs exploit cooperativity to inhibit kinase homo- and hetero-dimerization or select for active and inactive conformational states. A molecular understanding of binding cooperativity could lead to the development of new kinase-specific inhibitors, opening up novel therapeutic possibilities.
The CRISPR-Cas system provides adaptive immunity for prokaryotes against mobile genetic elements (MGEs) such as bacteriophages and plasmids. As a countermeasure, MGEs have evolved various anti-CRISPR (Acr) mechanisms tha...The CRISPR-Cas system provides adaptive immunity for prokaryotes against mobile genetic elements (MGEs) such as bacteriophages and plasmids. As a countermeasure, MGEs have evolved various anti-CRISPR (Acr) mechanisms that neutralize the CRISPR-mediated immunity. Canonical Acr proteins block target binding of Cas proteins in a stoichiometric or enzymatic manner. New findings reveal that Acr also disintegrates functional Cas complexes, induces promiscuous target binding, and mimics Cas proteins and crRNA with defective mutations. Here, we summarize a broad repertoire of structural and functional mechanisms underlying CRISPR-centric competition, highlighting recent discoveries of molecular machinery that modulates CRISPR function.
Protein-nucleic acid interactions are vital to gene regulation and disease, yet have long been considered "undruggable." Recent advances are reshaping this paradigm, enabling therapeutic targeting of DNA- and RNA-binding...Protein-nucleic acid interactions are vital to gene regulation and disease, yet have long been considered "undruggable." Recent advances are reshaping this paradigm, enabling therapeutic targeting of DNA- and RNA-binding proteins. In this review, we highlight four major strategies: (1) direct disruption of protein-nucleic acid binding, (2) stabilization of specific complexes or conformations, (3) targeted degradation of interaction partners, and (4) allosteric modulation. We explore key examples across transcription factors, RNA-binding proteins, and DNA repair proteins, and emphasize emerging chemical, structural, and computational techniques that are accelerating discovery. Together, by intervening directly in the gene regulatory machinery, these approaches expand the druggable genome and open new avenues for treating cancer, genetic disorders, and viral infections.
Sequence-dependent intrinsic conformational dynamics confer specificity to transcriptional factor recognition of genomic DNA. Their genome-scale investigation using all-atom simulations is challenging, and alternative ap...Sequence-dependent intrinsic conformational dynamics confer specificity to transcriptional factor recognition of genomic DNA. Their genome-scale investigation using all-atom simulations is challenging, and alternative approaches by coarse-graining DNA into beads-and-sticks or polymer models have their own limitations. One parallel approach is what we describe here as a dictionary-based approach. This has been shown to explain several transcriptional events in biological systems but has been inadequately reviewed. These approaches represent studies based on a finite number of DNA fragments and their corresponding conformational properties, scaled up to genomes by pooling nearby fragments and machine learning models. This article aims to organize efforts made in generating these models and their recent successful applications to stimulate further development of this approach.
Epigenetic memory-the stable inheritance of a cellular state over cell generations-has long been associated with chromatin modifications. But individual modifications are very dynamic. How can they carry information acro...Epigenetic memory-the stable inheritance of a cellular state over cell generations-has long been associated with chromatin modifications. But individual modifications are very dynamic. How can they carry information across cell generations? Recent theoretical work suggests the answer might lie, in part, in the three-dimensional organization of the genome. Cooperation between marks brought together by genome folding can correct epigenetic errors, making stable memory units out of unstable marks. If marks direct the phase separation of chromatin, the resulting bidirectional coupling between marks and structure provides a mechanism for many of these units to operate independently along the genome. Models of bidirectional coupling have helped identify elements, such as formation of a dense compartment, 3D mark spreading, and limited enzyme, which may be key to stable epigenetic memory. An analogy between these 3D models and a classic model of associative memory hints at a way chromatin could perform sophisticated information processing.
DNA-encoded library (DEL) technology has enabled efficient discovery of both non-covalent and covalent inhibitors, with covalent binders typically identified via covalent DELs (CoDELs) containing diverse electrophilic wa...DNA-encoded library (DEL) technology has enabled efficient discovery of both non-covalent and covalent inhibitors, with covalent binders typically identified via covalent DELs (CoDELs) containing diverse electrophilic warheads. Recent developments have expanded CoDEL applications beyond cysteine to residues like lysine, tyrosine, arginine, and glutamic acid. The integration of CoDEL with activity-based protein profiling (ABPP) has further enabled the identification of potential protein targets for CoDEL screening using residue-selective warheads. Additionally, proteome profiling with fully-functionalized tags has guided target identification for focused DELs with privileged structures. This review highlights recent advances in CoDEL technologies for targeting both cysteine and non-cysteine residues, and discusses how proteomics facilitates hit discovery through CoDELs and focused DELs.
Cells have evolved multiple pathways to preserve genome integrity, with homologous recombination (HR) playing a central role in the accurate repair of DNA-double strand breaks (DSBs) by using homologous templates. Severa...Cells have evolved multiple pathways to preserve genome integrity, with homologous recombination (HR) playing a central role in the accurate repair of DNA-double strand breaks (DSBs) by using homologous templates. Several proteins are involved in HR, and their mutations have been associated with cancer initiation and progression. In this review, we present an overview of recent structural insights into the HR pathway, highlighting the pivotal role of structural approaches in elucidating this complex and finely regulated DNA repair machinery, with the aim of advancing understanding and informing future research in the field.
Allosteric modulation offers an increasingly attractive route for precise intervention in enzymatic pathways. This review outlines emerging strategies for the identification and exploitation of allosteric sites, emphasiz...Allosteric modulation offers an increasingly attractive route for precise intervention in enzymatic pathways. This review outlines emerging strategies for the identification and exploitation of allosteric sites, emphasizing computational frameworks that integrate evolutionary, structural, and dynamic features with machine learning models. We discuss how perturbation-based simulations, network analyses, and deep mutational data are reshaping our understanding of allosteric regulation. In parallel, advances in experimental techniques have enabled validation of cryptic and functionally relevant pockets across diverse enzyme families. We further catalog FDA-approved allosteric modulators of enzymes, highlighting therapeutic designs that leverage distal regulation to enhance specificity and overcome resistance. Taken together, these developments reveal the growing utility of allostery in drug design and underscore its potential to expand the therapeutic target space beyond conventional binding sites.
Laine E, Grudinin S, Klypa R
… +1 more, Beauchêne IC
Curr Opin Struct Biol
· 2025 Dec · PMID 40987097
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A few years after AlphaFold revolutionised the field of protein structure prediction, the new frontiers and limitations in structural biology have become clearer. Predicting protein-nucleic acid interactions currently st...A few years after AlphaFold revolutionised the field of protein structure prediction, the new frontiers and limitations in structural biology have become clearer. Predicting protein-nucleic acid interactions currently stands as one of the major unresolved challenges in the field. This knowledge gap stems from the scarcity and limited diversity of experimental data, as well as the unique geometric, physicochemical, and evolutionary properties of nucleic acids. Despite these challenges, innovative ideas and promising methodological developments have emerged for both predicting protein-nucleic acid complex structures and designing nucleic acids capable of binding to specific protein conformations. This review presents these recent advances and discusses promising avenues, including the integration of high-throughput profiling data, the development of more rigourous and richer evaluation benchmarks, and the discovery of biologically meaningful regulatory and structural signals using self-supervised learning.
Curr Opin Struct Biol
· 2025 Dec · PMID 40974823
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Specific values of nucleosome spacing have long been associated with distinct chromatin organization, but recent studies reveal surprising structural and functional consequences of small changes in regular linker DNA len...Specific values of nucleosome spacing have long been associated with distinct chromatin organization, but recent studies reveal surprising structural and functional consequences of small changes in regular linker DNA length. This opinion article revisits experimental and modeling studies addressing the classic 10n versus 10n + 5 spacing, highlighting how this 5 bp difference can alter nucleosome orientation, fiber topology, and higher-order chromatin behavior. We underscore how differences in model parameters and system design yield different trends for the effect of linker DNA lengths on chromatin architecture. However, chromatin structure in vivo reflects the heterogeneous nucleosome spacing in combination with other cellular variables like salt conditions, epigenetic marks, and protein and RNA binding, which work together to shape gene folding and direct gene regulation.
The rapid evolution of microorganisms and cancer cells makes it difficult to treat tumours and infectious diseases, because resistance to drugs is the rule rather than the exception. Structures or models of protein-drug...The rapid evolution of microorganisms and cancer cells makes it difficult to treat tumours and infectious diseases, because resistance to drugs is the rule rather than the exception. Structures or models of protein-drug complexes help to understand how mutations lead to resistance and to design better drugs. However, it is difficult to reason how small changes in the structure lead to drug resistance. Thus, protein and drug dynamics need to be considered. Strategies to increase drug residence are sought after to increase the efficacy of drugs. Computational methods to calculate the effect of mutations on drug binding and residence times are being developed and improved, but are challenging. A priori prediction of a mutation's effect on drug binding is an even greater challenge. On the other hand, knowledge about protein-drug complexes has led to the development of multiple design strategies that aim to reduce mutation-driven drug resistance.
Protein function arises from the interplay of structure, dynamics, and biomolecular interactions. Despite advances in cryo-EM and AI-based structure prediction, capturing dynamic and energetic features remains a challeng...Protein function arises from the interplay of structure, dynamics, and biomolecular interactions. Despite advances in cryo-EM and AI-based structure prediction, capturing dynamic and energetic features remains a challenge. Biophysical methods like NMR, EPR, HDX-MS, SAXS, and cryo-EM provide valuable but often indirect signals. Connecting these to molecular mechanisms requires integrative approaches that combine experiments with physics-based simulations, revealing both stable structures and transient, functionally important intermediates. This review highlights recent advances in integrative modeling using the maximum entropy principle to build dynamic ensembles from diverse data while addressing uncertainty and bias. These methods help resolve heterogeneity and interpret low-resolution data. We conclude by exploring how integrative modeling, enhanced sampling, and AI-driven tools enable new insights into slow, large-scale conformational changes.