Journal of Molecular Biology最新文献

Selecting cell lines with specific Single Nucleotide Polymorphism (SNP) genotypes is a critical bottleneck in functional genomics, often requiring advanced bioinformatic skills. To address this, we developed CLISGen (Cell Lines SNP Genotypes), a database with a user-friendly web application that simplifies access to SNP genotypes in over 1,000 cancer cell lines from the Cancer Cell Line Encyclopedia. CLISGen integrates and harmonizes data from Whole-Genome, Whole-Exome, and RNA sequencing, enriching it with contextual information like copy number alterations and genetic ancestry. The platform allows users to search for specific variants or variants in specific genes or genomic regions and filter results by tissue type or data quality, providing intuitive graphical and tabular outputs. By eliminating a major experimental bottleneck, CLISGen offers researchers a powerful resource to efficiently select suitable cell models for studying the link between genetic variation and cancer. CLISGen is freely available at https://bcglab.cibio.unitn.it/clisgen.

LysR-type transcriptional regulators (LTTRs) are a diverse family of proteins that regulate various cellular processes, including motility in bacteria. In Escherichia coli, the LTTR LrhA represses flagellar biosynthesis by inhibiting the flhDC operon. However, the structural basis underlying this regulation has remained unclear. Here, we determined both a high-resolution crystal structure and a cryo-EM reconstruction of LrhA, revealing a predominant and stable tetrameric organization with pronounced structural variability in its effector-binding region. Structural and biochemical analyses demonstrate that mutations in these variable regions perturb the oligomeric equilibrium of LrhA, shifting the balance between tetrameric and dimeric species. This shift correlates with enhanced DNA binding affinity and stronger repression of the flhDC promoter. While ligand binding may similarly modulate LrhA activity, our data primarily support a model in which alterations in oligomeric state mediated by the variable regions regulate LrhA function. Together, these findings provide a structural framework for understanding how LrhA controls bacterial motility and offer broader insights into oligomerization-based regulation within the LTTR family.

This manuscript reflects the early stages of my ongoing journey into weak biomolecular interactions, beginning with my study of Vav1, a guanine nucleotide exchange factor. In this initial work, I uncovered how weak intramolecular interactions regulate protein activation, providing critical insights into their role in cellular processes like signal transduction. However, my understanding of weak interactions took an unexpected turn during research on the Nck/NWASP complex, when we serendipitously discovered that weak, multivalent interactions drive liquid-liquid phase separation (LLPS), a process essential for cellular organization. This unanticipated finding led to the development of the CoPIC platform, which enables high-throughput detection of weak interactions within living cells. Though the studies on Vav1 and LLPS are independent, both underscore the profound role of weak interactions in regulating cellular dynamics. This ongoing journey continues to challenge and deepen my understanding of how weak interactions orchestrate the complexity of biological systems. This personal trajectory exemplifies how pursuing seemingly focused mechanistic questions can unexpectedly reveal broader principles-here, that weak interactions are not peripheral but central architects of cellular complexity and adaptability.

Professor Jinpeng Sun has long been dedicated to pharmacological research on G protein-coupled receptors (GPCRs), achieving systematic advances in areas such as ligand-receptor recognition, drug target validation, and membrane receptor-based drug development. In 2007, he earned his Ph.D. in Molecular Pharmacology from the Albert Einstein College of Medicine, after which he conducted postdoctoral research in the laboratory of Nobel Laureate Professor Robert J. Lefkowitz at Duke University, a pioneering figure in GPCR biology. In 2011, Professor Sun established his independent research group in China, where he has since pursued in-depth investigations into GPCR pharmacology. To address key bottlenecks in GPCR drug discovery, such as the unclear pathophysiological hubs of complex diseases and the difficulty in designing selective drugs, Professor Sun's team proposed that the dynamic and multiple interactions among ligands, receptors, and intracellular (membrane) effectors are important players in microenvironment establishment that drive or modulate disease progresses. Based on this conceptual framework, they developed a suite of innovative methodologies, including endogenous ligand capture technology, highly sensitive multipathway GPCR activity profiling systems, and microscale biophysical activation platforms. Using these tools, the team successfully identified membrane receptors for several critical hormones and metabolites-such as glucocorticoids, androgens, progesterone, and ceramides-resolving several long-standing questions in pharmacology. They were the first to discover the GPCR responsible for the sense of balance and elucidated the molecular mechanisms through which GPCRs sense mechanical force, odors, pruritic stimuli, and acidic or alkaline environments, substantially expanding the known functional scope of GPCR biology. Furthermore, by integrating chemical biology with signaling assays, Professor Sun's group introduced theoretical models such as the "flute model" of functional coding of GPCR phosphorylation and "proline regions docking and sorting" for GPCR biased signaling. Exploiting AI-guided ligand design, Sun's group developed over 20 selective lead compounds targeting the GPCRs involved in psychiatric, metabolic, cardiovascular, and aging-related disorders. Several candidates have completed preliminary pharmacokinetic and toxicity studies, demonstrating strong translational potential. This article systematically summarizes the key scientific contributions from Professor Sun Jinpeng's laboratory over the past decade, highlighting their impact on receptor-ligand paring, signaling mechanism elucidation, tool development, and rational drug design, and discusses their implications for the future of precision medicine.

Nascent helicity in polypeptides and unfolded proteins arises from local structure formation and represents one of the earliest events in a protein folding reaction. Nascent helicity may also influence the physical properties of intrinsically disordered regions. For this reason, there has been great interest in statistical mechanical models that describe the coil→helix transitions that lead to nascent helicity. These models, collectively called helix-coil models, have been empirically parameterized using an extensive data set of circular dichroism (CD) measurements of natural and designed peptides that form various degrees of nascent helicity. The purpose of A Bayesian Statistical Engine to Infer HeLicity (ABSEIL) (https://abseil.oit.duke.edu/) is to allow users to submit polypeptide sequences to: 1) predict the overall helicity of the sequence; 2) predict the helicity of each residue; and 3) enumerate the ensemble of helix-coil configurations in order of their relative populations. The tool also allows users to search the database of peptide CD experiments on which the predictive model was trained. The website architecture allows for anonymous usage and enables administrative management. The web application server is managed by the Duke Office of Information Technology (OIT) system administrators and conforms to OIT's security and operational best practices.

The U.S. Department of Energy's Systems Biology Knowledgebase (KBase; www.kbase.us) is an open, collaborative platform that integrates data, models, and analysis tools to accelerate discovery in microbiology, plant biology, and environmental systems. Recently, KBase expanded as a comprehensive, multi-omics ecosystem. KBase enables representation of scientific samples, long-read sequence analysis, protein structure integration, and scalable modeling of microbial communities across diverse environments. KBase also generates digital notebooks as citable, executable research objects that link data, methods, and interpretation. KBase also supports a global education community focused on training the next generation of scientists to use high-performance computational tools. Together, these advances position KBase as a central hub for open, reproducible systems biology. In turn, this enables us to integrate many of the emerging advances in data federation, semantic interoperability, and agent-assisted analysis, paving the way for KBase to support the next generation of AI-driven discovery tools.

In response to a variety of signals outside of cells, G protein-coupled receptors (GPCRs) play essential roles in cell signal transduction by relaying the extracellular signals to the intracellular side through various signaling mechanisms, which govern diverse physiological and pathological processes. These receptors are involved in many diseases and comprise the largest drug target family. However, the molecular mechanisms underlying the GPCR signal transduction are poorly understood, which hinders the drug discovery with only a small portion of receptors having drugs marketed. Over the past decade, our laboratory has been focused on the ligand recognition and functional modulation mechanisms of different GPCRs, aiming for better understanding of the physiology and pathology of this receptor superfamily and new clues to carry out drug development. Through extensive structural and functional studies, we uncovered diverse interaction patterns of GPCRs in recognizing various ligands, including small molecules, peptides, and proteins. These molecular details not only reveal key factors that define ligand selectivity and receptor specificity, but also provide insights into allosteric modulation, ligand promiscuity, and intrinsic activation. Our knowledge about the GPCR modulations were further extended by investigating the conformational rearrangements and dynamics of GPCRs upon activation and coupling to downstream signaling transducers. With different molecular architectures, different receptors exhibit distinct patterns in regulating their activities and abilities to stimulate various signaling pathways, which are key for understanding biased signaling. These findings demonstrate the diversity and complexity of GPCR signaling and would enable development of novel drugs with improved efficacy and reduced side effects.

Metal ions play critical structural, regulatory, and enzymatic roles in proteins, making their binding essential for biological processes. Experimental identification of metal-binding sites is resource-intensive and limited in scalability. Recent advances in protein language models have transformed computational predictions, yet current tools do not address how residue-level metal-binding probabilities change upon mutation. To fill this gap, mCSM-metal leverages embeddings from ESMBind with our graph-based structural signatures to accurately predict the effects of single or multiple point mutations on the binding of seven essential ions (Zn²⁺, Ca²⁺, Mg²⁺, Mn²⁺, Fe³⁺, Co²⁺, Cu²⁺). Our model achieves accuracies, F1-scores, and Matthews Correlation Coefficient values up to 0.97, 0.97, and 0.95, outperforming other approaches. The webserver provides an interactive platform to assess and visualize local and long-range impacts of mutations on metal-ion binding, offering new avenues for applications in structural biology, disease modelling, and protein engineering. The web application is freely available at: https://biosig.lab.uq.edu.au/mcsm_metal/.

Mitoxantrone (MIT) is a chemotherapeutic drug widely used for its DNA intercalation and inhibition of topoisomerase. In this work, we show that MIT also affects cytoplasmic RNA-ribosome organization. In human cancer cells, MIT induced stress granules (SGs) that contained large ribosomal subunit proteins, including eL8, together with polyadenylated mRNA. These MIT-induced SGs were different from arsenite-induced SGs: they formed without eIF2α phosphorylation, mTOR inhibition, or 4E-BP1 activity, and they remained stable in the presence of cycloheximide and after drug withdrawal. In vitro assays further demonstrated that MIT promotes ribosome aggregation in a concentration- and salt-dependent manner. Taken together, our results identify a distinct type of ribosome-enriched SGs that form through RNA-ribosome condensation rather than classical translational stress pathways. This mechanism provides a direct example of how a clinically used drug can reorganize cytoplasmic RNA-protein complexes, with possible consequences for mRNA regulation, cancer therapy, and neurodegenerative disease.

PH0140, a hypothetical protein from the Feast/Famine Regulatory Protein (FFRP) family in Pyrococcus horikoshii OT3, is predicted to play a role in transcriptional regulation in response to exogenous amino acids. Previous in-silico studies suggest that PH0140 has a binding preference for exogenous tryptophan and regulates transcription through DNA recognition and allostery. However, its structure and regulatory mechanism remain largely unexplored. In this study, we determined the crystal structure of PH0140 at a resolution of 2.0 Å, revealing that it binds to exogenous isoleucine through distinct structural features: a unique C-terminal loop region near the Effector-Binding Domain (EBD) and a β-strand (β4) with notable structural deviations from homologous proteins. Size-exclusion chromatography showed that PH0140 forms oligomers in the presence of exogenous isoleucine and tryptophan. Furthermore, the amino acids binding was characterized through isothermal titration calorimetry (ITC). Studies suggest that exogenous tryptophan has a better binding affinity than isoleucine. To explore the structural basis of this ligand effect, we modeled an octameric PH0140-DNA complex containing a 111-bp promoter fragment and performed multiple molecular dynamics simulations. The simulations revealed that the octameric assembly undergoes a conformational opening to interact with the DNA promoter^TTTTregion. The hydrophobic driven force on the EBD results in distortion of β-strand (β4) into loop (Chain G) to adopt an open conformation, which facilitates interaction with the promoter^TTTT region for transcription regulation.