For more than 50 years, the linear sequence and the multiple sequence alignment have been the foundational data structures of protein science, and they remain central to homology search, phylogenetic inference, covariance-based contact prediction, and modern protein language models. However, relational and graph-based representations are increasingly being adopted alongside sequence-based methods to capture biological relationships that linear data structures express only implicitly. Proteins fold as three-dimensional residue interaction networks, evolve through high-dimensional genotype networks defined by mutational connectivity, and operate within cellular protein-protein interaction graphs. Here, we review how graph theory is being used to describe and understand these relationships across protein science, with an emphasis on what these methods offer biochemists working on enzyme superfamilies, protein engineering, drug targets, and functional annotation. We trace the development of these ideas from early theoretical topologies, through statistical coupling and the structural network analyses, to the geometric and graph-like representations used in recent machine-learning-driven advances. Throughout, we emphasise that graphs do not replace sequences or MSAs but provide a complementary representation for biochemical relationships that are difficult to express in one dimension.
Biomolecular condensates are membrane-less compartments formed through phase separation that concentrate and organize biomolecules within cells. Far from being static droplets, they are dynamic molecular assemblies whose internal dynamics, structure, mechanics, and composition can evolve over time during cellular aging, disease-associated aggregation, or engineered material formation. These changes are often described using state descriptors such as liquid-like, gel-like, aged, or solid-like, yet similar apparent states can be interpreted differently depending on whether they are assessed in terms of morphology, dynamics, structure, mechanical response, or composition. The present review article summarizes an experimental framework used to probe biomolecular condensates and their transitions across complementary dimensions while minimizing perturbation to the system. A diverse suite of label-free optical, scattering, spectroscopic, mechanical, and microfluidic approaches is discussed, highlighting how integrated strategies can connect these readouts to enable more rigorous interpretations of condensate behavior. This framework is intended to guide researchers to move beyond broad material descriptors toward understanding the mechanisms underlying condensate evolution, the resolution of phase transitions, and their implications for biological function, pathology, and biomaterial design.
N6-methyladenosine (m6A) is a prevalent RNA modification that regulates multiple aspects of RNA metabolism, including RNA localization, stability, decay, and translation. m6A deposition is catalyzed by distinct methyltransferase complexes, including the METTL3/14 complex and METTL16, which recognize different RNA sequence motifs. The biological effects of m6A are mediated by effector proteins that selectively recognize m6A-modified RNA. To identify previously uncharacterized m6A binding proteins, we developed an RNA-binding protein domain array to systematically screen for candidate m6A effectors. Using this approach, we identified the spliceosomal protein SF3B4 as a potential m6A reader. RNA pull-down assays using m6A-modified RNA probes demonstrated selective enrichment of endogenous SF3B4, supporting its ability to recognize m6A RNA. To define the RNA targets of SF3B4, we performed SF3B4 RIP-seq alongside m6A RIP-seq, followed by RIP-qPCR validation of overlapping targets. Motif analysis revealed that SF3B4 preferentially associates with the conserved GRAGRA (R=A/G) motif, consistent with the RNA sequence recognized by the METTL16 methyltransferase. Notably, transcripts of the BCR and MET oncogenes were identified as shared targets of SF3B4 and METTL16. Together, these findings identify SF3B4 as a previously unrecognized m6A effector and suggest that it participates in RNA metabolic processes downstream of METTL16-mediated m6A modification.
E3 ligases catalyze the final step in transferring ubiquitin to protein substrates, but resulting intermediates are characterized by conformational flexibility. Recent work by Chandler and colleagues ( Biochem J. (2026) 483, 1115-1130) demonstrates that photocross-linking activity-based probes can trap transient conformations of the intermediates, providing a valuable tool to map interfaces during the ubiquitin transfer process.
Heterotrimeric G proteins (αβγ) function as molecular switches that regulate intracellular signaling downstream of G protein-coupled receptors (GPCRs). Gα subunits cycle between an inactive GDP-bound state and an active GTP-bound state that engages downstream effectors. The duration of Gα-mediated signaling is primarily governed by Regulators of G Protein Signaling (RGS) proteins, which accelerate GTP hydrolysis as GTPase-activating proteins (GAPs). To characterize these multi-specific Gα interactions at the individual residue level, we employed computational energy calculations across Gα-partner complexes, focusing on residues critical for RGS interactions. This analysis showed that specific residues within the Gα "switch regions", conserved across the Gi and Gq subfamilies, play a central role in RGS recognition and GAP activity. Substantial energy contributions arise mainly from conserved residues within the switch I and II in the Gα GTPase domain. However, switch I interactions are dominated by electrostatic and non-polar contributions from the protein backbone, while switch II interactions are dominated by strong electrostatic side-chain contributions. Mutagenesis experiments confirmed that switch I side chains are not required for RGS GAP activity, but disrupting the tight switch I interface with RGSs abolished GAP activity. In contrast, alanine substitutions in individual switch II residues severely reduced RGS GAP activity, while switch III played only a minor role. Our findings offer residue-level insights into the conserved interactions underlying RGS/effector regulation of G proteins and provide a basis for engineering Gα subunits with tailored interaction profiles, developing inhibitors of Gα-RGS binding, or enabling rewiring of G protein-coupled signaling pathways in vivo.
It has been recognized a long time ago that the hedgehog (Hh) and Wnt signaling pathways have numerous similarities that suggest their common evolutionary origin. Although the Hh and Wnt proteins are unrelated, they are similar in that they carry lipid modifications that are critical for their interaction with their receptors. In our earlier work we have shown that Wnt inhibitory factor 1 (WIF1), originally identified as a Wnt antagonist also binds to and inhibits the signaling activity of sonic hedgehog (Shh), raising the possibility that the lipid moieties of these unrelated morphogens play a dominant role in their interaction with WIF1. In the present work we have compared the interactions of human WIF1 protein with lipidated and non-lipidated forms of human Shh using surface plasmon resonance spectroscopy and reporter assays monitoring the signaling activity of human Shh. Our studies have shown that human WIF1 protein has a modest but significantly higher affinity for lipidated than non-lipidated Shh, indicating that lipid modifications of Shh contribute to but are not essential for interaction with WIF1.
The spliceosome is emerging as a key regulatory hub in Toxoplasma gondii, yet the molecular basis of spliceosomal protein interactions remains largely unexplored. TgCyp23, a predicted nuclear cyclophilin (Cyp) from T. gondii, shares sequence similarity and catalytic properties with the human spliceosomal cyclophilin H (hCypH) that interacts with the splicing factors PRP4 and PRP18. Here, we investigated whether TgCyp23 engages in analogous interactions with the T. gondii orthologs TgPRP4 and TgPRP18 using peptides corresponding to their predicted Cyp-binding regions. High-resolution crystal structures of TgCyp23 in complex with a TgPRP4-derived peptide, including a ternary complex with the Cyp inhibitor cyclosporin A, reveal that spliceosomal partner recognition occurs outside the catalytic site, which remains fully accessible. NMR analyses extend this binding mode to TgPRP18, demonstrating that peptides derived from both spliceosomal factors engage the same surface in solution. Isothermal titration calorimetry shows that TgCyp23 binds both peptides with low- to mid-micromolar affinity, while circular dichroism and molecular dynamics support a folding-upon-binding mechanism. Notably, binding of either peptide does not affect the peptidyl-prolyl isomerase activity of TgCyp23 or its sensitivity to cyclosporin A, indicating that partner recognition and catalytic function are mechanistically separable. Together, these findings identify a non-catalytic interaction surface in TgCyp23 that mediates recognition of spliceosomal factors. The similar interaction mode observed for TgCyp23 and hCypH suggests that spliceosomal partner recognition may be conserved and supports a role for TgCyp23 as a spliceosome-associated Cyp in T. gondii, providing a structural framework for understanding Cyp interactions within the parasite spliceosome.
Replication of human mitochondrial DNA (mtDNA) is essential for the maintenance of oxidative phosphorylation and cellular energy homeostasis. Impairment of this process leads to mtDNA deletions, depletion, and point mutations that underlie a broad spectrum of mitochondrial diseases, as well as contributing to neurodegeneration, aging, and cancer. The core human mitochondrial replisome, composed of DNA polymerase γ (Polγ), the replicative helicase Twinkle, and the mitochondrial single-stranded DNA-binding protein (mtSSB), is the main complex responsible for replicating the mitochondrial genome through a highly coordinated yet still incompletely understood mechanism. Mutations in the nuclear genes encoding these proteins represent the most common cause of inherited disorders affecting mtDNA maintenance, underscoring the importance of understanding their coordinated molecular function. Recent advances in cryo-electron microscopy and single-molecule approaches have provided unprecedented insight into the structural organization and dynamic operation of the core components of the mitochondrial replisome. These complementary methods are establishing a quantitative mechanistic framework for understanding how the mitochondrial replisome initiates, progresses, and regulates the replication of the light and heavy strands of mtDNA. In the present review, we integrate recent structural and single-molecule findings to describe the mechanisms governing the activity of Polγ, Twinkle, and mtSSB at the mitochondrial replication fork, and discuss remaining challenges toward reconstructing a complete mechanistic model of human mtDNA replication.
Intracellular pH regulation of PIP2 aquaporins, primarily mediated by a histidine sensor in loop D, is essential for controlling water transport. While the C-terminal (CT) domain is involved, the precise contribution of the N-terminal (NT) and other flexible regions has been debated. Given that PIPs are small proteins that lack large regulatory domains yet achieve finely tuned gating, we sought to investigate how local structural flexibility and inter-region communication contribute to this process. To this end, we introduced alanine substitutions into conserved proline motifs in BvPIP2;2, targeting a contiguous NT PPP motif (BvPIP2;2-AAA) and two isolated prolines in loop D (BvPIP2;2-P194A and BvPIP2;2-P204A). Using heterologous expression in Xenopus laevis oocytes, we evaluated channel function and localization, while the structural impact of the mutations was analyzed with molecular dynamics (MD) simulations on full-length structural models. While BvPIP2;2-P204A impaired membrane trafficking, both BvPIP2;2-AAA and BvPIP2;2-P194A mutants localized to the plasma membrane and remained functional. Functional assays revealed that the BvPIP2;2-AAA mutant exhibited a marked alkaline shift in pH0.5, greater than that observed for BvPIP2;2-P194A. MD simulations showed that the NT mutation reduced flexibility, reorganized the network of interactions between NT and CT regions, and altered the environment and the pKa of the canonical pH sensor, His202. In contrast, the P194A mutation produced more modest structural and functional changes. These findings support a model in which transitions in structural flexibility within intracellular regions, particularly the NT region, govern communication between dynamic segments and modulate the electrostatic environment of the pH sensor, thereby fine-tuning pH gating in plant aquaporins.
The discovery of the C4 pathway laid the foundations of a revolution in plant biochemistry. The Hatch and Slack paper published in the Biochemical Journal described a specialized pathway of photosynthetic metabolism that minimizes the loss of carbon and nitrogen to maximize efficiency in hot, dry environments. Since that time, there has been an enormous increase in our understanding of the anatomical and biochemical modifications that facilitate C4 photosynthesis in different species. A key feature is the concentration of CO2 in the vicinity of the carboxylating enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, thereby increasing photosynthetic efficiency by minimizing photorespiration. C4 photosynthesis is an excellent model for complex trait evolution in response to environmental change. The C4 pathway represents one of the most convergent of evolutionary phenomena, having evolved independently many times over in diverse families of angiosperms. Climate change is changing the balance between C3 and C4 vegetation types, influencing how ecosystems process carbon and respond to isotope discrimination. Moreover, the pioneering work of Hatch and Slack has formed the biochemical backbone for the C4 rice project, which is one of the most ambitious metabolic engineering initiatives to be attempted in plant science.
The bacterial anti-phage immune system is complex, diverse, and in several important cases ancestral to that found in eukaryotes, including humans. One example is CBASS (cyclic oligonucleotide based anti-phage signalling system), a widespread bacterial defence that signals phage presence in the cell via cyclic nucleotide second messengers, activating ancillary effectors to combat infection. CBASS is homologous and ancestral to the eukaryotic cGAS/STING pathway for antiviral defence. The heart of the system is a nucleotide cyclase known as a cGAS/DncV-like nucleotidyltransferase, which is activated by phage infection. The mechanisms of activation of CBASS cyclases are diverse and in most cases not fully understood at a molecular level. Moreover, it is vital to keep these signal-generating enzymes fully inactive in the absence of phage infection to avoid auto-toxicity. Here, we report a structural and mechanistic study of a CBASS cyclase from Bacillus cereus. Using crystal structures of key reaction intermediates, coupled with kinetic analyses, we show that the substrate, ATP, plays a fundamental role in the inhibition of the non-activated form of the enzyme in vitro. We provide a molecular explanation for this regulation and explore the implications for the regulation of these important defence systems in bacterial immunity.
The endoplasmic reticulum (ER) hosts several integral membrane enzymes responsible for post-translational modifications of proteins entering the secretory pathway. These include protein glycosylation and the attachment of glycosylphosphatidylinositol (GPI) anchors. At the ER membrane, protein glycosylation is catalyzed by glycosyltransferases from the C-superfamily (GT-C), which use lipid donor substrates to attach a complex oligosaccharide to asparagine residues (N-glycosylation), or a single mannose unit to threonine, serine (O-mannosylation), or tryptophan (C-mannosylation) residues. In contrast, the attachment of GPI anchors to acceptor proteins is catalyzed by the multimeric enzyme transamidase, which cleaves a C-terminal GPI signal peptide of the acceptor protein and replaces it with a GPI anchor. In the present review, we will discuss recent mechanistic studies that shed light on the architecture of these membrane protein machineries and on how they recognize their substrates and catalyze protein glycan modifications at the ER membrane.
In some purple phototrophic bacteria, the enzyme spheroidene monooxygenase (CrtA) catalyses the final step of carotenoid biosynthesis, introducing a keto group at the C2 position of spheroidene to produce spheroidenone. CrtA from the model purple bacterium Cereibacter (previously Rhodobacter) sphaeroides has a C-terminal extension consisting of a disordered, proline-rich sequence followed by a short region containing a significant proportion of glycine residues; the role of this extension is not understood. In addition to the accumulation of spheroidene, a C. sphaeroides ΔcrtA mutant generated in a previous study had a slower growth rate and made fewer photosynthetic complexes than the wild-type strain. We show here that these phenotypes are largely due to a polar effect of the crtA deletion on the downstream bchID bacteriochlorophyll biosynthesis genes. We generated a crtA mutant where bchID expression was not interrupted and used this background to test a series of CrtA C-terminal truncations to identify the minimal enzyme that retains spheroidene monooxygenase activity. Using structural modelling, we identify a histidine residue that acts as the axial ligand to a heme group required for spheroidene monooxygenase activity, and two threonine residues found proximal to the bound carotenoid. We show that the subtle photoheterotrophic growth phenotype of this new ΔcrtA mutant is due to the loss of the C-terminus of the enzyme rather than the altered carotenoid content of this strain. We further demonstrate that the C-terminal extension appears to mediate the association of CrtA with other membrane proteins, forming a range of high-molecular-mass complexes.
PSD-95/DLG/ZO-1 (PDZ) domains canonically recognize short C-terminal peptide motifs, but numerous non-canonical binding mechanisms have also been described. We previously identified a direct interaction between the cell polarity protein SCRIB and the RAS effector AFDN, mediated by the SCRIB PDZ1 and AFDN FHA domains, though a structural basis remained unresolved. Here, we use AlphaFold3 predictions to model the SCRIB:AFDN complex, revealing that binding is driven by a conserved β-hairpin motif C-terminal to the AFDN FHA domain. This β-hairpin engages the canonical peptide-binding groove of SCRIB PDZ1, contributing an antiparallel strand to extend the PDZ β-sandwich, inserting a phenylalanine into the hydrophobic pocket, and forming contacts that recapitulate a canonical PDZ:peptide complex. We validate the interface with nuclear magnetic resonance spectroscopy (NMR) and biochemical assays and show that point mutations at either side of the interface can abolish complex formation. Unexpectedly, disruption of full-length AFDN:SCRIB binding in cells results in loss of interaction with the KRAS oncoprotein. The identified β-hairpin motif is highly conserved across deuterostomes but absent from most invertebrate AFDN orthologs, including Drosophila melanogaster - the organism in which AFDN/Canoe and SCRIB/Scribbled functions have been most extensively characterized. The complex closely resembles the only previously described PDZ:β-hairpin structure, between the PDZ domain of Syntrophin and a motif from nNOS/NOS1. A proteome-wide search identifies hundreds of candidate internal PDZ-binding sites conforming to this β-hairpin motif, suggesting the mechanism may be significantly more widespread than currently appreciated.
The bone-morphogenetic protein (BMP)-SMAD signal transduction pathway regulates fundamental cellular processes such as fate specification, tissue patterning, and stem cell homeostasis across metazoans and has a conserved signaling architecture. However, the quantitative dynamics of SMAD signaling and regulatory strategies governing pathway activity show a wide range of variation across developmental and stem-cell systems. In the present review, we summarize insights from six major biological contexts—Drosophila embryo, germline stem cells, and the larval and pupal wing discs; the Danio rerio (zebrafish) embryo; and human pluripotent stem cells—to compare how BMP signals are measured, manipulated, modeled, and integrated. We begin by outlining the canonical BMP signaling pathway and the mechanisms of BMP gradient formation across developmental systems, highlighting how conserved pathway components contribute to the formation of system-specific spatial profiles. We then summarize intracellular Smad dynamics and how endogenous pathway dynamics are measured through quantitative imaging of the phosphorylated Smad. Next, we examine how BMP signaling is interpreted through tiered transcriptional responses of downstream target genes. Next, we summarize the mechanistic and computational models of integrated gradient formation, signal transduction, and gene regulation across the presented systems. These insights reveal unifying design principles and performance objectives that govern BMP-SMAD signaling across species and cell types and frame open questions for future cross-species and translational studies.
Protein S (PROS1) is a vitamin K-dependent plasma glycoprotein that was originally described as a non-enzymatic cofactor of activated protein C in the regulation of blood coagulation. Over the past three decades, PROS1 has emerged as a pleiotropic signaling molecule with functions that extend far beyond hemostasis. PROS1 is a ligand for the TAM family receptor tyrosine kinases TYRO3 and MERTK and, as such, regulates diverse cellular processes including proliferation, migration, phagocytosis, vascular homeostasis, and immune modulation. These activities are mediated through both circulating and locally produced PROS1 and are pathological context-dependent. In the present review, we first summarize our current knowledge on the regulation of PROS1 gene expression and protein structure. We then discuss its established anticoagulant mechanisms alongside emerging activated protein C-independent functions. Finally, we examine key regulatory roles of protein S-TAM signaling in the vessel wall, central nervous system, inflammation, and cancer. Collectively, these findings position PROS1 as a molecular integrator of coagulation, immunity, and tissue homeostasis and underscore its relevance as both a biomarker and a potential therapeutic target in a broad range of pathophysiological contexts.
Heparan sulfate proteoglycans are central modulators of cell-cell communication, largely through the information encoded in their sulfation patterns. Among extracellular regulators of this 'heparan sulfate code', the endosulfatases SULF1 and SULF2 have emerged as unique enzymes with the capacity to selectively remove 6-O-sulfate groups from heparan sulfate (HS) glucosamine residues. This activity distinguishes them from canonical lysosomal sulfatases and positions them as critical editors of HS from the extracellular matrix and cell-surface. Recent biochemical studies have highlighted their distinctive domain organization, extensive post-translational modifications, and finely tuned substrate specificity, revealing that 6-O-desulfation is a non-random, highly regulated process. Functionally, SULFs influence major signaling pathways and thereby participate in diverse biological processes, such as development, tissue homeostasis, injury repair, inflammation, and tumor progression. Accumulating evidence also implicates SULF1 and SULF2 in disease pathogenesis and highlights them as promising, yet underexplored, therapeutic targets. In the present review, we provide an updated perspective on SULF biology, emphasizing recent advances in functional characterization, their roles as extracellular 'code editors', and the therapeutic opportunities that may arise from targeting their activity. We also address several key questions that remain unresolved and that are needed to understand this complex mechanism of regulation.
Chromatin remodeling regulator CECR2 (cat eye syndrome chromosome region candidate 2) plays critical roles in neurulation, spermatogenesis, DNA damage response, and cancer metastasis, yet the structural determinants underlying its recognition of acetylated histones remain poorly understood. Here, we characterize the histone-binding specificity and structural determinants of the CECR2 bromodomain using isothermal titration calorimetry, biochemical assays, and structure-guided computational modeling. The CECR2 bromodomain recognizes multiple monoacetylated histone H4 peptides, including H4K5ac, H4K8ac, H4K12ac, and H4K16ac, demonstrating engagement across the H4 N-terminal tail. Combinatorial acetylation differentially modulates binding: while H4K5acK8ac exhibits reduced affinity, H4K5acK12ac and H4K8acK12ac maintain binding comparable to monoacetylated peptides. The bromodomain also recognizes acetylated peptides from other histones, including H3K23ac and H2AK5ac. Mutational analysis identifies conserved pocket residues essential for acetyllysine recognition. Molecular docking suggests that a single acetyllysine occupies the canonical bromodomain pocket, while additional modifications extend along the protein surface, providing a structural basis for selective recognition of combinatorial marks. Comparative analysis with the BET family bromodomain BRD4-BD1 highlights differences in pocket architecture and ligand accommodation, with CECR2 displaying a more permissive binding environment that can accommodate larger acyllysine modifications. Together, these findings provide biochemical and modeled structural insight into histone recognition by the CECR2 bromodomain and establish a foundation for understanding how it contributes to chromatin engagement.
Malaria is caused by Plasmodium parasites, and its clinical symptoms are a result of parasite invasion of red blood cells and the subsequent cycles of replication and proliferation. In human populations, Plasmodium vivax is responsible for the most widely distributed recurring malaria infections whereas Plasmodium falciparum inflicts the most mortality and morbidity. One well-characterized family of adhesins involved in red blood cell invasion is the reticulocyte-binding-like protein homolog family, known as the RBL superfamily that includes the PfRh family in P. falciparum and PvRBP family in P. vivax. Here we report a collection of nanobodies against three members of this adhesin family, PfRh5, PfRh4, and PvRBP2b. Nanobodies against these Plasmodium adhesins bind with high affinity across several epitopes and can block receptor engagement and inhibit parasite invasion of red blood cells. Using computational design, we generated stabilized PfRh4 variants that encompass the conserved scaffold present in the PfRh and PvRBP families of adhesins and show that several variants with improved expression retained binding to mouse monoclonal antibodies, nanobodies, and Complement Receptor 1, the human receptor for PfRh4. We also observed that most of the inhibitory nanobodies against the three antigens recognized the conserved structural scaffold that define this family of adhesins. These results demonstrate the potential of nanobodies to block malaria parasite invasion into red blood cells.
