Structural Biology Approaches Research Articles

Single-molecule imaging and molecular dynamics simulations reveal early activation of the MET receptor in cells

Embedding of cell-surface receptors into a membrane defines their dynamics but also complicates experimental characterization of their signaling complexes. The hepatocyte growth factor receptor MET is a receptor tyrosine kinase involved in cellular processes such as proliferation, migration, and survival. It is also targeted by the pathogen Listeria monocytogenes, whose invasion protein, internalin B (InlB), binds to MET, forming a signaling dimer that triggers pathogen internalization. Here we use an integrative structural biology approach, combining molecular dynamics simulations and single-molecule Förster resonance energy transfer (smFRET) in cells, to investigate the early stages of MET activation. Our simulations show that InlB binding stabilizes MET in a conformation that promotes dimer formation. smFRET reveals that the in situ dimer structure closely resembles one of two previously published crystal structures, though with key differences. This study refines our understanding of MET activation and provides a methodological framework for studying other plasma membrane receptors.

Orphan GPCRs in Neurodegenerative Disorders: Integrating Structural Biology and Drug Discovery Approaches.

Neurodegenerative disorders, particularly Alzheimer's and Parkinson's diseases, continue to challenge modern medicine despite therapeutic advances. Orphan G-protein-coupled receptors (GPCRs) have emerged as promising targets in the central nervous system, offering new avenues for drug development. This review focuses on the structural biology of orphan GPCRs implicated in these disorders, providing a comprehensive analysis of their molecular architecture and functional mechanisms. We examine recent breakthroughs in structural determination techniques, such as cryo-electron microscopy and X-ray crystallography, which have elucidated the intricate conformations of these receptors. The review highlights how structural insights inform our understanding of orphan GPCR activation, ligand binding and signaling pathways. By integrating structural data with molecular pharmacology, we explore the potential of structure-guided approaches in developing targeted therapeutics toward orphan GPCRs. This structural-biology-centered perspective aims to deepen our comprehension of orphan GPCRs and guide future drug discovery efforts in neurodegenerative disorders.

Mechanism and regulation of kinesin motors.

Kinesins are a diverse superfamily of microtubule-based motors that perform fundamental roles in intracellular transport, cytoskeletal dynamics and cell division. These motors share a characteristic motor domain that powers unidirectional motility and force generation along microtubules, and they possess unique tail domains that recruit accessory proteins and facilitate oligomerization, regulation and cargo recognition. The location, direction and timing of kinesin-driven processes are tightly regulated by various cofactors, adaptors, microtubule tracks and microtubule-associated proteins. This Review focuses on recent structural and functional studies that reveal how members of the kinesin superfamily use the energy of ATP hydrolysis to transport cargoes, depolymerize microtubules and regulate microtubule dynamics. I also survey how accessory proteins and post-translational modifications regulate the autoinhibition, cargo binding and motility of some of the best-studied kinesins. Despite much progress, the mechanism and regulation of kinesins are still emerging, and unresolved questions can now be tackled using newly developed approaches in biophysics and structural biology.

Robust approach for production of the human oncology target Aurora kinase B in complex with its binding partner INCENP

Protein kinases are key players in many eukaryotic signal transduction cascades and are as a result often linked to human disease. In humans, the mitotic protein kinase family of Aurora kinases consist of three members: Aurora A, B and C. All three members are involved in cell division with proposed implications in various human cancers. The human Aurora kinase B has in particular proven challenging to study with structural biology approaches, and this is mainly due to difficulties in producing the large quantities of active enzyme required for such studies. Here, we present a novel and E. coli-based production system that allows for production of milligram quantities of well-folded and active human Aurora B in complex with its binding partner INCENP. The complex is produced as a continuous polypeptide chain and the resulting fusion protein is cleaved with TEV protease to generate a stable and native heterodimer of the Aurora B:INCENP complex. The activity, stability and degree of phosphorylation of the protein complex was quantified by using a coupled ATPase assay, 31P NMR spectroscopy and mass spectrometry. The developed production system enables isotope labeling and we here report the first 1H–15N-HSQC of the human Aurora B:INCENP complex. Our developed production strategy paves the way for future structural and functional studies of Aurora B and can as such assist the development of novel anticancer drugs targeting this important mitotic protein kinase.

Structural Plasticity within 3-Hydroxy-3-Methylglutaryl Synthases Catalyzing the First Step of β-Branching in Polyketide Biosynthesis Underpins a Dynamic Mechanism of Substrate Accommodation.

Understanding how enzymes have been repurposed by evolution to carry out new functions is a key goal of mechanistic enzymology. In this study we aimed to identify the adaptations required to allow the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) enzymes of primary isoprenoid assembly to function in specialized polyketide biosynthetic pathways, where they initiate β-branching. This role notably necessitates that the HMG synthases (HMGSs) act on substrates tethered to noncatalytic acyl carrier protein (ACP) domains instead of coenzyme A, and accommodation of substantially larger chains within the active sites. Here, we show using a combination of X-ray crystallography and small-angle X-ray scattering, that a model HMGS from the virginiamycin system exhibits markedly increased flexibility relative to its characterized HMGCS counterparts. This mobility encompasses multiple secondary structural elements that define the dimensions and chemical nature of the active site, as well the catalytic residues themselves. This result was unexpected given the well-ordered character of the HMGS within the context of an HMGS/ACP complex, but analysis by synchrotron radiation circular dichroism demonstrates that this interaction leads to increased HMGS folding. This flexible to more rigid transition is notably not accounted for by AlphaFold2, which yielded a structural model incompatible with binding of the native substrates. Taken together, these results illustrate the continued necessity of an integrative structural biology approach combining crystallographic and solution-phase data for elucidating the mechanisms underlying enzyme remodeling, information which can inform strategies to replicate such evolution effectively in the laboratory.

Residue-Specific Epitope Mapping of the PD-1/Nivolumab Interaction Using X-ray Footprinting Mass Spectrometry.

X-ray footprinting coupled with mass spectrometry (XFMS) presents a novel approach in structural biology, offering insights into protein conformation and dynamics in the solution state. The interaction of the cancer-immunotherapy monoclonal antibody nivolumab with its antigen target PD-1 was used to showcase the utility of XFMS against the previously published crystal structure of the complex. Changes in side-chain solvent accessibility, as determined by the oxidative footprint of free PD-1 versus PD-1 bound to nivolumab, agree with the binding interface side-chain interactions reported from the crystal structure of the complex. The N-linked glycosylation sites of PD-1 were confirmed through an LC-MS/MS-based deglycosylation analysis of asparagine deamidation. In addition, subtle changes in side-chain solvent accessibility were observed in the C'D loop region of PD-1 upon complex formation with nivolumab.

How Can the Diterpene Synthase CotB2V80L Alter the Product Profile?

AbstractStructural diversity of diterpenes is mediated by the enigmatic family of diterpene synthases. The overall enzymatic contribution hereby lies in a carefully concerted chemistry of highly reactive carbocation intermediates mainly guided by aromatic and polar amino acid side chains and the pyrophosphate cofactor. To date several studies aimed to shed light on the mechanism underlining terpene synthases chemistry. Specifically, the diterpene synthase CotB2 serves as model enzyme for detailed mutagenesis studies. Here we investigate the catalytic mechanism of CotB2 variant V80L in a holistic, biochemical, structural, and computational biology approach. We were able to identify an altered product profile compared to CotB2WT for the substrates geranylgeranyl diphosphate and farnesyl diphosphate. Moreover, we solved the crystal structure, and shed further light on terpene synthase chemistry by modelling of the substrate and intermediate binding.

Structural characterization of TIR-domain signalosomes through a combination of structural biology approaches.

The TIR (Toll/interleukin-1 receptor) domain represents a vital structural element shared by proteins with roles in immunity signalling pathways across phyla (from humans and plants to bacteria). Decades of research have finally led to identifying the key features of the molecular basis of signalling by these domains, including the formation of open-ended (filamentous) assemblies (responsible for the signalling by cooperative assembly formation mechanism, SCAF) and enzymatic activities involving the cleavage of nucleotides. We present a historical perspective of the research that led to this understanding, highlighting the roles that different structural methods played in this process: X-ray crystallography (including serial crystallography), microED (micro-crystal electron diffraction), NMR (nuclear magnetic resonance) spectroscopy and cryo-EM (cryogenic electron microscopy) involving helical reconstruction and single-particle analysis. This perspective emphasizes the complementarity of different structural approaches.

A new mode of inhibition.

Complementary structural biology approaches reveal how an agonist and a covalent inhibitor simultaneously bind to a nuclear receptor.

Integration of AlphaFold with Molecular Dynamics for Efficient Conformational Sampling of Transporter Protein NarK.

Transporter proteins carry their substrate across the cell membrane by changing their conformation. Thus, conformational dynamics are crucial for transport function. However, clarifying the complete transport cycle is challenging even with the current structural biology approach. Molecular dynamics (MD) simulation is a computational approach that can provide the time-resolved conformational dynamics of transporter proteins in atomic details but suffers from a high computational cost. Here, we integrate state-of-the-art protein structure prediction AI, AlphaFold2 (AF2), with MD simulation to reduce the computational cost. Focusing on the transporter protein NarK, we first show that AF2 sampled broad conformations of NarK, including the inward-open, occluded, and outward-open states. We also applied the coevolution-informed mutation in AF2, identifying state-shifting mutations. Then, we show that MD simulations from AF2-generated outward-open conformation, which is experimentally unresolved, captured the essence of the conformational state. We also found that MD simulations from AF2-generated intermediates showed transient dynamics like a transition state connecting two conformational states. This study paves the way for efficient conformational sampling of transporter proteins.

What Structural Biology Tells Us About the Mode of Action and Detection of Toxicants.

The study of the adverse effects of chemical substances on living organisms is an old and intense field of research. However, toxicological and environmental health sciences have long been dominated by descriptive approaches that enable associations or correlations but relatively few robust causal links and molecular mechanisms. Recent achievements have shown that structural biology approaches can bring this added value to the field. By providing atomic-level information, structural biology is a powerful tool to decipher the mechanisms by which toxicants bind to and alter the normal function of essential cell components, causing adverse effects. Here, using endocrine-disrupting chemicals as illustrative examples, we describe recent advances in the structure-based understanding of their modes of action and how this knowledge can be exploited to develop computational tools aimed at predicting properties of large collections of compounds.

Structure analysis of the telomere resolvase from the Lyme disease spirochete Borrelia garinii reveals functional divergence of its C-terminal domain.

Borrelia spirochetes are the causative agents of Lyme disease and relapsing fever, two of the most common tick-borne illnesses. A characteristic feature of these spirochetes is their highly segmented genomes which consists of a linear chromosome and a mixture of up to approximately 24 linear and circular extrachromosomal plasmids. The complexity of this genomic arrangement requires multiple strategies for efficient replication and partitioning during cell division, including the generation of hairpin ends found on linear replicons mediated by the essential enzyme ResT, a telomere resolvase. Using an integrative structural biology approach employing advanced modelling, circular dichroism, X-ray crystallography and small-angle X-ray scattering, we have generated high resolution structural data on ResT from B. garinii. Our data provides the first high-resolution structures of ResT from Borrelia spirochetes and revealed active site positioning in the catalytic domain. We also demonstrate that the C-terminal domain of ResT is required for both transesterification steps of telomere resolution, and is a requirement for DNA binding, distinguishing ResT from other telomere resolvases from phage and bacteria. These results advance our understanding of the molecular function of this essential enzyme involved in genome maintenance in Borrelia pathogens.

Structural details of helix-mediated TDP-43 C-terminal domain multimerization.

The primarily disordered C-terminal domain (CTD) of TAR DNA binding protein-43 (TDP-43), a key nuclear protein in RNA metabolism, forms neuronal inclusions in several neurodegenerative diseases. A conserved region (CR, spanning residues 319-341) in CTD forms transient helix-helix contacts important for its higher-order oligomerization and function that are disrupted by ALS-associated mutations. However, the structural details of CR assembly and the explanation for several ALS-associated variants' impact on phase separation and function remain unclear due to challenges in analyzing the dynamic association of TDP-43 CTD using traditional structural biology approaches. By employing an integrative approach, combining biophysical experiments, biochemical assays, AlphaFold2-Multimer (AF2-Multimer), and atomistic simulations, we generated structural models of helical oligomerization of TDP-43 CR. Using NMR, we first established that the native state of TDP-43 CR under physiological conditions is α-helical. Next, alanine scanning mutagenesis revealed that while hydrophobic residues in the CR are important for CR assembly, phase separation and TDP-43 nuclear retention function, polar residues down regulate these processes. Finally, pairing AF2-Multimer modeling with AAMD simulations indicated that dynamic, oligomeric assemblies of TDP-43 that are stabilized by a methionine-rich core with specific contributions from a tryptophan/leucine pair. In conclusion, our results advance the structural understanding of the mechanisms driving TDP-43 function and provide a window into the initial stages of its conversion into pathogenic aggregates.

The antibiotic resistance crisis and the development of new antibiotics.

The Global Burden of Disease report of 2019 estimated 14 million infection-related deaths, making it the second leading cause of death after ischaemic heart disease. Bacterial pathogens accounted for 7.7 million deaths and deaths attributable to bacterial antibiotic resistance amounted to 1.3 million, describing a clear demand for novel antibiotics. Antibiotic development had its golden age in 1930-1960. Following failures in the screening of chemical libraries for novel antibiotics at the beginning of this century, the high cost of launching new antibiotics (estimated at US$ 1.4 billion per registered drug) and difficulties in achieving a return of investment for novel antibiotics, pharmaceutical industry has mostly left the field. The current Lilliput review analyses the question whether scientific or economic hurdles prevented the registration of new antibiotics. Scientifically, substantial progress has been achieved over recent years to define the chemical properties needed to overcome the permeation barrier in Gram-negative pathogens; in extending the chemical space of antibiotic candidates by full modular synthesis of suitable molecules; by extending bioprospecting to previously 'unculturable' bacteria or unusual bacteria; by attacking bacterial targets on the outer bacterial membrane; and by looking for support from structural biology, genomics, molecular genetics, phylogenetic analyses and deep machine learning approaches. However, these research activities were mostly conducted by academic researchers and biotech companies with limited financial resources. It thus seems that the development of new antibiotics, frequently described as the drying of the pipeline, is less limited by lack of scientific insight than by lack of the mobilization of the monetary resources needed to bring these discoveries to the market despite recent financial push and pull efforts of the public sector.

Master corepressor inactivation through multivalent SLiM-induced polymerization mediated by the oncogene suppressor RAI2

While the elucidation of regulatory mechanisms of folded proteins is facilitated due to their amenability to high-resolution structural characterization, investigation of these mechanisms in disordered proteins is more challenging due to their structural heterogeneity, which can be captured by a variety of biophysical approaches. Here, we used the transcriptional master corepressor CtBP, which binds the putative metastasis suppressor RAI2 through repetitive SLiMs, as a model system. Using cryo-electron microscopy embedded in an integrative structural biology approach, we show that RAI2 unexpectedly induces CtBP polymerization through filaments of stacked tetrameric CtBP layers. These filaments lead to RAI2-mediated CtBP nuclear foci and relieve its corepressor function in RAI2-expressing cancer cells. The impact of RAI2-mediated CtBP loss-of-function is illustrated by the analysis of a diverse cohort of prostate cancer patients, which reveals a substantial decrease in RAI2 in advanced treatment-resistant cancer subtypes. As RAI2-like SLiM motifs are found in a wide range of organisms, including pathogenic viruses, our findings serve as a paradigm for diverse functional effects through multivalent interaction-mediated polymerization by disordered proteins in healthy and diseased conditions.

UPF1 helicase orchestrates mutually exclusive interactions with the SMG6 endonuclease and UPF2.

Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. Here, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by different conformational states connected to the RNA-binding status of UPF1. Our findings rationalize a key event in metazoan NMD and advance our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.

Beyond the VSG layer: Exploring the role of intrinsic disorder in the invariant surface glycoproteins of African trypanosomes.

In the bloodstream of mammalian hosts, African trypanosomes face the challenge of protecting their invariant surface receptors from immune detection. This crucial role is fulfilled by a dense, glycosylated protein layer composed of variant surface glycoproteins (VSGs), which undergo antigenic variation and provide a physical barrier that shields the underlying invariant surface glycoproteins (ISGs). The protective shield's limited permeability comes at the cost of restricted access to the extracellular host environment, raising questions regarding the specific function of the ISG repertoire. In this study, we employ an integrative structural biology approach to show that intrinsically disordered membrane-proximal regions are a common feature of members of the ISG super-family, conferring the ability to switch between compact and elongated conformers. While the folded, membrane-distal ectodomain is buried within the VSG layer for compact conformers, their elongated counterparts would enable the extension beyond it. This dynamic behavior enables ISGs to maintain a low immunogenic footprint while still allowing them to engage with the host environment when necessary. Our findings add further evidence to a dynamic molecular organization of trypanosome surface antigens wherein intrinsic disorder underpins the characteristics of a highly flexible ISG proteome to circumvent the constraints imposed by the VSG coat.

Dynamic localization of the chromosomal passenger complex in trypanosomes is controlled by the orphan kinesins KIN-A and KIN-B.

The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess unique kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles nor how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in Trypanosoma brucei, the kinetoplastid parasite that causes African sleeping sickness. KIN-A and KIN-B form a scaffold for the assembly of the remaining CPC subunits. We show that the C-terminal unstructured tail of KIN-A interacts with the KKT8 complex at kinetochores, while its N-terminal motor domain promotes CPC translocation to spindle microtubules. Thus, the KIN-A:KIN-B complex constitutes a unique 'two-in-one' CPC localization module, which directs the CPC to kinetochores from S phase until metaphase and to the central spindle in anaphase. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora kinases in early eukaryotes.

The SPATA5-SPATA5L1 ATPase complex directs replisome proteostasis to ensure genome integrity

Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.

A Molecular Dynamics Investigation of Ligand-Mediated PPARγ Transrepression

Background and Hypothesis: PPARγ is a nuclear receptor expressed in most cells of the body and exerts metabolic function, including adipocyte differentiation and insulin sensitization. This mechanism is termed transactivation, where ligand binding leads to transcription of target genes. However, PPARγ also has a transrepression activity, where ligand binding inhibits other transcriptional pathways, leading to regulation of the immune system. While the molecular mechanisms of transactivation are well understood, there is little research on the mechanisms of transrepression. Previous studies have implied that these activity states act independently and can be selected for in small molecule design campaigns, but this has yet to be accomplished. Therefore, the aim of this project was to investigate if distinct molecular interactions exist between PPARγ transactivation versus transrepression using a combined computational and structural biology approach.
 Methods: Molecular dynamics (MD) simulations were performed using the CHARMM molecular modeling software to produce 300 ns simulations of PPARγ-ligand complexes. Simulations were analyzed to identify potential protein-ligand interactions, and distances were measured between atoms of interest and compared.
 Results: Although PPARγ’s binding site is large, structural analyses showed that most ligands remained near their initial starting position within the binding pocket throughout an MD simulation. Starting coordinates were taken from previously solved crystal structures or via manual docking based on molecular similarities. Four residues were identified to consistently interact with most of the ligands: Arg280, Ser342, Glu259, and Cys285. These residues differ from previously reported interactions associated with PPARγ transactivation.
 Conclusion: Our results suggest that these residues may play a role in PPARγ transrepression, but these conclusions are preliminary and should be confirmed through further investigation. Future research would include performing replicate simulations and mutational analyses to further explore the involvement of these residues in PPARγ transrepression. Additional PPARγ ligands should also be explored.