Large-scale Conformational Changes Research Articles

Computational design of prefusion-stabilized Herpesvirus gB trimers.

In the absence of effective vaccines, human-infecting members of the Herpesvirus family cause considerable morbidity and mortality worldwide. Herpesvirus infection relies on receptor engagement by a gH/gL glycoprotein complex which induces large-scale conformational changes of the gB glycoprotein to mediate fusion of the viral and host membranes and infection. The instability of all herpesvirus gBs have hindered biochemical and functional studies, thereby limiting our understanding of the infection mechanisms of these pathogens and preventing vaccine design. Here, we computationally stabilized and structurally characterized the Epstein-Barr virus prefusion gB ectodomain trimer, providing an atomic-level description of this key therapeutic target. We show that this stabilization strategy is broadly applicable to other herpesvirus gB trimers and identified conformational intermediates supporting a previously unanticipated mechanism of gB-mediated fusion. These findings provide a blueprint to develop vaccine candidates for these pathogens with major public health burden.

Non-averaged single-molecule tertiary structures reveal RNA self-folding through individual-particle cryo-electron tomography

Large-scale and continuous conformational changes in the RNA self-folding process present significant challenges for structural studies, often requiring trade-offs between resolution and observational scope. Here, we utilize individual-particle cryo-electron tomography (IPET) to examine the post-transcriptional self-folding process of designed RNA origami 6-helix bundle with a clasp helix (6HBC). By avoiding selection, classification, averaging, or chemical fixation and optimizing cryo-ET data acquisition parameters, we reconstruct 120 three-dimensional (3D) density maps from 120 individual particles at an electron dose of no more than 168 e–Å−2, achieving averaged resolutions ranging from 23 to 35 Å, as estimated by Fourier shell correlation (FSC) at 0.5. Each map allows us to identify distinct RNA helices and determine a unique tertiary structure. Statistical analysis of these 120 structures confirms two reported conformations and reveals a range of kinetically trapped, intermediate, and highly compacted states, demonstrating a maturation folding landscape likely driven by helix-helix compaction interactions.

Cryo-EM Structures of the Plasmodium falciparum Apicoplast DNA Polymerase

The apicoplast DNA polymerase (apPol) from Plasmodium falciparum is essential for the parasite’s survival, making it a prime target for antimalarial therapies. Here, we present cryo-electron microscopy structures of the apPol in complex with DNA and incoming nucleotide, offering insights into its molecular mechanisms. Our structural analysis reveals that apPol contains critical residues for high-fidelity DNA synthesis, but lacks certain structural elements to confer processive DNA synthesis during replication, suggesting the presence of additional accessory factors. The enzyme exhibits large-scale conformational changes transitioning upon DNA and nucleotide binding, particularly within the fingers and thumb subdomains. These movements reveal potential allosteric sites that could serve as targets for drug design. Our findings provide a foundation for advancing the understanding of apPol’s unique functional mechanisms and potentially offering new avenues for the development of novel inhibitors and therapeutic interventions against malaria.

Magnesium Regulates RNA Ring Dynamics and Folding in Subgenomic Flaviviral RNA.

Mosquito-borne flaviviruses including dengue, Zika, yellow fever, and regional encephalitis produce a large amount of short subgenomic flaviviral RNAs during infection. A segment of these RNAs named as xrRNA1 features a multi-pseudoknot (PK)-associated structure, which resists the host cell enzyme (XRN1) from degrading the viral RNA. We investigate how this long-range RNA PK folds in the presence of counterions, specifically in a mix of monovalent (K+) and divalent (Mg2+) salts at physiological concentrations. In this study, we use extensive explicit solvent molecular dynamics (MD) simulations to characterize the RNA ion environment of the folded RNA conformation, as determined by the crystal structure. This allowed us to identify the precise locations of various coordinated RNA-Mg2+ interactions, including inner-sphere/chelated and outer-sphere coordinated Mg2+. Given that RNA folding involves large-scale conformational changes, making it challenging to explore through classical MD simulations, we investigate the folding mechanism of xrRNA1 using an all-atom structure-based RNA model with a hybrid implicit-explicit treatment of the ion environment via the dynamic counterion condensation model, both with and without physiological Mg2+ concentration. The study reveals potential folding pathways for this xrRNA1, which is consistent with the results obtained from optical tweezer experiments. The equilibrium and free energy simulations both capture a dynamic equilibrium between the ring-open and ring-close states of the RNA, driven by a long-range PK interaction. Free energy calculations reveal that with the addition of Mg2+ ions, the equilibrium shifts more toward the ring-close state. A detailed analysis of the free energy pathways and ion-mediated contact probability map highlights the critical role of Mg2+ in bridging G50 and A33. This Mg2+-mediated connection helps form the long-range PK which in turn controls the transition between the ring-open and ring-close states. The study underscores the critical role of Mg2+ in the RNA folding transition, highlighting specific locations of Mg2+ contributing to the stabilization of long-range PK connections likely to enhance the robustness of Xrn1 resistance of flaviviral xrRNAs.

A membrane-associated conveyor belt controls the rotational direction of the bacterial type 9 secretion system.

Many bacteria utilize the type 9 secretion system (T9SS) for gliding motility, surface colonization, and pathogenesis. This dual-function motor supports both gliding motility and protein secretion, where rotation of the T9SS plays a central role. Fueled by the energy of the stored proton motive force and transmitted through the torque of membrane-anchored stator units, the rotary T9SS propels an adhesin-coated conveyor belt along the bacterial outer membrane like a molecular snowmobile, thereby enabling gliding motion. However, the mechanisms controlling the rotational direction and gliding motility of T9SS remain elusive. Shedding light on this mechanism, we find that in the gliding bacterium Flavobacterium johnsoniae , deletion of the C-terminus of a conveyor belt protein controls, and in fact, reverses the rotational direction of T9SS from counterclockwise (CCW) to clockwise (CW). Largescale conformational changes at the interface of the T9SS ring with the C-terminus of the conveyer belt, as well as those of the ring protein themselves, in concert with a CW bias of the stators general rotation brings forth a 'tri-component gearset' model: the conveyor belt controls the conformation of the T9SS ring, and thereby its rotational direction. Consequently, the CW rotating stator either push the outer edge of the T9SS rings, causing its CCW rotation or press against the inner surface of the rings, resulting in CW rotation. This regulatory mechanism exemplifies how an outer membrane associated conveyor belt adjusts the rotational direction of its driver, the T9SS, thus providing adaptive sensory feedback to control the motility of a molecular snowmobile.

Utilizing Molecular Dynamics Simulations, Machine Learning, Cryo-EM, and NMR Spectroscopy to Predict and Validate Protein Dynamics.

Protein dynamics play a crucial role in biological function, encompassing motions ranging from atomic vibrations to large-scale conformational changes. Recent advancements in experimental techniques, computational methods, and artificial intelligence have revolutionized our understanding of protein dynamics. Nuclear magnetic resonance spectroscopy provides atomic-resolution insights, while molecular dynamics simulations offer detailed trajectories of protein motions. Computational methods applied to X-ray crystallography and cryo-electron microscopy (cryo-EM) have enabled the exploration of protein dynamics, capturing conformational ensembles that were previously unattainable. The integration of machine learning, exemplified by AlphaFold2, has accelerated structure prediction and dynamics analysis. These approaches have revealed the importance of protein dynamics in allosteric regulation, enzyme catalysis, and intrinsically disordered proteins. The shift towards ensemble representations of protein structures and the application of single-molecule techniques have further enhanced our ability to capture the dynamic nature of proteins. Understanding protein dynamics is essential for elucidating biological mechanisms, designing drugs, and developing novel biocatalysts, marking a significant paradigm shift in structural biology and drug discovery.

Disorder-to-order active site capping regulates the rate-limiting step of the inositol pathway

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD+-dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol pathway. Previous structural studies focused on the detailed molecular mechanism, neglecting large-scale conformational changes that drive the function of this 240 kDa homotetrameric complex. In this study, we identified the active, endogenous MIPS in cell extracts from the thermophilic fungus Thermochaetoides thermophila. By resolving the native structure at 2.48 Å (FSC = 0.143), we revealed a fully populated active site. Utilizing 3D variability analysis, we uncovered conformational states of MIPS, enabling us to directly visualize an order-to-disorder transition at its catalytic center. An acyclic intermediate of G6P occupied the active site in two out of the three conformational states, indicating a catalytic mechanism where electrostatic stabilization of high-energy intermediates plays a crucial role. Examination of all isomerases with known structures revealed similar fluctuations in secondary structure within their active sites. Based on these findings, we established a conformational selection model that governs substrate binding and eventually inositol availability. In particular, the ground state of MIPS demonstrates structural configurations regardless of substrate binding, a pattern observed across various isomerases. These findings contribute to the understanding of MIPS structure-based function, serving as a template for future studies targeting regulation and potential therapeutic applications.

Combining quantitative proteomics and interactomics for a deeper insight into molecular differences between human cell lines.

Cellular functional pathways have evolved through selection based on fitness benefits conferred through protein intra- and inter-molecular interactions that comprise all protein conformational features and protein-protein interactions, collectively referred to as the interactome. While the interactome is regulated by proteome levels, it is also regulated independently by, post translational modification, co-factor, and ligand levels, as well as local protein environmental factors, such as osmolyte concentration, pH, ionic strength, temperature and others. In modern biomedical research, cultivatable cell lines have become an indispensable tool, with selection of optimal cell lines that exhibit specific functional profiles being critical for success in many cases. While it is clear that cell lines derived from different cell types have differential proteome levels, increased understanding of large-scale functional differences requires additional information beyond abundance level measurements, including how protein conformations and interactions are altered in certain cell types to shape functional landscapes. Here, we employed quantitative in vivo protein cross-linking coupled to mass spectrometry to probe large-scale protein conformational and interaction changes among three commonly employed human cell lines, HEK293, MCF-7, and HeLa cells. Isobaric quantitative Protein Interaction Reporter (iqPIR) technologies were used to obtain quantitative values of cross-linked peptides across three cell lines. These data illustrated highly reproducible (R2 values larger than 0.8 for all biological replicates) quantitative interactome levels across multiple biological replicates. We also measured protein abundance levels in these cells using data independent acquisition quantitative proteomics methods. Combining quantitative interactome and proteomics information allowed visualization of cell type-specific interactome changes mediated by proteome level adaptations as well as independently regulated interactome changes to gain deeper insight into possible drivers of these changes. Among the biggest detected alterations in protein interactions and conformations are changes in cytoskeletal proteins, RNA-binding proteins, chromatin remodeling complexes, mitochondrial proteins, and others. Overall, these data demonstrate the utility and reproducibility of quantitative cross-linking to study systems-level interactome variations. Moreover, these results illustrate how combined quantitative interactomics and proteomics can provide unique insight on cellular functional landscapes.

Scaffold-enabled high-resolution cryo-EM structure determination of RNA.

Cryo-EM structure determination of protein-free RNAs has remained difficult with most attempts yielding low to moderate resolution and lacking nucleotide-level detail. These difficulties are compounded for small RNAs as cryo-EM is inherently more difficult for lower molecular weight macromolecules. Here we present a strategy for fusing small RNAs to a group II intron that yields high resolution structures of the appended RNA, which we demonstrate with the 86-nucleotide thiamine pyrophosphate (TPP) riboswitch, and visualizing the riboswitch ligand binding pocket at 2.5 Å resolution. We also determined the structure of the ligand-free apo state and observe that the aptamer domain of the riboswitch undergoes a large-scale conformational change upon ligand binding, illustrating how small molecule binding to an RNA can induce large effects on gene expression. This study both sets a new standard for cryo-EM riboswitch visualization and offers a versatile strategy applicable to a broad range of small to moderate-sized RNAs, which were previously intractable for high-resolution cryo-EM studies.

Nanomechanical collective vibration of SARS-CoV-2 spike proteins.

The development of effective therapeutics against COVID-19 requires a thorough understanding of the receptor recognition mechanism of the SARS-CoV-2 spike (S) protein. Here the multidomain collective dynamics on the trimer of the spike protein has been analyzed using normal mode analysis (NMA). A common nanomechanical profile was identified in the spike proteins of SARS-CoV-2 and its variants. The profile involves collective vibrations of the receptor-binding domain (RBD) and the N-terminal domain (NTD), which may mediate the physical interaction process. Quantitative analysis of the collective modes suggests a nanomechanical property involving large-scale conformational changes, which explains the difference in receptor binding affinity among different variants. These results support the use of intrinsic global dynamics as a valuable perspective for studying the allosteric and functional mechanisms of the S protein. This approach also provides a low-cost theoretical toolkit for screening potential pathogenic mutations and drug targets.

Time-Resolved Ion Mobility Mass Spectrometry to Solve Conformational Changes in a Cryptochrome.

Many biological mechanisms rely on the precise control of conformational changes in proteins. Understanding such dynamic processes requires methods for determining structures and their temporal evolution. In this study, we introduce a novel approach to time-resolved ion mobility mass spectrometry. We validated the method on a simple photoreceptor model and applied it to a more complex system, the animal-like cryptochrome from Chlamydomonas reinhardtii (CraCRY), to determine the role of specific amino acids affecting the conformational dynamics as reaction to blue light activation. In our setup, using a high-power LED mounted in the source region of an ion mobility mass spectrometer, we allow a time-resolved evaluation of mass and ion mobility spectra. Cryptochromes like CraCRY are a widespread type of blue light photoreceptors and mediate various light-triggered biological functions upon excitation of their inbuilt flavin chromophore. Another hallmark of cryptochromes is their flexible carboxy-terminal extension (CTE), whose structure and function as well as the details of its interaction with the photolyase homology region are not yet fully understood and differ among different cryptochromes types. Here, we addressed the highly conserved C-terminal domain of CraCRY, to study the effects of single mutations on the structural transition of the C-terminal helix α22 and the attached CTE upon lit-state formation. We show that D321, the putative proton acceptor of the terminal proton-coupled electron transfer event from Y373, is essential for triggering the large-scale conformational changes of helix α22 and the CTE in the lit state, while D323 influences the timing.

Testing the feasibility of targeting a conserved region on the S2 domain of the SARS-CoV-2 spike protein

The efficacy of vaccines against the SARS-CoV-2 virus significantly declines with the emergence of mutant strains, prompting investigation into the feasibility of targeting highly conserved but often cryptic regions on the S2 domain of spike protein. Using tools from molecular dynamics, we find that exposure of a conserved S2 epitope located in the central helices below the receptor binding domains would require large-scale motion beyond receptor binding domain up-down motion, but, along the reaction coordinates we explored, it is unlikely to be exposed by such large-scale dynamic fluctuations of the S1 domain without any external facilitating factors, despite some previous computational evidence suggesting transient exposure of this region. Furthermore, glycans, particularly those on N165 and N234, hinder S2-exposing opening dynamics, and thus stabilize spike in addition to immunologically shielding the protein surface. Although the S2 epitope region examined here is central to large-scale conformational changes during viral entry, free energy landscape analysis obtained using the path coordinate formalism reveals no inherent “loaded spring” effect, suggesting that a vaccine immunogen would tend to present the epitope in a prefusion-like conformation and may be effective in neutralization. These findings contribute to a deeper understanding of the dynamic origins of the function of the spike protein, as well as further characterizing the feasibility of the S2 epitope as a therapeutic target.

Structural transitions modulate the chaperone activities of Grp94

Hsp90s are ATP-dependent chaperones that collaborate with co-chaperones and Hsp70s to remodel client proteins. Grp94 is the ER Hsp90 homolog essential for folding multiple secretory and membrane proteins. Grp94 interacts with the ER Hsp70, BiP, although the collaboration of the ER chaperones in protein remodeling is not well understood. Grp94 undergoes large-scale conformational changes that are coupled to chaperone activity. Within Grp94, a region called the pre-N domain suppresses ATP hydrolysis and conformational transitions to the active chaperone conformation. In this work, we combined in vivo and in vitro functional assays and structural studies to characterize the chaperone mechanism of Grp94. We show that Grp94 directly collaborates with the BiP chaperone system to fold clients. Grp94's pre-N domain is not necessary for Grp94-client interactions. The folding of some Grp94 clients does not require direct interactions between Grp94 and BiP in vivo, suggesting that the canonical collaboration may not be a general chaperone mechanism for Grp94. The BiP co-chaperone DnaJB11 promotes the interaction between Grp94 and BiP, relieving the pre-N domain suppression of Grp94's ATP hydrolysis activity. In structural studies, we find that ATP binding by Grp94 alters the ATP lid conformation, while BiP binding stabilizes a partially closed Grp94 intermediate. Together, BiP and ATP push Grp94 into the active closed conformation for client folding. We also find that nucleotide binding reduces Grp94's affinity for clients, which is important for productive client folding. Alteration of client affinity by nucleotide binding may be a conserved chaperone mechanism for a subset of ER chaperones.

Archaeal GPN-loop GTPases involve a lock-switch-rock mechanism for GTP hydrolysis.

GPN-loop GTPases have been found to be crucial for eukaryotic RNA polymerase II assembly and nuclear trafficking. Despite their ubiquitous occurrence in eukaryotes and archaea, the mechanism by which these GTPases mediate their function is unknown. Our study on an archaeal representative from Sulfolobus acidocaldarius showed that these dimeric GTPases undergo large-scale conformational changes upon GTP hydrolysis, which can be summarized as a lock-switch-rock mechanism. The observed requirement of SaGPN for motility appears to be due to its large footprint on the archaeal proteome.

Glycosylation and Crowded Membrane Effects on Influenza Neuraminidase Stability and Dynamics.

All protein simulations are conducted with varying degrees of simplification, oftentimes with unknown ramifications about how these simplifications affect the interpretability of the results. In this work, we investigated how protein glycosylation and lateral crowding effects modulate an array of properties characterizing the stability and dynamics of influenza neuraminidase. We constructed three systems: (1) glycosylated neuraminidase in a whole virion (i.e., crowded membrane) environment, (2) glycosylated neuraminidase in its own lipid bilayer, and (3) unglycosylated neuraminidase in its own lipid bilayer. We saw that glycans tend to stabilize the protein structure and reduce its conformational flexibility while restricting the solvent movement. Conversely, a crowded membrane environment encouraged exploration of the free energy landscape and a large-scale conformational change, while making the protein structure more compact. Understanding these effects informs what factors one must consider in attempting to recapture the desired level of physical accuracy.

Crystal structures of human and mouse ketohexokinase provide a structural basis for species- and isoform-selective inhibitor design.

A molecular understanding of the proteins involved in fructose metabolism is essential for controlling the current spread of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism starts with the phosphorylation of D-fructose to fructose 1-phosphate by ketohexokinase (KHK). KHK exists in two alternatively spliced isoforms: the hepatic and intestinal isoform KHK-C and the peripheral isoform KHK-A. Here, the structure of apo murine KHK (mKHK), which differs from structures of human KHK in overall conformation, is reported. An isoform-selective ligand, which offers a 50-fold higher potency on mKHK and human KHK-A compared with KHK-C, is further characterized. In mKHK, large-scale conformational changes are observed upon ligand binding. The structures suggest a combined strategy for the design of species- and isoform-selective KHK inhibitors.

Redox driven B12-ligand switch drives CarH photoresponse

CarH is a coenzyme B12-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes.

Quinone Catalysis Modulates Proton Transfer Reactions in the Membrane Domain of Respiratory Complex I.

Complex I is a redox-driven proton pump that drives electron transport chains and powers oxidative phosphorylation across all domains of life. Yet, despite recently resolved structures from multiple organisms, it still remains unclear how the redox reactions in Complex I trigger proton pumping up to 200 Å away from the active site. Here, we show that the proton-coupled electron transfer reactions during quinone reduction drive long-range conformational changes of conserved loops and trans-membrane (TM) helices in the membrane domain of Complex I from Yarrowia lipolytica. We find that the conformational switching triggers a π → α transition in a TM helix (TM3ND6) and establishes a proton pathway between the quinone chamber and the antiporter-like subunits, responsible for proton pumping. Our large-scale (>20 μs) atomistic molecular dynamics (MD) simulations in combination with quantum/classical (QM/MM) free energy calculations show that the helix transition controls the barrier for proton transfer reactions by wetting transitions and electrostatic effects. The conformational switching is enabled by re-arrangements of ion pairs that propagate from the quinone binding site to the membrane domain via an extended network of conserved residues. We find that these redox-driven changes create a conserved coupling network within the Complex I superfamily, with point mutations leading to drastic activity changes and mitochondrial disorders. On a general level, our findings illustrate how catalysis controls large-scale protein conformational changes and enables ion transport across biological membranes.

Structural basis of the T4 bacteriophage primosome assembly and primer synthesis

The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer length is defined are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates. We show that gp41 alone is an open spiral, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the ring closure and activates the helicase. Helicase activation exposes a cryptic hydrophobic surface to recruit the gp61 primase. The primase binds the helicase in a bipartite mode in which the N-terminal Zn-binding domain and the C-terminal RNA polymerase domain each contain a helicase-interacting motif that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Our study reveals the T4 primosome assembly process and sheds light on the RNA primer synthesis mechanism.

Principles of human pre-60S biogenesis.

During the early stages of human large ribosomal subunit (60S) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60S particles by an unknown mechanism. Here, we report a series of cryo-electron microscopy structures of human nucleolar and nuclear pre-60S assembly intermediates at resolutions of 2.5 to 3.2 angstroms. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how guanosine triphosphatases and adenosine triphosphatase couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA-processing complex, the rixosome, couples large-scale RNA conformational changes with pre-ribosomal RNA processing by the RNA degradation machinery. Our ensemble of human pre-60S particles provides a rich foundation with which to elucidate the molecular principles of ribosome formation.