Biophysics and Physicobiology

Retinal is a chromophore covalently bound to various photoreceptors. Its photo-induced isomerization triggers a series of structural changes named photocycle, leading to diverse biological functions. Despite tremendous advances in structural biology and artificial intelligence-driven structure prediction, it remains challenging to analyze all photocyclic intermediates. Here, we present an optimized computational approach to calculate RSBH+ isomerization and its induced structural changes based on a classical molecular mechanics approach using quantum mechanically improved retinal force field parameters. Isomerization is induced by an excited state restraint which is subsequently relaxed to allow the return to the electronic ground state. We applied this approach to the key protein of optogenetics, Channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Besides the reformation of the alltrans/CN-anti ground state, we observed the production of a mixture of two isomeric states 13-cis/CN- anti and 13-cis/CN-syn. These findings agree with the previously found branched photocycle model based on experimental data. Our calculations show an asymmetric potential energy landscape of the excited state leading to a corresponding isomerization state distribution. Unlike earlier publications, our procedure describes the retinal photoisomerization on the natural timescale of 500 fs. As our newly derived retinal force field parameter set precisely relies on quantum biological knowledge, it assists to improve the refinement of experimental structure biological data. Our readily customizable strategy provides mechanistic insights at high spatio-temporal resolution, which permits accurate structural predictions of early photocycle intermediates. These insights will stimulate the rational design of optogenetic tools thus providing improved diagnostic and therapeutic approaches for neuronal and other diseases. HighlightsO_LIuniversal method to study molecular mechanism of optogenetic tools C_LIO_LIretinal photo-isomerization calculation in real time C_LIO_LIprediction of branched photo cycle agrees with experimental IR spectroscopic results C_LIO_LIdetected asymmetric excited state potential energy landscape C_LIO_LIassists to improve structural model refinement of retinal proteins C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=116 HEIGHT=200 SRC="FIGDIR/small/707937v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@71f7fdorg.highwire.dtl.DTLVardef@503482org.highwire.dtl.DTLVardef@1a77120org.highwire.dtl.DTLVardef@1f410a0_HPS_FORMAT_FIGEXP M_FIG C_FIG

The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is a respiratory chain enzyme found in pathogenic bacteria, including Vibrio cholerae, and is essential for energy metabolism by generating a transmembrane Na+ gradient that drives ATP synthesis and flagellar motility. Because the molecular structure of Na+-NQR is unrelated to the corresponding mitochondrial H-pumping NADH-quinone oxidoreductase (respiratory complex I), it is a promising antibiotic target. Although it has been shown that Na+ pumping is mediated by an alternating-access conformational change in the NqrD/E subunits, coupled to redox switching of a cofactor, the thermodynamics and kinetics of the conformational transition, including the free-energy profile and the rate-limiting steps, remain unclear. Here, we construct redox-state-dependent Markov state models (MSMs) from extensive molecular dynamics (MD) trajectories in the oxidized and reduced states to quantify the conformational free-energy landscapes and primary transition pathway. To accelerate conformational sampling, MD simulations are initiated from diverse NqrD/E conformations generated by AlphaFold. Our analysis clarifies how the NqrD/E conformation is regulated by the redox state and by Na+ binding to achieve Na+ translocation. This study provides a quantitative framework for understanding ion-pumping mechanisms of redox-driven membrane proteins.

Single molecule methods have become prevalent tools in elucidating molecular processes across various life science fields over the past three decades, driving breakthroughs in understanding their underlying molecular mechanisms. In our study, we employed two single-molecule methods, Forster Resonance Energy Transfer (smFRET) and high-resolution optical tweezers, to investigate the transcription of Mycobacterium tuberculosis RNA polymerase (MtbRNAP) from initiation through to termination. We aim to provide a set of comprehensive biophysical tools to deepen our current understanding of MtbRNAP and its transcription factors. These experimental assays represent an important step towards unraveling the molecular dynamics and interactions that support transcription in Mycobacterium tuberculosis.

Nuclear Pore Complexes (NPCs) are large protein complexes in eukaryotic cells that span the double-membrane of the nucleus and regulate bi-directional transport between nucleus and cytoplasm. T h e NPC core is lined by intrinsically disordered protein chains called nucleoporins (Nups) which form a selective barrier where large macromolecules (cargoes) need to bind to nuclear transport receptors (NTRs) such as Karyopherins (Kaps) to cross. Previous experimental results have suggested that not only Nups but Kaps, too, are important in the transport process of other NTRs/NTR-cargo complexes. In this work, we assess the role of Kaps in the transport of other NTRs (specifically, NTF2s) through the NPC, a process referred to as the "Kap-centric transport model". Here, using coarse-grained MD simulation we show that Kaps are able to direct NTF2s into the Nup meshwork, which leads to their increased flow. Our results also suggest that NTRs follow specific lanes inside the pore to maximize efficient transport.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are are a family of voltage-gated, cyclic-nucleotide modulated Na+/K+ channels that regulate spontaneous rhythmic electrical activity in both the heart and the brain. Understanding differences in the responsiveness to cyclic adenosine monophosphate (cAMP) modulation between HCN isoforms would offer insight into the specific binding interactions that drive channel activation. Using all-atom molecular dynamics (MD) simulations and the free-energy perturbation (FEP) approach, we determined the absolute binding free energy of cAMP to the the cyclicnucleotide-binding domain (CNBD) of HCN isoforms 1-4. By studying the free-energy of ligand binding to the various isoforms of HCN, our study advances the understanding of HCN channel activation and modulation mechanisms. Overall, our work offers insight into explaining differences in channel sensitivity across the isoforms of HCN.

Targeted protein degradation is an emerging approach that utilizes cellular degradation pathways to inhibit a target protein. Small molecules such as molecular glues or PROTACs can be used to mediate the formation of a ternary complex with an E3 ligase and the target protein, which can dramatically enhance the degradation process. This approach is promising for cancer therapy, where degradation of oncogenic proteins can lead to cancer cell toxicity. To design new molecular glues, it is important to develop methods that predict how well a given molecule stabilizes a protein-protein interaction. However, conventional molecular dynamics simulations face challenges in capturing the long-timescale binding and unbinding events that would be used to evaluate this stabilization. In this study, we developed a strategy that allows us to evaluate the stability of protein-protein interactions in the presence of a glue molecule using weighted ensemble simulations in combination with weakened protein-protein interactions. Using this strategy, we generated unbinding trajectories of the DCAF15-RBM39 system with small molecules E7820, Indisulam, and several other Indisulam analogs. We were able to observe distinctly different behaviors between systems with different glues, which was in agreement with their reported EC50 values. We believe this approach could aid drug discovery efforts by expanding the set of druggable targets and improving the success rate of molecular glue development.

Lebers Hereditary Optic Neuropathy (LHON) is a rare genetic condition and severe neurological disorder characterized by dysfunctional mitochondria under extreme oxidative stress, resulting in retinal ganglion cell death and subsequent rapid bilateral loss of central vision. The m.14484T>C mutation in the ND6 subunit of mitochondrial complex I is known for inducing LHON, and is a prevalent LHON-associated mutation, yet its mechanism of impairment at the molecular level is currently unresolved. In this study, we explore the biophysical underpinnings of this mutation and its role in LHON through disruption of human complex I function. We consider, using atomistic simulations, the differential thermodynamics and kinetics of coenzyme Q10 binding between the mutant and wild-type forms, altered dynamics of the complex upon mutation, and key interactions between coenzyme Q10 and complex I binding sites. The hydrogen bond network present near and within the coenzyme Q10 binding domain, along with proper hydration of E-channel residues that couple redox chemistry to proton pumping, is found to be critical for complex I stability and quinone binding, which the ND6-centered mutation disrupts.

Transition metal based compounds are promising therapeutic agents, particularly in cancer treatment. However, predicting their binding sites remains a major challenge. In this work, we investigate the applicability of two tools, Metal3D and Metal1D, for this purpose. Although originally trained to predict zinc ion binding sites only, both predictors successfully identify several experimentally observed binding sites for transition metal complexes directly from apo protein structures. At the same time, we highlight current limitations, such as the sensitivity to side-chain conformations, and discuss possible strategies for improvement. This work provides a first step toward establishing a robust computational pipeline in which rapid and low-cost predictors are able to identify putative hotspots for transition metal binding, which can then be refined using more accurate but computationally demanding methods. Author summaryTransition metals play a crucial role as therapeutic agents, especially in cancer therapy. However, the prediction of their binding site locations is challenging, as accurate computational methods often require time-consuming simulations, making them impractical when many possible binding sites must be explored. In this work, we explored the capability of two binding site predictors, originally developed to locate metal ions in proteins, to identify binding sites for more complex covalently-bound transition metal based agents. We found that these tools can often identify the experimentally-known binding regions, even when starting from the apo structure, in which the protein does not already contain the metal compound. At the same time, our results show clear limitations in more challenging cases, particularly when the binding involves only a single amino acid or when the binding site undergoes major structural rearrangements upon binding. Overall, our study shows that fast predictors can provide valuable early insights in the investigation of the binding sites of covalently-bound transition metal based compounds. When combined with more accurate simulation techniques, they can help focus computational efforts and ultimately support the rational design of transition metal based drugs.

Polyethylene terephthalate (PET) is a commonly used plastic worldwide and reducing its prevalence is crucial to improving environmental pollution. PETase that degrades PET plastic have received a lot of attention recently. This paper evaluates the ester hydrolysis process under both acidic and basic conditions, and shows that the local environment of the protein active site takes advantage of both. High pH in the protein buffer creates a better nucleophile to attack the ester through a proton shuttle channel in the protein, while local hydrogen bonds to the carbonyl of the ester stabilizes the intermediate/transition state of the hydrolysis reaction. With the understanding at the atomic level, we propose two engineering directions that can potentially improve the reactivity of the PETase: 1) increase the alkaline stability of the protein in general; 2) perturb the local hydrogen bond network to increase the partial charge on the PET carbonyl to be hydrolyzed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/703441v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@151b69borg.highwire.dtl.DTLVardef@1abb95dorg.highwire.dtl.DTLVardef@116a225org.highwire.dtl.DTLVardef@ef2bb1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Protein-DNA complexes are involved in vital cellular functions like gene regulation, replication, transcription, packaging, rearrangement, and damage repair. In this work, streamlined geometric formalism for computing the absolute binding free energy was used to obtain chemical accurate in silico estimation of binding free energy of three Protein-DNA complexes. Additionally, molecular interactions between Protein and DNA involved hydrogen bonds, electrostatic, van der Waals, and hydrophobic interactions. Using this formalism, researcher can obtain the absolute binding free energy for a Protein-DNA complex with remarkable accuracy and modest computational cost.

GPI-anchored proteins are crucial cell surface proteins with diverse, organism-specific functions, in eukaryotes. They are produced when the GPI transamidase (GPIT), a five-subunit membrane-bound enzyme complex, attaches a pre-formed GPI anchor to the C-terminal end of nascent proteins on the lumenal face of the endoplasmic reticulum. This process requires the removal of a C-terminal signal sequence (SS) on the substrate protein by the action of an endopeptidase subunit of the GPIT, Gpi8/ PIG-K. Using an AMC-tagged peptide in a cell free (post-mitochondrial fraction) assay, this manuscript studies the steady state kinetics of enzymatic cleavage of the substrate by GPIT of the human pathogenic fungus, C. albicans. We show that Mn+2 enhances activity by improving substrate binding but plays no direct role in substrate cleavage per se. Molecular dynamics simulations suggest that the divalent cation binds at a site away from the active site but provides compactness and stability to Gpi8. It also enables a conformation in which a flexible loop (219-244 residues) in the vicinity of the catalytic pocket is able to interact with and position the scissile bond for cleavage by Cys202. Steady state kinetics also indicate that peptides of lengths 7-mer to 9-mer are better bound than 4-mer or 15-mer peptide substrates. A bulky residue at the site of cleavage reduces the catalytic activity of the GPIT. This is the first detailed steady state kinetics study on the endopeptidase activity of a GPIT from any organism.

Time-resolved resonance Raman spectroscopy with continuous-wave excitation is a fundamental technique that has contributed substantially to the understanding of structure and dynamics of bacteriorhodopsin and related retinal proteins. However, the underlying principles were developed about fifty years ago for instrumentation that is hardly in use any more. Thus, the adaptation of the technique to current state-of-the art equipment is needed to satisfy the increasing demand for the spectroscopic characterization of microbial retinal proteins. In this work, we focus on pump-probe time-resolved resonance Raman experiments with a confocal spectrometer using a rotating cell. We discuss the boundary conditions that fulfill the fresh sample conditions and the photochemical innocence of the probe beam as a prerequisite for studying parent or intermediate states of retinal proteins that undergo a cyclic photoinduced reaction sequence. For the measurements of intermediate states and reaction kinetics, pump-probe experiments are required in which the two laser beams hit the flowing sample with a defined but variable delay time. An appropriate set-up for such two-beam experiments with a confocal spectrometer is proposed and tested in time-resolved experiments of bacteriorhodopsin. The comparison with the results obtained with previous classical slit spectrometers with 90-degree-scattering illustrates the advantages and disadvantages of the confocal arrangement. It is shown that modern confocal spectrometers substantially decrease the spectra acquisition time but require a more demanding optical set-up. Furthermore, the extent of photoconversion by the pump beam is lower than for the 90-degree-scattering arrangement which lowers the accuracy of kinetic measurements.

Kinase family proteins constitute the second largest protein class targeted in drug development efforts, most prominently to treat cancer, but also several other diseases associated with kinase dysfunction. In this work we focus on type II kinase inhibitors which bind to the "classical" inactive conformation of the protein kinase catalytic domain where the DFG motif has a "DFG-out" orientation and the activation loop is folded. Many Tyrosine kinases (TKs) exhibit strong binding affinity with a wide spectrum of type II inhibitors while serine/threonine kinases (STKs) often bind more weakly. Recent work suggests this difference is largely due to differences in the folded to extended conformational equilibrium of the activation loop between TKs vs. STKs. The binding affinity of a type II inhibitor to its kinase target can be decomposed into a sum of two contributions: (1) the free energy cost to reorganize the protein from the active to inactive state, and (2) the binding affinity of the type II inhibitor to the inactive kinase conformation. In previous work we used a Potts statistical energy potential based on sequence co-variation to thread sequences over ensembles of active and inactive kinase structures. The threading function was used to estimate the free energy cost to reorganize kinases from the active to classical inactive conformation, and we showed that this estimator is consistent with the results of molecular dynamics free energy simulations for a small set of STKs and TKs. In the current study, we analyze the results of a large-scale study of the binding affinities of 50 type II inhibitors to 348 kinases, of which the results for 16 of the 50 type II inhibitors were reported in an earlier study (the "Davis dataset"); the binding data for the remaining 34 type II inhibitors to the panel of 348 kinases were recently obtained (the "Schrodinger dataset"). We use the Potts statistical energy model to investigate the contribution of protein reorganization to the selectivity of the large kinase panel against the set of 50 type II inhibitors, and find that protein reorganization makes a significant contribution to the selectivity. The AUC of the receiver-operator characteristic curve is [~]0.8. We report the results of an internal "blind test", that shows how Potts threading energies can provide more accurate estimates of kinase selectivity than corresponding predictions using experimental results of small sample size. We discuss why two STK phylogenetic kinase families, STE and CMGC, appear to contain many outliers, and how to improve the ability to predict kinase selectivity with a more complete analysis of the kinase conformational landscape. We compare the performance of Potts threading for predicting binding properties of the large set of (50) Type II inhibitors to 348 kinases, with those of a sequence-based purely machine learning model, DeepDTAGen, a publicly available machine learning model that was trained on the complete Davis dataset, including both Type I and Type II kinase inhibitors. We observe that DeepDTAGen performs well on binding predictions for the 16 type II inhibitors in the Davis dataset, but performs poorly on binding predictions for the 34 type II inhibitors against 348 kinases in the Schrodinger dataset.

We have conducted all atom molecular dynamics simulations of POPC and DPPC lipid bilayers using AMBER Lipid21 force field with eight different water models, including SPC/E, TIP3P, TIP3P-FB, TIP4P-FB, TIP4P-Ew, TIP4P/2005, TIP4P-D, and OPC, to identify the most compatible one without any modification. A number of parameters have been computed in order to understand the structure of the lipid bilayer: Area per lipid, Isothermal compressibility modulus, average Volume per lipid, electron density profile, bilayer thickness, X-ray and neutron scattering form factors, deuterium order parameter, and radial distribution function. The estimated Area per lipid, Isothermal compressibility factor, volume per lipid and bilayer thickness are highly consistent with experimental results for the SPC/E water model, indicating its suitability with the AMBER Lipid21 force field, insted of any modification. The bilayer electron density profiles of both the lipid bilayers demonstrate a little augmentation of water penetration with respect to the membrane surface for TIP4P-D water model. However, the experimental X-ray and neutron scattering form factors are aligning well with the simulated results for all studied water models, and TIP4P-D shows better for X-ray data. The deuterium order parameter for lipid acyl chains value less than 0.25 for all observed water models, depicting their disorderness for both the lipid bilayers. The lateral diffusion and reorientation autocorrelation function of the lipid molecules in both the bilayers are computed to reveal their dynamics across all water models. In comparison to other water models, the simulated trajectories predict better structure and reasonably fair dynamic properties for the SPC/E water model. The TIP4P-Ew water model reproduces the lateral diffusion co-efficient in close agreement with experiment. Reorientational dynamics for both the lipids in the bilayers for eight different water models are observed; the presence of slow and slowest time components corresponds to the lipid axial motion (wobble motion) and Twist/Splay motions. So, in view of the overall performance of the different water models with the AMBER Lipid21 all atom force field in reproducing membrane physical properties, the SPC/E water model appears to be an optimal choice.

Whole-cell patch-clamp studies often fail to observe the expected effect of melatonin on the IK1 current in cardiomyocytes, which may be due to cytoplasmic dialysis and the loss of key components of the intracellular signaling system. The aim of this study was to develop a simple theoretical model to estimate the expected effect on the IK1 inward-rectifying potassium current in an experiment with intact melatonin signaling. The modeling was performed using a well-established model of rat cardiomyocyte electrophysiology (Pandit et al., 2001). The maximum conductance of IK1 (gK1) channels was chosen as the target for modulation, consistent with the established mechanism of direct receptor-mediated increase in potassium conductance under the action of melatonin.Realistic modulation values were used for the modeling. The -50% value for the antagonist effect of 1 M luzindole was obtained by direct calculation from our experimental data. The +20% value for the agonist effect (melatonin) was determined by generalizing literature data and reflects the typical expected strength of signaling pathway modulation, rather than being strictly tied to a specific concentration.It was shown that modulation of gK1 in the specified ranges leads to significant changes in IK1 amplitude in the physiologically important range of resting potentials. The developed model serves as a "computational benchmark" for validating experimental protocols, allowing one to distinguish methodological artifacts from a true lack of effect.

Protein kinases regulate signaling by recognizing short sequence motifs, and how these motifs bind influences both specificity and therapeutic strategies that target kinase pathways. Peptide-based inhibitors that engage substrate-recognition regions are attracting interest, but designing them requires an understanding of how a flexible peptide approaches and settles into the bound pose. Traditional studies have focused on the bound pose and affinities, whereas the steps that link the initial encounter with the bound pose have been explored less thoroughly because the relevant intermediates are too short-lived to capture experimentally and evolve on timescales that standard molecular dynamics cannot readily access. Here, we focused on Abl kinase and Abltide, the experimentally identified optimal substrate peptide for Abl kinase, and examined the sequence of events linking initial encounter to the bound pose using two-dimensional replica exchange (gREST/REUS), which selectively enhances flexibility in the peptide and its binding interface while also sampling progression along a distance coordinate. The resulting simulations yielded a detailed binding landscape, revealing five distinct encounter regions outside the substrate-binding site and six intermediate states that may connect the initial approach to the bound pose. Some encounter regions and intermediate states participate in the dominant binding pathways. During this process, EF/G/{beta}11 hydrophobic patch, together with G helix negative patch, plays a central role in guiding Abltide toward the substrate-binding site. These findings provide mechanistic insight into substrate recognition by protein kinases and offer a foundation for the rational design of peptide-based inhibitors.

Group I Introns are non-coding regions of pre-mRNA that catalyze their splicing from the RNA sequence by folding to a specific structure. We used computer simulations to study the folding mechanism of the P4-P6 domain in the Tetrahymena thermophila group I intron, focusing on the GAAA tetraloop-receptor (TL-R) interaction, which is a ubiquitous tertiary interaction in RNA structures. We show that the intron folds via a multistep pathway, populating seven states with distinct tertiary contacts. Under physiological Mg2+ concentrations ([Mg2+]), the loop-bulge-P4 tertiary interaction is essential to stabilize the docked TL-R complex, whereas in high [Mg2+], the TL-R complex is stable by itself. The solvated Mg2+ ions modulate the TL-R docking-undocking dynamics and stabilize non-native intermediate states. The condensation of Mg2+ in the major grooves of the TL and R helices is critical for them to attain specific stiffness essential for their facile docking. The results highlight the critical role of Mg2+ ions in facilitating TL-R interaction formation, which stabilizes long-range tertiary contacts in RNA structures. For Table of Contents Use Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/700762v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@142852org.highwire.dtl.DTLVardef@1632ad1org.highwire.dtl.DTLVardef@190021aorg.highwire.dtl.DTLVardef@17a1261_HPS_FORMAT_FIGEXP M_FIG C_FIG

The process of DNA transcription leads to the generation of torsional stress, which must be resolved for smooth progression of the transcription machinery. In Saccharomyces cerevisiae, DNA topoisomerase I (Top1), a type IB topoisomerase, plays a critical role in relaxing supercoils and mitigating the topological strain associated with transcription. While several proteins from the transcription machinery have been reported to interact with yeast Top1, detailed characterization and functional relevance of these interactions have remained underexplored. This gap is partly due to the absence of a complete three-dimensional structure of the full-length enzyme, which hinders structure-based computational analyses of its interactome. In this study, we present a template-based model of full-length yeast Top1. Leveraging this model, we investigated its molecular interaction with Rpc82, a key subunit of RNA polymerase III enzyme, responsible for transcribing small non-coding RNAs such as tRNAs and 5S rRNA. Through molecular docking and molecular dynamics simulations, critical residues at the Top1-Rpc82 interface were identified that likely mediate their interaction. Our findings provide new insights into the structural basis of Top1s association with RNA polymerase III and its potential role in regulating Pol III-mediated transcription. The Top1 model developed here offers a valuable framework for future in silico studies aimed at elucidating the broader interactome and regulatory mechanisms of this essential enzyme.

Synaptic vesicle glycoproteins 2 (SV2) are integral membrane proteins essential for neurotransmitter release and are implicated in neurological disorders including epilepsy and Parkinsons disease. In the attempt to develop a ligand selective for SV2C, and in collaboration with UCB, UCB-F was identified as a potential candidate. However, the affinity of UCB-F to SV2C was found to be temperature dependent, decreasing by about 10-fold from +4 to 37 degrees. UCB1A was subsequently identified as SV2C ligand displaying in vitro a 100-fold selectivity for SV2C compared with SV2A. In this study we investigated whether the binding of UCB-1A to SV2A and SV2C was affected by the temperature. A combination of experimental binding assay data and molecular dynamics (MD) simulations were used. The binding studies revealed that UCB1A affinity for SV2A decreased significantly at 37 {degrees}C compared with 4 {degrees}C, whereas binding to SV2C remained largely unchanged. MD simulations reproduced these observations, namely that ligand RMSD values at 310 K showed that UCB1A binding fluctuated markedly in the SV2A complex, with many trajectories exceeding the 3.0 [A] stability cutoff, whereas UCB1A remained relatively well-anchored in SV2C under the same conditions. Structural analysis showed that, while UCB1A adopts a conserved binding pose across all isoforms stabilized by {pi}- {pi} stacking and a hydrogen bond with Asp, SV2C possesses a unique stabilizing feature. In SV2C, Tyr298 is less exposed to the solvent and engages in a persistent hydrogen bond with Asparagine, a structural feature that reinforces pocket stability and limits temperature-induced destabilization. This interaction is absent in SV2A, consistent with its greater temperature sensitivity. Together, these findings provide a mechanistic explanation for the experimentally observed temperature independence of UCB1A binding to SV2C. More broadly, the results highlight the importance of incorporating physiologically relevant temperatures into SV2 ligand evaluation and demonstrate how combining experiments with simulations can uncover isoform-specific mechanisms of ligand recognition and stability.

The KRAS oncogene, central to cellular signaling via MAPK and PI3K-AKT pathways, is a notorious cancer driver frequently activated in pancreatic, colorectal, and lung carcinomas. Regulation of human KRAS oncogene expression is important due to its capital role in cell growth, proliferation, and survival. Misregulation of its expression contributes directly to the development and progression of multiple types of cancer. In previous studies, the role of G-quadruplexes elements in both the promoter and 5 UTR regions have shown to play important roles in KRAS expression, particularly when these G4s elements interact with regulatory protein hnRNPA1. In this study, we reveal that KRAS expression is also modulated at the post-transcriptional level through the formation of RNA G-quadruplexes (rG4s) situated at the 5 untranslated region (5UTR) of the mRNA. Biophysical and binding studies were carried out to probe the interaction. Through isothermal titration calorimetry (ITC), we quantified a strong binding affinity between the UP1 domain of hnRNPA1 and short-nucleotide RNA segments capable of adopting different G-quadruplex fold. The binding interaction is characterized by a favorable Gibbs free energy change in the range of {Delta}G {approx} -32 to -34 kJ/mol, suggesting a specific and energetically favorable association. One-dimensional and two-dimensional 1H-15N HSQC NMR spectroscopy revealed pronounced chemical shift changes in residues of both RNA recognition motifs (RRMs) of UP1, signifying direct contact with the rG4 structure.