Salt Bridge Research Articles

Reshaping UDP-binding pocket of bacterial sucrose synthase to improve efficiency of UDP-glucose production.

Reshaping UDP-binding pocket of bacterial sucrose synthase to improve efficiency of UDP-glucose production.

Engineering and structural elucidation of a Sac7d-derived IgG Fc-specific affitin and its application for the light-controlled affinity purification of antibodies.

While protein A affinity chromatography is widely established for antibody purification, the acidic elution conditions often lead to protein aggregation and deamidation. Here, we describe an alternative approach for the purification of antibodies utilizing an engineered binding protein based on the archaebacterial Sac7d scaffold in combination with light-controlledα-CD affinity chromatography (Excitography). Starting from a published affitin molecule, we engineered a monomeric protein version (C3A24) by substituting the unpaired thiol side chain Cys24 within the binding site by Ala and, unexpectedly, its binding activity towards the human IgG1 Fc region was even improved (KD= 76 nM). X-ray analysis of the co-crystallized C3A24 with a recombinant human Fc fragment revealed a 2:1 stoichiometry, with a binding site at the junction between the CH2 and CH3 domains. Interestingly, this binding site coincides with the ones of protein A, protein G and the neonatal Fc receptor (FcRn). The affitin/Fc interaction is dominated by a network of hydrogen bonds whereas, unpredicted by the initial affitin design, the two C-terminal Lys residues are also involved via a salt bridge and another hydrogen bond. Using the Azo-tagged C3A24, we purified clinically relevant antibodies from cell culture medium in a single step under physiological buffer conditions.

Mining Thermophile Genomes for New PETases with Exceptional Thermostabilities using Sequence Similarity Networks.

Enzymatic hydrolysis of polyethylene terephthalate (PET) is a promising technology for advancing a circular PET economy. Several PET-degrading α/β hydrolases have been identified, but the full potential of this enzyme family to catalyze PET hydrolysis remains largely unexplored. To address this, sequence similarity networks were employed to investigate the α/β hydrolase fold-5 subfamily (IPR029059) for new PETases. Priority was given to sequences from thermophiles, as thermostable enzymes are likely more suitable for industrial applications. Ten enzymes with ~20% sequence identity to the well-known LCC-PETase were identified, and seven were successfully overexpressed and purified for in vitro characterization. Each enzyme catalyzed the hydrolysis of p-nitrophenyl butyrate, a mimic of trimeric PET, and emulsified PET nanoparticles. Notably, three enzymes were also capable of hydrolyzing PET films. Novel PETases exhibited melting temperatures (Tm) exceeding 55 °C and only modest losses of activity after incubation at 70 °C for 24 hours. The crystal structure of AroC (Tm = 85 °C) was resolved to 2.2 Å, revealing several salt bridges that likely confer thermostability, and a unique loop that is conserved amongst the PETases described here. These novel enzymes will enable engineering campaigns to generate thermostable and catalytically efficient PETases for use as industrial biocatalysts.

Discovery of New Inhibitors of Human Activated Protein C to Treat Hemophilia

Background: Activated Protein C (APC) is a plasma serine protease with antithrombotic function. APC acts as an anticoagulant by promoting the degradation of factors Va and VIIIa, thus inhibiting the formation of thrombin. Specific inhibition of APC has been proposed to benefit hemophilia therapy. Methods: We used chromogenic tripeptide substrate hydrolysis assay to screen a series of arginine and arginine-like containing small molecules to identify inhibitors of APC. Similar hydrolysis assays were used to determine selectivity against other serine proteases and blood-clotting enzymes. Molecular modeling was exploited to illustrate the binding of the most potent and selective inhibitor onto the putative binding site. Results: We identified inhibitor 2 as a potent inhibitor with an IC50 value of 1.1 μM. The molecule demonstrated >100-fold selectivity against thrombin, factor XIa, and neutrophil elastase, >50-fold selectivity against factor XIIIa, 10-fold selectivity against factor Xa, and 8-fold selectivity against human plasmin. Molecular modeling reveals that inhibitor 2 binds to the active site of APC with the best-docked structure, indicating that one protonated amidino group establishes a salt bridge to the side chain carboxylate of Asp189 residue. Another inhibitor was identified, yet it was not as selective to APC. Importantly, inhibitor 2 demonstrates favorable physicochemical, pharmacokinetic, and drug-likeness properties. Conclusion: Inhibitor 2 is a selective and potent inhibitor of APC that serves as a powerful lead for the development of hemophilia therapy.

Designing and Evaluation of a Novel IL-1RA Fusion Cytokine to Enhance the Pharmacokinetics and Receptor Affinity for Better Therapeutic Intervention in Inflammatory Disorders.

The extended IL-1 activity is implicated in autoimmune disorders, such as rheumatoid arthritis, diabetes mellitus, and Parkinson's disease, as well as delayed wound healing. Additionally, it can result in cytokine storms during pathogenic infections. The regulation was carried out by Interleukin-1 receptor antagonist (IL-1RA), a key anti-inflammatory molecule. IL-1RA serves as a decoy protein that competes with Interleukin-1 receptors (IL-1RI and IL-1RII) for binding, effectively counteracting the activity of Interleukin- 1 (IL-1). The deficiency was substantiated by commercially available recombinant IL-1RA called Anakinra. The main problem with the existing drug is that it has less pharmacokinetics and reduced binding affinity to its receptor, which requires frequent administration of the drug. To overcome these drawbacks, we have designed a new fusion protein by adding an Fc fragment of Human IgGI fused with IL-1RA using a linker in between, and the design aimed to transport the protein into the N-glycosylation pathway. These characteristic features increase the pharmacokinetics, solubility, and binding efficiency of the protein. As the protein was designed to be expressed in a eukaryotic system, to understand the possibility of the proposed hypothesis, we used machine learning-based AlphaFold2 to model the protein structure and molecular simulation studies to understand the functional integrity of the designed protein. The in silico results showed that the modeled fusion protein structure has very good binding to its receptor with the support of 21 H bonds and 7 salt bridges and maintained the binding stability over the MD simulations. These findings support fusion protein's potential as a promising and stable therapeutic candidate.

Structural characterization of codon 129 polymorphism in prion peptide segments (PrP127-132) using the Markov State Models.

Structural characterization of codon 129 polymorphism in prion peptide segments (PrP127-132) using the Markov State Models.

Designing lysyl hydroxylase inhibitors for oral submucous fibrosis - Insights from molecular dynamics.

Designing lysyl hydroxylase inhibitors for oral submucous fibrosis - Insights from molecular dynamics.

Probing the Dynamics of Yersinia Adhesin A (YadA) in Outer Membranes Hints at Requirements for β-Barrel Membrane Insertion.

The vast majority of cells are protected and functionalized by a dense surface layer of glycans, proteoglycans, and glycolipids. This surface represents an underexplored space in structural biology that is exceedingly challenging to recreate in vitro. Here, we investigate β-barrel protein dynamics within an asymmetric outer membrane environment, with the trimeric autotransporter Yersinia adhesin A (YadA) as an example. Magic-angle spinning NMR relaxation data and a model-free approach reveal increased mobility in the second half of strand β2 after the conserved G72, which is responsible for membrane insertion and autotransport, and in the subsequent loop toward β3. In contrast, the protomer-protomer interaction sites (β1i-β4i-1) are rigid. Intriguingly, the mobility in the β-strand section following G72 is substantially elevated in the outer membrane and less so in the detergent environment of microcrystals. A possible source is revealed by molecular dynamics simulations that show the formation of a salt bridge involving E79 and R76 in competition with a dynamic interplay of calcium binding by E79 and the phosphate groups of the lipids. An estimation of overall barrel motion in the outer membrane and detergent-containing crystals yields values of around 41 ns for both. The global motion of YadA in the outer membrane has a stronger rotational component orthogonal to the symmetry axis of the trimeric porin than in the detergent-containing crystal. In summary, our investigation shows that the mobility in the second half of β2 and the loop to β3 required for membrane insertion and autotransport is maintained in the final folded form of YadA.

The effect of cysteine oxidation on conformational changes of SARS-CoV-2 spike protein using atomistic simulations

The SARS-CoV-2 Spike (S) protein plays a central role in viral entry into host cells, making it a key target for therapeutic interventions. Oxidative stress, often triggered during viral infections, can cause oxidation of cysteine in this protein. Here we investigate the impact of cysteine oxidation, specifically the formation of cysteic acid, on the conformational dynamics of the SARS-CoV-2 S protein using atomistic simulations. In particular, we examine how cysteine oxidation influences the transitions of the S protein’s receptor-binding domain (RBD) between “down” (inaccessible) and “up” (accessible) states, which are critical for host cell receptor engagement. Using solvent-accessible surface area (SASA) analysis, we identify key cysteine residues susceptible to oxidation. The results of targeted molecular dynamics (TMD) and umbrella sampling (US) simulations reveal that oxidation reduces the energy barrier for RBD transitions by approximately 30 kJ mol−1, facilitating conformational changes and potentially enhancing viral infectivity. Furthermore, we analyze the interactions between oxidized cysteine residues and glycans, as well as alterations in hydrogen bonds and salt bridges. Our results show that oxidation disrupts normal RBD dynamics, influencing the energy landscape of conformational transitions. Our work provides novel insights into the role of cysteine oxidation in modulating the structural dynamics of the SARS-CoV-2 S protein, highlighting potential targets for antiviral strategies aimed at reducing oxidative stress or modifying post-translational changes. These findings contribute to a deeper understanding of viral infectivity and pathogenesis under oxidative conditions.

Computational discovery of potential human carbonic anhydrase IX inhibitors

This study explores the potential of nitro compounds as inhibitors of Carbonic Anhydrase IX (CAIX) for cancer therapy. Molecular docking and virtual screening were employed to identify lead molecules, focusing on their binding affinities and interactions with CAIX. Six compounds, namely D1 (−8.295 kcal/mol), D2 (−10.980 kcal/mol), D3 (−10.963 kcal/mol), D4 (−7.170 kcal/mol), D5 (−7.368 kcal/mol), and D6 (−8.705 kcal/mol), were selected based on their promising docking scores and Molecular Mechanics-Generalized Born Surface Area (MMGBSA) results. Molecular dynamics simulations further confirmed the stability of the CAIX-lead compound complexes, with low root-mean-square deviation (RMSD) values and minimal fluctuations in protein and ligand structures. The analysis of root-mean-square fluctuations (RMSF) highlighted the overall stability of these complexes, with localized fluctuations. Detailed investigation of protein-ligand contact dynamics unveiled strong and consistent interactions, including hydrogen bonds, ionic interactions, water bridges, and salt bridges. These novel interactions shed light on the dynamic behavior of the CAIX-lead compound complexes. Briefly, this study establishes a robust framework for advancing D1, D2, and D3 as promising CAIX inhibitors in cancer treatment. The meticulous analysis and comprehensive approach applied to these primary compounds yield substantial insights into their potential as therapeutic agents, setting the stage for forthcoming experimental validation and clinical utilization.

Structural Insights Into the Mechanisms of Recognition of Recombinant Full-Length Antibodies for the Detection of Staphylococcus aureus Enterotoxins C1, C2, and C3.

Food poisoning caused by Staphylococcus aureus (S. aureus) and its enterotoxins has become a global food safety issue. Herein, three types of Staphylococcal enterotoxins (SE), including SEC1, SEC2, and SEC3, were successfully expressed and immunized in mice to prepare monoclonal antibodies (mAbs). We screened a pair of mAbs 16E12-9B7 from 12 strains that could simultaneously recognize SEC1, SEC2, and SEC3. Furthermore, the genes from hybridoma cells 9B7 and 16E12 were extracted, amplified, and inserted into expression vectors to obtain recombinant antibodies (rAbs), whose affinities were consistent with those of ascites antibodies. The paired rAbs 16E12-9B7 were applied to a gold immunochromatographic strip (GICS) system to enable the rapid detection of SEC1, SEC2, and SEC3 with visual limits of detection (vLOD) of 2, 0.5, and 2 ng/mL in milk samples. Noticeably, we found that hydrogen bonds and salt bridges played a significant role in these antigen-antibody interactions. The key sites for 9B7 were ASN28 and SER31 in complementarity determining region (CDR) and the key sites for 16E12 were SER32 in the 16E12 variable light chain (VL) and ARG100, SER101, and TYR102 in the 16E12 variable heavy chain (VH). The analysis of key rAbs sites has potential in the screening of mutant antibodies.

Dynamic Mechanism of Norepinephrine Reuptake and Antidepressants Blockade Regulated by Membrane Potential.

During nerve signaling, changes in membrane potential are key to regulating neuronal activity. The norepinephrine transporter (NET) plays a crucial role in the reuptake of norepinephrine (NE), which is essential for maintaining neurotransmitter homeostasis. However, the impact of membrane potential on NET function has long been understudied. Despite the great biological significance of NET, the dynamic molecular mechanisms of NE transport and the blockade effects of antidepressants on this process remain unclear. Here, we reveal the structural, electrostatic, and dynamic characteristics of the NET-NE/antidepressants systems, indicating the dynamic voltage dependence of the NET function. By analyzing the structure and electrostatic properties of the central binding pocket, we find that a hydrophobic network stabilizes the localization of NE, while the dynamic hydrogen bond and salt bridge network plays a crucial role in facilitating the inward transport of NE. Changes in membrane potential significantly affect the reuptake of NE through an electrostatically driven substrate transport pathway, primarily influencing the substrate entrance, the hydrophilic channel leading to the central site, and the exit region. The hyperpolarized state favors NE reuptake, exhibiting a marked preference for inward movement, which aligns with the physiological need for neurons to regulate neurotransmitter concentration in the synaptic cleft via reuptake. Conversely, in the depolarized state, which corresponds to the generation of nerve impulses, NE reuptake may not peak. Furthermore, antidepressants, with their larger molecular size and longer charged amino groups, initially anchor to the essential residue E382 required for NE reuptake. They subsequently occupy the same binding pathway as NE, creating spatial hindrance that effectively blocks NE binding to the central pocket. Additionally, their binding/dissociation behaviors exhibit significant voltage dependence. Under the hyperpolarized state, antidepressants can better block NE entry through more flexible electrostatic and hydrophobic interactions with NET, while the depolarized state raises the binding barrier for antidepressants, facilitating their dissociation. And with this work, a computational strategy for membrane protein-ligand is proposed to emphasize that considering the effects of electric fields in the calculations can reveal more underlying mechanisms and key interactions.

Conserved lipid-facing basic residues promote the insertion of the porin OmpC into the E. coli outer membrane.

Almost all integral membrane proteins that reside in the outer membrane (OM) of gram-negative bacteria contain a closed amphipathic β sheet ("β barrel") that serves as a membrane anchor. The membrane integration of β barrel structures is catalyzed by a highly conserved heterooligomer called the barrel assembly machine (BAM). Although charged residues that are exposed to the lipid bilayer are infrequently found in outer membrane protein β barrels, the β barrels of OmpC/OmpF-type trimeric porins produced by Enterobacterales contain multiple conserved lipid-facing basic residues located near the extracellular side of the OM. Here, we show that these residues are required for the efficient insertion of the Escherichia coli OmpC protein into the OM in vivo. We found that the mutation of multiple basic residues to glutamine or alanine slowed insertion and reduced insertion efficiency. Furthermore, molecular dynamics simulations provided evidence that the basic residues promote the formation of hydrogen bonds and salt bridges with lipopolysaccharide, a unique glycolipid located exclusively in the outer leaflet of the OM. Taken together, our results support a model in which hydrophilic interactions between OmpC and LPS help to anchor the protein in the OM when the local environment is perturbed by BAM during membrane insertion and suggest a surprising role for membrane lipids in the insertion reaction.IMPORTANCEThe assembly (folding and membrane insertion) of bacterial outer membrane proteins (OMPs) is an essential cellular process that is a potential target for novel antibiotics. A heterooligomer called the barrel assembly machine (BAM) plays a major role in catalyzing OMP assembly. Here, we show that a group of highly conserved lipid-facing basic residues in Escherichia coli OmpC, a member of a major family of abundant OMPs known as trimeric porins, is required for the efficient integration of the protein into the outer membrane (OM). Based on our work and previous studies, we propose that the basic residues form interactions with a unique OM lipid (lipopolysaccharide) that promotes the insertion reaction. Our results provide strong evidence that interactions between specific membrane lipids and at least a subset of OMPs are required to supplement the activity of BAM and facilitate the integration of the proteins into the membrane.

Exploring the potential antidepressant mechanisms of ibuprofen and celecoxib based on network pharmacology and molecular docking.

Exploring the potential antidepressant mechanisms of ibuprofen and celecoxib based on network pharmacology and molecular docking.

Sphenocentrum jollyanum furanoditerpenes activates Glucagon-like peptide 1 receptor (GLP-1R): a computational evaluation

Sphenocentrum jollyanum is a major bio-resource used in the folkloric treatment of diabetes, unfortunately without knowledge of how or the mechanism of action. The present study focuses on using molecular docking to identify the component responsible for the anti-diabetic claim of the fruit as well as check the ADMET properties of the phytochemicals. A library of 23 phytochemicals that have been previously characterized from Sphenocentrum jollyanum fruit was generated and docked, using Autodock Vina, into 3D structures of dual-specificity tyrosine phosphorylation regulated kinase 1a (DYRK1A), peroxisome proliferator-activated receptor alpha (PPAR-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), pancreatic alpha-amylase, dipeptidyl peptidase 4 (DPP4), glucagon-like peptide 1 receptor (GLP-1R), renal sodium-dependent glucose transporter (SGLUT 2). From the docking result, GLP-1R was identified as a major target for two key S. jollyanum furanoditerpenes (columbin and isocolumbin) with docking scores of -10.7 and -10.9 Kcal/mol respectively while the reference ligand (danuglipron) characterized with the GLP-1R had a score of -11.0 Kcal/mol. The protein-ligand interaction between columbin and GLP-1R showed several interactions such as hydrogen bond (H-bond), hydrophobic interactions and salt bridges, all within the extracellular domain (ECD), transmembrane domain (TM) of the receptor which is similar to that of danuglipron. Also, the ADMET profiling result was favourable as both ligands (columbin and isocolumbin) passed the Lipinski rule of 5. The results suggest that columbin identified from the docking result is most likely responsible for the antidiabetic claim of Sphenocentrum jollyanum, further investigation is needed to ascertain this claim.

Design and Characterization of pH-Responsive DGEA-Derived Peptide Scaffolds: A Comprehensive Molecular Dynamics Simulation Study.

Peptide-based, functionally active, stimuli-responsive biomaterials hold immense potential for diverse biomedical applications. Functionally active motifs of extracellular matrix (ECM) proteins, when conjugated with self-assembling peptides (SAP) or polymers, demonstrate significant promise in the development of such bioactive scaffolds. However, synthesis complexity, high associated costs, limited functionality, and potential immune responses present significant challenges. This study explores collagen-I-derived DGEA motif-based SAPs, incorporating modifications such as salt bridge pairing, charged and polar residues, hydrophobic residues, amyloidogenic sequences, and non-ECM motifs, to develop stimuli-responsive, functionally active scaffolds. Extensive molecular dynamics (MD) simulations, totaling 16.7 μs, were conducted on 20 systematically designed peptide systems. These simulations also characterized the stimuli-responsive properties of the peptides, focusing on pH and temperature responsiveness. Among the 20 designs, three peptide systems─DGEA-SBD, DGEA-SBE (salt-bridge modifications), and DGEA-F4 (with hydrophobic residue addition at the C-terminus)─successfully formed large, stable, and bioactive scaffolds. These systems exhibited enhanced aggregation (greater than 90%) and improved interpeptide hydrogen bonding (more than 30 bonds) while maintaining the accessibility of functional motifs (60-70% availability) compared to the unmodified DGEA motif. Notably, the DGEA-SBD and DGEA-SBE peptides showed a transition from small, unstable, uneven gel-like structures to large, stable, uniform, and functionally active scaffolds as the pH shifted from 3.0 to physiological pH. Comprehensive MD simulation studies demonstrated that these designed peptides exhibit increased aggregation and enhanced interpeptide hydrogen bonding while retaining their functional activity under various physiological conditions, highlighting their promising potential for biomedical applications.

Mechanistic Insights into the Inhibitory and Destabilizing Effects of K353 Acetylation on Tau Peptides and Protofibrils.

Misfolding and aggregation of microtubule-associated tau protein is implicated in a variety of neurodegenerative disorders (named tauopathies), including Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE). AD is the most common type of dementia associated with aging, and CTE is a special tauopathy that mostly affects contact sports athletes (such as those active in American football and boxing). Experimental studies have found that tau acetylated on residue K353 exhibited a declined aggregation propensity; however, the underlying molecular mechanism remains elusive. In this study, we performed replica exchange and conventional molecular dynamics simulations of acetylated and unacetylated tau protein models in an explicit solvent. Our results revealed that the acetylated R4 (the fourth microtubule-binding repeat domain) dimer showed less β structure and more disordered conformations than the unacetylated one. K353 acetylation weakened peptide-peptide interactions and interrupted the salt-bridge network, thus inhibiting R4 dimerization. Besides, K353 acetylation reduced the β-sheet structure probability and induced loosely packed conformations of R3-R4 (the third and fourth microtubule-binding repeat regions) protofibrils. The replacement of the charged group by acyl on K353 resulted in the loss of K353-D358 salt bridges, leading to the enlargement of the β6-β7 angle and the distance between the carboxyl-terminal and β-turn region, finally eliciting an opened "H" configuration. Our work provided a clear picture of the inhibitory mechanisms of K353 acetylation on tau at the microscopic level, which may be helpful in the development of new therapeutics against tauopathies from the perspective of post-translational modification (PTMs).

Structural basis of disease mutation and substrate recognition by the human SLC2A9 transporter

Urate provides ~50% of the reducing potential in human and primate plasma which is key to detoxifying reactive oxygen by-products of cellular metabolism. Urate is the endpoint of purine metabolism in primates, and its concentration in plasma is a balance between excretion from kidney and intestine, and subsequent reabsorption in and through cells of kidney proximal tubules to maintain a regulated concentration in plasma. SLC2A9 is the primary transporter that returns urate from the basolateral side of kidney tubule cells back to plasma. A shorter splice variant of SLC2A9 is directed to the apical surface where several transporters recapture urate from the tubule back into cells. Too high a concentration in plasma causes hyperuricemia, is linked to gout, and favors kidney stone formation. To understand the molecular basis of uric acid transport and the role of disease-causing mutations in SLC2A9, we determined structures of human SLC2A9 in its apo form, and its urate-bound form by cryo-EM, at resolution of 3.3 Å and 4.1 Å respectively. Both structures are captured in an inward open conformation. Using the inward-facing structure as a template we modeled the outward-facing conformation to understand the alternating access mechanism. Alternative salt bridge pairs on the cytoplasmic side suggest a mechanism that can balance the energetics of the inward open and outward open states. The location of disease-causing mutants suggests their role in impacting function. Our structures elucidate the molecular basis for urate selectivity and transport and provide a platform for future structure-based drug discovery aimed at reducing plasma urate levels in diseases of hyperuricemia and gout.

Chloride-Dependent Cation Transport via SLC12 Carriers at Atomic Resolution.

The SLC12 family of genes encodes electroneutral Cl--dependent cation transporters (i.e., Na-Cl, K-Cl, Na-K-2Cl cotransporters), which play significant roles in maintaining cell and body homeostasis. Recent resolution of their structures at the atomic level provides a new understanding how these transporters operate in health and disease and how they are targeted for therapeutic intervention. Overall, the SLC12 transporter cryo-EM structures confirm some key features established by traditional biochemical and molecular methods, such as the presence of 12 transmembrane domains and the formation of a functional dimer. Study of these structures also uncovers previously unknown features, such as the presence of strategic salt bridges that explain why transporters are stabilized in specific conformations. The cryo-EM structures show similarities with other transport protein structures, especially regarding the position of the cations. The structures also pose challenging questions regarding the number of ions bound and the strict electroneutrality that is conventional understanding.

Elucidating allosteric signal disruption in PBP2a: impact of N146K/E150K mutations on ceftaroline resistance in methicillin-resistant Staphylococcus aureus.

Ceftaroline (CFT) effectively combats methicillin-resistant Staphylococcus aureus (MRSA) by binding to the allosteric site on penicillin-binding protein 2a (PBP2a) and activating allosteric signals that remotely open the active pocket. However, the widespread clinical use of CFT has led to specific mutations, such as N146K/E150K, at the PBP2a allosteric site, which confers resistance to CFT in MRSA by disrupting the transmission of allosteric signals. Herein, computational simulations were employed to elucidate how the mutations disrupt the transmission of allosteric signals, thereby enhancing the resistance of MRSA to CFT. Specifically, the mutations alter the salt bridge network and electrostatic environment, resulting in a dynamic setting and decreased binding affinity of CFT within the allosteric pocket. Additionally, dynamical network analysis and the identification of allosteric pathways revealed that the reduced binding affinity diminishes the propagation of allosteric signals to the active site. Further evaluations demonstrated that this diminished signaling reduces the openness of the active pocket in the mutant systems, with "gatekeeper" residues and functional loops remaining partially closed. Redocking experiments confirmed that mutations lead to decreased docking scores and unfavorable docking poses for CFT within the active pocket. These findings highlight the complex interactions between structural changes induced by mutations and antibiotic resistance, providing crucial insights for developing new therapeutic strategies against MRSA resistance.