搜索结果：Journal of structural biology

Age acceleration in survivors of breast cancer is a critical issue because cancer and its treatment can increase structural and numerical chromosomal aberrations, while simultaneously shortening telomere length and changing ageing phenotype. Therefore, the current study will be using machine learning architectures to accurately predict the factors that contribute to age acceleration among survivors of breast cancer. The Cancer Survivors' Trajectories of Ageing Research (C*STAR) is a hospital-based cross-sectional study involving multi-ethnic Malaysian survivors of breast cancer and a non-breast cancer control group, frequency-matched by age group (±5 years), sex and ethnicity. The three main stages of this study will be conducted in the predictive model development. First, a set of validated questionnaires will be used to collect the data on modifiable factors of ageing phenotypes and behavioural determinants of health. Second, 3 mL non-fasting blood samples will be collected, and lymphocytes will be isolated to determine telomere length using real-time PCR as a biomarker of age acceleration. Lastly, a machine learning architecture will be deployed to identify modifiable factors that may contribute to age acceleration in survivors of breast cancer and controls, with these factors used as input and ageing biomarkers of telomere length as output. The study outcomes may serve as guidance to enhance the quality of life of survivors of breast cancer and hinder the recurrence of cancer while ageing successfully. Ethical approval was obtained from the Research Ethics Committee, Universiti Kebangsaan Malaysia (JEP-2022-700) to carry out this study. Written informed consent will be obtained from each survivor of breast cancer and each cancer-free woman prior to participation. The results of this study will be published for future research and clinical applications.

Mutations within the transmembrane domains (TMDs) of single-pass transmembrane receptors often cause aberrant, ligand-independent receptor signaling associated with diverse malignancies, but their mechanism of action remains largely unknown. These TMD mutations are generally not targetable as they are buried in the membrane. Here, we determined the mechanism of a gain-of-function (GOF) TMD mutation of interleukin-7 receptor (IL-7R) associated with T cell acute lymphoblastic leukemia and addressed the possibility of directly targeting the TMD mutation by using rationally designed transmembrane helices to restore order to uncontrolled signaling. We find that the GOF mutation of IL-7R severely shifts the TMD homodimerization interface, causing the receptor to homodimerize in a geometry that activates downstream signaling independent of ligand. Designed transmembrane helices that interfere with the new interface, delivered with mRNA technology, selectively block ligand-independent but not ligand-dependent signaling. Our study provides a conceptual framework for understanding and repairing disease-causing TMD mutations of single-pass cytokine receptors.

With the increasing use of genetic sequencing to investigate inborn errors of immunity, rare variants are frequently identified, yet their clinical relevance often remains uncertain. Establishing pathogenicity requires a multidisciplinary approach that integrates genetic, structural, functional, and clinical data. Here, we used such a strategy to investigate a previously unreported hemizygous missense variant - alanine (A) to threonine (T) at residue 518 - in Toll-like receptor 8 (TLR8), identified in 2 male siblings with recurrent infections and systemic inflammation, characterized by a proinflammatory immune signature and B cell dysregulation. Functional studies showed that the TLR8 A518T variant enhanced NF-κB activation and increased secretion of proinflammatory cytokines compared with WT TLR8 upon stimulation, consistent with a gain-of-function effect. Protein degradation and turnover assays revealed reduced abundance of the mutant TLR8 protein due to faster turnover and increased proteasomal degradation. Computational modeling predicted enhanced structural stabilization of the active TLR8 homodimer interface via additional water-mediated hydrogen bonds introduced by the A518T substitution. Together, these findings integrating structural modeling with functional assays identify a novel TLR8 ligand-specific gain-of-function mutation resulting in complex immunopathology in 2 siblings.

Thymidine (deoxythymidine triphosphate) plays very important role in DNA synthesis, replication and repair. Therefore, thymidine synthesis pathway enzymes are crucial for the survival of the organism and hence are potent therapeutic targets. Thymidylate kinase (TMPK) is at the junction of de novo synthesis pathway and salvage pathway of thymidine synthesis. TMPK is widely recognized as a potential therapeutic target. Inhibiting TMPKs would be an effective technique for discovering medications to treat infectious disorders like bacterial and parasite infections. The slight variation in active sites between human TMPK (hTMPK) and pathogen TMPKs provide support for the development of specific inhibitors. Here, we report the crystal structure of thymidylate kinase from Helicobacter pylori (HpTMPK) at 2.5 Å. The three-dimensional structure of HpTMPK depicts two conserved regions DRX motif and P loop. The highly flexible LID region was absent in HpTMPK crystal structure. Chemdiv library was screened against HpTMPK and the compounds were shortlisted based on the docking scores. Our in-vitro enzyme inhibition study shows that compound F725_0025 exhibits the best inhibition with an IC50 of 84 µM and a strong affinity of 10.7 µM. It inhibits Helicobacter pylori (H. pylori) with an IC50 value of 30.14 µM. According to the growth curve of H. Pylori in the presence of inhibitory chemicals, F725_0025 may be a promising lead therapeutic molecule to combat H. pylori infection.

Structural variants (SVs) may increase SOX3 expression in the gonads and have been observed in individuals with ovotesticular differences in sex development (OT-DSD) and XX testicular differences in sex development (T-DSD). Most of the SVs found in OT-DSD individuals are whole-gene duplications, and to date, only one SV affecting SOX3 expression by a positional effect has been described. We report an individual raised as a female with SRY-negative OT-DSD. Karyotype analysis showed a pericentric inversion in one of the X chromosomes - 46,X, inv(X)(p22;q27). The breakpoints and fusion were mapped using optical genome mapping (OGM) and short-read whole genome sequencing. One of the breakpoints was mapped on Xq27.1 (genomic position chrX:140,420,874 - GRCh38), 82 kb downstream of the SOX3 gene. This breakpoint was predicted to interrupt a topological associate domain (TAD) affecting 24 enhancer-promoter interactions of SOX3. RNA sequencing (RNA-seq) of a formalin-fixed paraffin-embedded (FFPE) sample of the gonads confirmed increased SOX3 expression. The present study is the first to analyze gene expression in gonadal tissues from an OT-DSD individual, and the first reporting an inversion-based mechanism leading to XX OT-DSD. Additionally, an X-inactivation assay on DNA extracted from the gonads revealed random inactivation. These findings support the hypothesis that inappropriate SOX3 expression may result from the positional effects of SVs, leading to OT-DSD in 46,XX individuals. Structural variants are large alterations in DNA (greater than 50 base pairs) that modify chromosome structure. These alterations can create breakpoints that lead to chromosomal reorganization. Additionally, they may alter the expression of genes located within or near the affected region, due to changes in the genomic position of these genes. When such alterations occur in XX individuals and involve the SOX3 gene or nearby regions, they can lead to overexpression of SOX3. This may result in changes in sexual development, leading to conditions known as ovotesticular differences in sex development (OT-DSD) and XX testicular differences in sex development (T-DSD). In the present study, we investigated an XX individual with OT-DSD who presented with an inversion on one of the X chromosomes, initially detected by chromosomal analysis. Using more advanced techniques, we precisely identified the disrupted region and found that one of the breakpoints was located near the SOX3 gene. Expression analysis demonstrated overexpression of SOX3, which correlates with the clinical features observed in this individual.

Glycosaminoglycans (GAGs), linear polysaccharides with varying degrees of sulfation, are key structural components of the endothelial glycocalyx. The endothelial glycocalyx helps maintain the integrity of the blood-brain barrier (BBB); however, its degradation or shedding contributes to BBB dysfunction. Understanding the relationship between GAG levels and BBB dysfunction requires a robust, high-throughput strategy for GAG quantification. In previous studies using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), GAGs were typically enzymatically or chemically depolymerized into disaccharides before analysis. Intact GAGs are characterized by heterogeneous molecular weights, diverse sulfation patterns, and multiple charge states, making direct MS analysis challenging. To overcome these challenges, we employed a MSALL-based liquid chromatography-triple-quadrupole/time-of-flight mass spectrometry (LC-Q-TOF MS) technique. To our knowledge, this is the first quantification of intact GAGs by LC-MS/MS without chemical or enzymatic digestion. Additionally, a unique sample preparation method, involving concentration followed by phenol and dichloromethane extraction, was developed to overcome the electrostatic interactions between GAGs and proteins. Method validation demonstrated a linear detection range for GAGs at concentrations of 50-5000 ng/mL with limit of detection (LOD) of 20 ng/mL and lower limit of quantification (LLOQ) of 50 ng/mL. As a proof of principle, analysis of CSF from 83 stroke patients and 42 control subjects revealed elevated GAG levels in the stroke group, corresponding to 83.13% specificity and 76.19% sensitivity in distinguishing stroke-induced BBB dysfunction from controls. These findings suggest that GAG quantification can serve as an effective measure of glycocalyx shedding and that GAG may represent a potential biomarker for BBB dysfunction.

European forests play an important role for climate change mitigation and biodiversity conservation. As they have been shaped by silviculture for centuries, it is important to understand how management practices affect forest structure and in turn influence the role of forests in achieving both goals. We analyzed data on a wide range of temperate European forests encompassing the most widespread management regimes to understand the interplay of forest structure, aboveground carbon stocks, and the richness of several taxonomic groups. Using structural equation modeling, we identified the forest structural characteristics that are positively correlated with both carbon stocks and species richness. We found that stand age and tree species richness are related to other forest structural characteristics, which had positive links to carbon stocks in deadwood. Increasing stand age was associated with an increase in deadwood carbon stocks. There were no direct negative relationships between stand age or tree species richness and the richness of different taxonomic groups. An increasing richness of deadwood types had positive links with the species richness of birds, saproxylic beetles, and saproxylic fungi, as with deadwood carbon stocks. However, increases in the species richness of birds and understory vascular plants were negatively related to increasing carbon stocks in living wood, while beetle species richness was positively related to this carbon stock. Birds' species richness was directly and positively associated with increasing mean tree diameter. Conversely, a higher richness of tree species was indirectly linked to lower carbon stocks in living wood. Additionally, an increase in mean tree diameter was indirectly correlated with a decrease in bird and vascular plant species richness. Our findings highlight potential trade-offs between carbon stocks in living wood and the species richness of several taxonomic groups in European forests, while the species richness of some taxonomic groups was positively correlated to deadwood carbon stocks. Policies focused on increasing living biomass may not target both the climate and biodiversity crises. Instead, the diversity of deadwood emerges as a key factor in explaining the relationship between carbon storage and biodiversity, and should hence play a prominent role in forest management strategies and related policies.

Voltage-gated sodium (NaV) channel α-subunits are modulated by associated β-subunits affecting their localization, trafficking and gating behaviour. The β-subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules and the interactions between their extracellular Ig-domains may modify channel clustering. The full-length β3-subunit can form cis trimers on the plasma membrane. The atomic resolution structure of a deglycosylated trimeric β3-subunit Ig-domain has been solved by X-ray crystallography. However, it is not clear whether this particular trimeric Ig-domain structure is plausible for cell-expressed, glycosylated β3-subunits. Here we use glycan profiling to confirm an extensive and heterogeneous pattern of β3-subunit glycosylation, with the majority of glycans being bi- and tri-antennary structures with one or two terminal sialic acids. Two tryptic peptides of the β3 Ig-domain are predicted to contain potential N-linked glycosylation sites. When the isolated, glycosylated full-length β3-subunit was trypsin-digested and analysed by LC-MS/MS, only one of these peptides - containing an N-linked glycosylation site at residue N95 and located close to the trimer interface - was identified in its unmodified form, suggesting that residue N95 is under-glycosylated. All-atom molecular dynamics simulations of the glycosylated, membrane-bound full-length β3 trimer confirmed that glycans can be accommodated with the Ig-domain trimer and indeed, may contribute to protein-membrane and inter-protomer interactions within the full-length, membrane-embedded trimer. Further biochemical studies are warranted to explore the interactions between oligomeric β-subunits with corresponding α-subunit sodium channels.

Systemic lupus erythematosus (SLE) is a chronic, multi-organ autoimmune disease characterised by a highly heterogeneous presentation. Specific genetic variations predispose patients to the disease, and rare monogenic forms caused by single-gene variations have been identified in a small percentage of patients, often with early disease onset. In this study, we used exome sequencing in a large cohort of patient with juvenile-onset SLE to gain insight into the genetic basis of juvenile SLE (jSLE). Patients were selected if disease onset occurred before the age of 18. We performed exome sequencing on 263 individuals across 172 distinct families. The majority of cases were solo exomes (n = 118), while others included affected duos, trios, or multiplex families (n = 18 + 5 + 1), as well as classical trios with unaffected parents (n = 30). A molecular diagnosis consistent with the clinical presentation was established in 17 patients from unrelated families (10%). Among them, we identified pathogenic or likely pathogenic variants in genes previously associated with monogenic lupus, including a novel C1QA variant as well as other lupus-associated genes (COPA, ADAR, TLR7, IKZF3, RELA, PTPN11, SERPING1). Strikingly, exome sequencing also revealed variants in immunodeficiency-associated genes (IRAK4, USB1), autoinflammatory disorders (PSTPIP1) and unexpected candidates like ETV6, and MAN1B1 revealing previously unrecognised pathways in SLE development. Syndromic features and very early-onset (before the age of 5) were strongly associated with a higher diagnostic yield, reaching nearly 33% in these subgroups. This study expands our understanding of causes of lupus, highlighting its genetic heterogeneity. It also supports the systematic use of genetic testing in cases of juvenile lupus, especially those with very early onset or syndromic features, regardless of the clinical presentation. Given the range of unexpected molecular diagnoses identified in this study, pangenomic analysis such as exome or genome sequencing appears to be the most appropriate approach in these cases. This work was supported by: The Institut National de la Santé et de la Recherche Médicale (INSERM); Government grants managed by the Agence Nationale de la Recherche (ANR) as part of the "Investment for the Future" program: Institut Hospitalo-Universitaire Imagine (ANR-10-IAHU-01), Recherche Hospitalo-Universitaire (ANR-18-RHUS-0010); The Centre de Référence Déficits Immunitaires Héréditaires (CEREDIH); The Fondation pour la Recherche Médicale (FRM: EQU202103012670, FDM202006011291); French and European grants managed by the ANR: ANR-14-CE14-0026 (Lumugène), ANR-21-CE17-0064 (SOCSIMMUNITY); The National Reference Center for Rheumatic, Autoimmune and Systemic Diseases in Children (RAISE).

Asgard archaea were pivotal in the origin of complex cellular life1. Heimdallarchaeia (a class within the phylum Asgardarchaeota) are inferred to be the closest relatives of eukaryotes. Limited sampling of these archaea constrains our understanding of their ecology and evolution2,3, including their role in eukaryogenesis. Here we use massive DNA sequencing of marine sediments to obtain 404 Asgardarchaeota metagenome-assembled genomes, including 136 new Heimdallarchaeia and several novel lineages. Analyses of their global distribution revealed they are widespread in marine environments, and many are enriched in variably oxygenated coastal sediments. Detailed metabolic reconstructions and structural predictions suggest that Heimdallarchaeia form metabolic guilds that are distinct from other Asgardarchaeota. These archaea encode hallmark proteins of an aerobic lifestyle, including electron transport chain complex (IV), haem biosynthesis and reactive oxygen species detoxification. Heimdallarchaeia also encode novel clades of respiratory membrane-bound hydrogenases with additional Complex I-like subunits, which potentially increase proton-motive force generation and ATP synthesis. Thus, we propose an updated Heimdallarchaeia-centric model of eukaryogenesis in which hydrogen production and aerobic respiration may have been present in the Asgard-eukaryotic ancestor. This expanded catalogue of Asgard archaeal genomic diversity suggests that bioenergetic factors influenced eukaryogenesis and constitutes a valuable resource for investigations into the origins and evolution of cellular complexity.

The PI3K/AKT/mTOR signaling pathway is crucial for regulating essential cellular processes such as growth, survival, metabolism, and protein synthesis. Dysregulation of this pathway is strongly associated with diseases like cancer, where it drives uncontrolled cell proliferation and survival. The mTOR kinase forms two multiprotein complexes, mTORC1 and mTORC2, which govern distinct signaling pathways. mTORC1, regulated by nutrients, controls protein synthesis, cell growth, and autophagy, while mTORC2 acts as a central node in phosphoinositide 3-kinase (PI3K) and Ras signaling, often disrupted in cancer and diabetes. AKT, recruited by PIP3 to the plasma membrane, is phosphorylated by PDK1 and mTORC2, enabling it to regulate various cellular functions. Notably, mTORC2 selectively phosphorylates AKT and PKC but no other closely related kinases targeted by mTORC1, reflecting a high degree of substrate specificity. This specificity is due to structural elements in AKT that interact with the mTORC2 subunit mSin1 as revealed by recent studies using semisynthetic probes, paving the way for the design of mTORC2-specific inhibitors. Given the pathway's significant role in disease progression, particularly cancer, targeting the AKT/mTOR axis holds considerable therapeutic promise. However, challenges remain due to the complex regulation and feedback mechanisms in this pathway. Emerging combination therapies show promise in overcoming these obstacles. This review highlights the intricate regulation of the AKT/mTOR pathway and its potential for developing targeted therapies.

For certain patients, breast reconstruction is an integral part of cancer recovery. The Association of Breast Surgery (ABS) and British Association of Plastic, Reconstructive and Aesthetic Surgeons (BAPRAS) published guidance which advocates for unrestricted access to these procedures in 2018. Despite this, in 2022, the charity Breast Cancer Now, published a report demonstrating widespread inequalities in the provision of breast reconstruction with many CCGs restricting access to reconstructive procedures. The 2022 transition from Clinical Commissioning Groups (CCGs) to Integrated Care Boards (ICBs) aimed to reduce regional variation. This study evaluates whether ICB policies have improved compliance with national ABS/BAPRAS guidance. A cross-sectional policy review of all 42 English ICBs was conducted between July and December 2025. Data was obtained via a Freedom of Information (FOI) request to all 42 ICBs alongside a review of any publicly available commissioning policies for breast reconstruction. Responses were received from 100% (42/42) of ICBs. While 71% (30/42) demonstrated alignment with national guidance, 29% (12/42) retained restrictions on access to breast reconstruction. These included caps on procedure counts (ranging from one to four surgeries) and rigid timelines for completion (2-5 years). Restrictive policies directly contradict commissioning guidance that permits multiple procedures to achieve satisfactory reconstructive outcomes. Structural reorganisation into ICBs has not eliminated the postcode lottery. Compared to prior audits, the proportion of restricted health systems has increased, suggesting regression rather than progress. Almost a third of health systems continue to enforce restrictions on procedure numbers and timelines, directly contravening ABS/BAPRAS guidance.

Previous research suggests interindividual variability in the location of the genital representation field and use-associated structural variation of genital field thickness associated with normative sexual activity in adult women. Using a sensory-tactile fMRI paradigm, we individually mapped genital fields of 128 women with and without exposure to childhood sexual abuse. We assessed whether structural variation of the individual genital field is driven by exposure to childhood sexual abuse or sexual frequency in the past year. We show that exposure to childhood sexual abuse associated with reduced thickness of individually-mapped genital cortex. Earlier abuse onset predicted greater reductions of genital field thickness. There was no effect of sexual frequency in the past year on genital field thickness. Classic neuroplasticity research indicates amplifying effects of stimulation on sensory cortex. In contrast, our results show long-lasting damaging effects of inappropriate stimulation during early development, emphasizing the need to protect children from sexual adversity.

Single amino acid substitutions in the ATP-binding domain of ACVRL1, a key receptor in the bone morphogenetic protein (BMP) signaling pathway, are frequently classified as variants of uncertain significance (VUS), complicating molecular diagnosis for pulmonary arterial hypertension (PAH) and Hereditary Hemorrhagic Telangiectasia (HHT). Since aberrant ATP binding disrupts downstream SMAD1/5/8 phosphorylation, we employed molecular dynamics (MD) simulations to quantitatively assess the functional impact of these variants. We first validated our approach on 20 known pathogenic/likely pathogenic variants within 5&#xc5; of the ATP-binding site, finding that 18 (90%) caused significant alterations in binding affinity (|d| &#x2265; 0.8, p&#xa0;<&#xa0;0.001). We then applied this protocol to all known VUS, conflicting, and unclassified variants within the same region, reclassifying 20 of 32 (63%) as likely pathogenic. Comprehensive in silico mutagenesis of all possible substitutions at ATP-binding pocket positions, combined with InterVar classification under HHT phenotype, enabled reclassification of 9 of 12 (75%) VUS as likely pathogenic. Finally, we demonstrated the applicability of this approach in two PAH patients with HHT carrying ACVRL1 VUS. This work establishes MD simulation of ATP-binding affinity as an effective and scalable tool for the functional interpretation of kinase variants, with broad potential for application across other disease-associated kinases.

The target of rapamycin complex 2 (TORC2) is a central node in signaling feedback loops, serving to maintain the biophysical homeostasis of the plasma membrane (PM). How TORC2 is regulated by mechanical perturbation of the PM is not well understood. To address this, we determined the cryo-electron microscopy structure of endogenous yeast TORC2 at up to 2.2 &#xc5; resolution. Our model refines the position and interactions of TORC2-specific subunits, providing a structural basis for the differential assembly of Tor2 into TORC2. Furthermore, we observe the insertion of the pleckstrin-homology domain of the Avo1 subunit into the Tor2 active site, providing a regulatory mechanism mediated by phosphoinositides. Structure-guided functional experiments reveal a potential TORC2 membrane-binding surface and a positively charged pocket in the Avo3 subunit that is necessary for TORC2 activation. Collectively, our data suggest that signaling phosphoinositides activate TORC2 by membrane-induced structural rearrangements via the concerted action of conserved regulatory subunits.

Cryopreserving the adult brain is challenging due to damage from ice formation, and traditional freezing methods fail to maintain neural architecture and function. Vitrification offers a promising alternative but has not been surveyed in the brain. Here, we demonstrate short-term recovery of the adult murine hippocampus after vitrification of brain slices and of the whole brain in situ. Key features of the hippocampus are preserved, including structural integrity, metabolic responsiveness, neuronal excitability, and synaptic transmission and plasticity. Notably, hippocampal long-term potentiation (LTP) was well preserved, indicating that the cellular machinery of learning and memory remains operational. These findings extend known biophysical limits for cerebral hypothermic shutdown by demonstrating recovery after complete cessation of molecular mobility in the vitreous state and thus contribute to achieving the objective of structural and functional preservation of neural tissue.

The Seh1-associated complex (SEAC; GATOR in mammals) transduces amino acid signals to the Target of Rapamycin Complex 1 (TORC1), a master regulator of cell growth. The SEAC is composed of two subcomplexes, SEACIT (GATOR1), an inhibitor of TORC1 that has GAP activity against Gtr1, and SEACAT (GATOR2), which appears to regulate SEACIT. However, the molecular details of this regulation are unclear. Here we determined the cryo-electron microscopy structure of the SEAC bound to its substrate, the EGOC (Ragulator-Rag), and studied its function in TORC1 amino acid signaling. A single SEAC can interact with two EGOC molecules via SEACIT, binding exclusively to the 'active' version of the EGOC, without involvement of SEACAT. The GAP activity of the SEACIT is essential for the regulation of TORC1 by amino acids and its loss phenocopies the lack of Gtr1-Gtr2, establishing the SEAC-EGOC complex as an amino acid-sensing hub. Compared to other SEACAT subunits, the loss of Sea2, or its N-terminal &#x3b2;-propeller domain, yielded strong defects in amino acid signaling to TORC1. Our results suggest that the Sea2 &#x3b2;-propeller recruits a GAP inhibitor to mediate fast amino acid signaling to TORC1, with additional pathways acting with slower kinetics.

Breast cancer disparities in outcomes remain a persistent challenge in the USA, with survival influenced by neighborhood context, access to resources, and the applicability of existing scientific evidence across populations. These disparities arise from complex, intersectional factors spanning social, structural, and biologic domains. While social determinants of health (SDOH) are strongly associated with breast cancer incidence and outcomes, the causality of biologic mechanisms underlying these associations remain incompletely understood. This review examines breast cancer disparities at the societal level and highlights emerging research that links social epidemiology with tumor biology, emphasizing the need for continued investigation using advanced genomic and epigenetic approaches to better understand and ultimately reduce inequities in breast cancer outcomes.

PSD95, a member of the membrane-associated guanylate kinase family, plays a key role in synaptic transmission. In this multidomain protein, the third PDZ domain has a complex regulatory mechanism that modulates its binding of carboxyl-terminal sequences. Phosphorylation of Tyr397, located in the additional &#x3b1;3 helix of this PDZ domain, has been shown to affect the domain's binding affinity. To explore the molecular basis of these changes in affinity, the crystal structure of the mutant Tyr397Glu, a point mutation intended to mimic phosphorylated tyrosine, has been determined. The crystal structure of this mutant reveals conformational changes induced by the introduction of a negative charge into the extra-domain &#x3b1;3 helix, suggesting communication between distant secondary-structure elements that may affect the binding affinity of this domain. Additionally, DSC folding studies show a noticeable decrease in the mutant's stability, indicating significant conformational changes. Altogether, the experimental results included in this work demonstrate that &#x3b1;3 is part of an electrostatic network that regulates stability and conformational changes at distant sites, including the &#x3b2;-hairpin at the binding site.