Biology subjects

Sandalova, T.

Publications and source records attributed to Sandalova, T..

9 recordsLinked to original sources

bioRxiv2025

Insight into Malt1 activation mechanism through synergetic approach of AlphaFold, MD Simulation and NMR dynamic analyses

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a central regulator of immune signalling, yet the conformational dynamics governing its activation remain poorly defined. Building on our earlier solution-state analysis of apo MALT1(PCASP-Ig3)339-719, which revealed domain flexibility, dynamic autoinhibition, and sensitivity to physiological ionic conditions, we combine NMR relaxation, molecular dynamics simulations, and ensemble modelling to delineate how solution environment reshapes its conformational landscape. Because most structural information derives from dimeric or inhibitor-bound states, the behaviour of monomeric, ligand-free MALT1 in physiological solution has remained unclear. Here, MD simulations show that low-salt conditions drive all trajectories toward a unified inactive-like ensemble, marked by inward rotation of W580 and coordinated rearrangements of Loop 2 and Loop 3, indicating that the inactive state is energetically favoured and its reactivation kinetically suppressed. Physiological ionic strength partially restores access to active-like loop motions, aligning with NMR evidence that sodium modulates catalytic readiness. In contrast, high-salt conditions rigidify the PCASP-Ig3 module, suppressing loop fluctuations and preventing active-inactive transitions, thereby strongly enriching the active-state population. Importantly, the combined MD-NMR analysis demonstrates that the NMR-initiated ensembles provide the most faithful representation of backbone and loop dynamics under low-salt conditions, capturing substrate-independent loop rearrangements, stable hydrophobic-core behaviour, and the intrinsic transitions that shape MALT1s conformational equilibrium. Together, these findings identify ionic strength as a key regulator of MALT1 conformational equilibria,, highlighting how loop dynamics and domain flexibility tune its proteolytic competence and providing a dynamic framework for future structure-based modulation of MALT1 activity.

molecular biology↗

bioRxiv2025

From Structure to Immunogenicity: Decoding Correlated Dynamics atthe Peptide MHC interface to Understand TCR Recognition

The interaction between a class I peptide-major histocompatibility complex (pMHC) and a T cell receptor (TCR) plays a central role in the elicitation of CD8+ T cell immune responses. As a result, considerable effort has been invested in understanding the structural, dynamic, and biophysical parameters that govern this recognition event, including designing altered peptide ligands (APLs) which seek to modulate the downstream signaling outcomes. However, dynamic links between modified peptide positions and distant residues have until yet been ill resolved. Using an integrative approach combining crystallographic ensemble and single models with atomistic molecular dynamics simulations and correlational analysis, we have established an approach that allows us to identify coupled dynamics between spatially distant residues at the pMHC interface. Furthermore, we constructed a network encoding the inter-residue couplings observed throughout the simulations. This computational workflow corroborates well with experimental data and leads to novel insights regarding the differential immunogenicity of the closely related peptides analyzed in this study. Ultimately, we present an intuitive and comprehensive strategy for decoding the linked dynamics at the pMHC interface allowing for mechanistic insights into the biophysical bases governing immunogenicity. One Sentence SummaryThe dynamics at the pMHC interface can be encoded as a biophysically relevant network to yield molecular insights into immunogenicity

immunology↗

bioRxiv2025

Mutational insights into human kynurenine aminotransferase 1: modulation of transamination and β-elimination activities across diverse substrates

Human kynurenine aminotransferase 1 (hKYAT1) plays a crucial role in the transamination of aromatic amino acids and kynurenine. This promiscuous homodimeric enzyme transaminates various amino acids into their corresponding alpha-keto acids. Additionally, hKYAT1 is known to catalyze the beta-elimination of cysteine-S-conjugates and cysteine-Se-conjugates. In this study, we performed mutational analyses of hKYAT1, targeting its catalytic, ligand-binding, and substrate-binding sites. The transamination activity of thirteen mutant variants was systematically evaluated against sixteen different amino acid substrates, including kynurenine, selenomethionine (SeMet), and Se-methylselenocysteine (MSC), as well as for the {beta}-elimination of SeMet and MSC. Our results demonstrate that mutations of residues E27 in the catalytic site and H279 in the substrate-stabilizing site significantly enhanced the transamination of several amino acids, including phenylalanine, tryptophan, histidine, and MSC. The H279F mutation increased transamination and {beta}-elimination of MSC by two- and 1.5-fold, respectively. Furthermore, mutation at the ligand-binding residues R398 and N185 abolished the transamination activity of hKYAT1. Interestingly, none of the tested mutations affected the transamination of L-kynurenine, a natural substrate of hKYAT1. These findings provide a foundation for the rational design of selective inhibitors with potential therapeutic applications.

bioengineering↗

bioRxiv2025

Development of DARPin T cell engagers for specific targeting of tumor-associated HLA/peptide complexes

The compromise between affinity and specificity in TCR-dependent targeting of HLA-restricted tumor-associated antigens presents a significant challenge in developing efficacious immunotherapies. As such, T cell engagers which circumvent these limitations are of particular interest. We have established a process to generate bispecific Designed Ankyrin Repeat Proteins (DARPins) that simultaneously target HLA-I molecules in complex with tumor-associated peptides and CD3{varepsilon}. High-affinity HLA-A*0201/NY-ESO1157-165-specific DARPins were isolated after only four rounds of in-vitro selection from naive DARPin libraries. Combining HLA-A*0201/NY-ESO1157-165-specific DARPins with a CD3{varepsilon}-specific DARPin created potent T cell engagers which elicited CD8+ T cell activation towards tumor targets with high peptide specificity, as confirmed by X-scanning mutagenesis and functional killing assays. The cryo-EM structure of a ternary DARPin/HLA-A*0201/NY-ESO1157-165 complex revealed a rigid and concave DARPin surface that binds to the entire length of the peptide-binding cleft, contacting both -helices and the peptide. The present results unveil promising immuno-oncotherapeutic approaches with the possibility of rapidly developing DARPins with high affinity and specificity to HLA/peptide targets that can be readily combined with a new generation of anti-CD3{varepsilon}-specific DARPins.

synthetic biology↗

bioRxiv2025

Accurate Protein Dynamic Conformational Ensembles: Combining AlphaFold, MD and Amide 15N(1H) NMR Relaxation.

Conformational heterogeneity is critical for protein function, but the validation of dynamic ensembles remains a challenge. In this study, we introduced an approach that integrates free MD simulations, using an AlphaFold-generated structure as the starting point, with experimental relaxation data to identify biologically relevant conformational ensembles. For the extracellular region of Streptococcus pneumoniae PsrSp, we found that only certain segments of the MD long trajectory aligned well with experimental data. The defined ensembles revealed two regions with increased flexibility that play important functional roles.

molecular biology↗

bioRxiv2024

Crystallographic and NMR studies of Streptococcus pneumonia LCP protein PsrSp indicate the importance of dynamics in four long loops for ligand specificity

The crystal structure of the extracellular region of the second pneumococcal LCP, a polyisoprenyl-teichoic acid-peptidoglycan teichoic acid transferase PsrSp, was determined and refined to 2.15[A] resolution. Despite the low sequence homology with other LCP proteins, the PsrSp maintains the fold of the LCP domain and the positions of the 15 residues suggested to participate in the transferase function are conserved. The empty tunnel found in the PsrSp between the central {beta}-sheet and three -helices is wide enough to accommodate polyisoprenyl-teichoic acid. Comparison of the crystallographic temperature factors of LCP from distinct bacteria demonstrated that the four long loops located close to the teichoic acid and peptidoglycan binding sites have different relative mobility. To compare the dynamics of the PsrSp in crystalline state and in solution, NMR spectra were recorded, and 88% of the residues were assigned in the 1H-15N TROSY HSQC spectra. Comparison of the secondary structure of the crystal structure of PsrSp with NMR data demonstrated a perfect concordance between the results using these two methods. Moreover, the relative mobility of the essential loops estimated from the crystallographic B-factor is in good agreement with order parameter S2, predicted from chemical shift. We hypothesize that the dynamics of these loops are important for the substrate promiscuity of LCP proteins.

microbiology↗

bioRxiv2024

Towards understanding of allostery in MALT1: a possible role of interdomain motions as revealed by NMR and AlphaFold

Mucosa-associated lymphoid tissue lymphoma-translocation protein 1 (MALT1) has emerged as an attractive target for the development of modulatory compounds, particularly in the treatment of lymphoma and other cancers. While the three-dimensional structure of MALT1(PCASP-Ig3)339-719 has been previously determined through X-ray analysis, its dynamic behaviour in solution has remained largely unexplored. We present here inaugural dynamic analyses of the apo MALT1(PCASP-Ig3)339-719 form along with its mutated variant, E549A. This investigation harnessed an array of NMR relaxation techniques, including longitudinal and transverse 15N auto-relaxation, heteronuclear NOE, transverse cross-correlated relaxation and NOE measurements between side-chain methyl groups. Our findings unequivocally confirm that MALT1(PCASP-Ig3)339-719 exists solely as a monomer in solution, and demonstrate that the two domains display semi-independent movements in relation to each other. Our extensive dynamic study, covering a range of time scales, along with the assessment of diverse conformational populations for MALT1(PCASP-Ig3)339-719, by Molecular Dynamic simulations, Alpha Fold modelling and PCA analysis, shed light at potential mechanisms underlying the allosteric regulation of this enzyme, and the specific importance of interdomain motions.

molecular biology↗

bioRxiv2021

PD-1 expression on NK cells can be related to cytokine stimulation and tissue residency

Although PD-1 was shown to be a hallmark of T cells exhaustion, controversial studies have been reported on the role of PD-1 on NK cells. Here, we found by flow cytometry and single cell RNA sequencing analysis that PD-1 can be expressed on MHC class I-deficient tumor-infiltrating NK cells in vivo. We also demonstrate distinct alterations in the phenotype of PD-1-deficient NK cells which in part could be attributed to a decrease in tumor-infiltrating NK cells in PD-1-deficient mice. NK cells from PD-1-deficient mice exhibited a more mature phenotype which might reduce their capacity to migrate and kill in vivo. Finally, our results demonstrate that PD-L1 molecules in membranes of PD-1-deficient NK cells migrate faster than in NK cells from wildtype mice, suggesting that PD-1 and PD-L1 form cis interactions with each other on NK cells.

immunology↗

bioRxiv2019

Tuning antiviral CD8 T-cell response via proline-altered peptide ligand vaccination

Viral escape from CD8+ cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8+ T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape. Author SummaryViral escape mutagenesis correlates often with disease progression and represents a major hurdle for vaccination-based therapies. Here, we have designed and developed a novel generation of altered epitopes that re-establish and enhance significantly CD8+ T cell recognition of a naturally occurring viral immune escape variant. Biophysical and structural analyses provide a clear understanding of the molecular mechanisms underlying this reestablished recognition. We believe that this approach can be implemented to currently available or novel vaccination approaches to efficiently restore T cell recognition of virus escape variants to control disease progression.

immunology↗