RESUMO
Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting integrins in the plasma membrane with the actomyosin cytoskeleton. To understand how talin function is regulated, we determined a cryoelectron microscopy (cryo-EM) structure of full-length talin1 revealing a two-way mode of autoinhibition. The actin-binding rod domains fold into a 15-nm globular arrangement that is interlocked by the integrin-binding FERM head. In turn, the rod domains R9 and R12 shield access of the FERM domain to integrin and the phospholipid PIP2 at the membrane. This mechanism likely ensures synchronous inhibition of integrin, membrane, and cytoskeleton binding. We also demonstrate that compacted talin1 reversibly unfolds to an â¼60-nm string-like conformation, revealing interaction sites for vinculin and actin. Our data explain how fast switching between active and inactive conformations of talin could regulate FA turnover, a process critical for cell adhesion and signaling.
Assuntos
Adesões Focais/metabolismo , Domínios e Motivos de Interação entre Proteínas , Talina/química , Talina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Sítios de Ligação , Adesão Celular/fisiologia , Microscopia Crioeletrônica , Citoesqueleto/metabolismo , Dimerização , Escherichia coli/metabolismo , Humanos , Integrinas/metabolismo , Modelos Moleculares , Ligação Proteica , Transdução de Sinais/fisiologia , Vinculina/metabolismoRESUMO
Sequential proteolysis of the amyloid precursor protein (APP) by γ-secretases generates amyloid-ß (Aß) peptides and defines the proportion of short-to-long Aß peptides, which is tightly connected to Alzheimer's disease (AD) pathogenesis. Here, we study the mechanism that controls substrate processing by γ-secretases and Aß peptide length. We found that polar interactions established by the APPC99 ectodomain (ECD), involving but not limited to its juxtamembrane region, restrain both the extent and degree of γ-secretases processive cleavage by destabilizing enzyme-substrate interactions. We show that increasing hydrophobicity, via mutation or ligand binding, at APPC99 -ECD attenuates substrate-driven product release and rescues the effects of Alzheimer's disease-associated pathogenic γ-secretase and APP variants on Aß length. In addition, our study reveals that APPC99 -ECD facilitates the paradoxical production of longer Aßs caused by some γ-secretase inhibitors, which act as high-affinity competitors of the substrate. These findings assign a pivotal role to the substrate ECD in the sequential proteolysis by γ-secretases and suggest it as a sweet spot for the potential design of APP-targeting compounds selectively promoting its processing by these enzymes.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Doença de Alzheimer/metabolismo , ProteóliseRESUMO
Alzheimer's disease (AD) pathogenesis has been linked to the accumulation of longer, aggregation-prone amyloid ß (Aß) peptides in the brain. Γ-secretases generate Aß peptides from the amyloid precursor protein (APP). Γ-secretase modulators (GSMs) promote the generation of shorter, less-amyloidogenic Aßs and have therapeutic potential. However, poorly defined drug-target interactions and mechanisms of action have hampered their therapeutic development. Here, we investigate the interactions between the imidazole-based GSM and its target γ-secretase-APP using experimental and in silico approaches. We map the GSM binding site to the enzyme-substrate interface, define a drug-binding mode that is consistent with functional and structural data, and provide molecular insights into the underlying mechanisms of action. In this respect, our analyses show that occupancy of a γ-secretase (sub)pocket, mediating binding of the modulator's imidazole moiety, is sufficient to trigger allosteric rearrangements in γ-secretase as well as stabilize enzyme-substrate interactions. Together, these findings may facilitate the rational design of new modulators of γ-secretase with improved pharmacological properties.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Humanos , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Inibidores e Moduladores de Secretases gama , Doença de Alzheimer/metabolismo , Imidazóis/uso terapêuticoRESUMO
One-third of the human proteome is comprised of membrane proteins, which are particularly vulnerable to misfolding and often require folding assistance by molecular chaperones. Calnexin (CNX), which engages client proteins via its sugar-binding lectin domain, is one of the most abundant ER chaperones, and plays an important role in membrane protein biogenesis. Based on mass spectrometric analyses, we here show that calnexin interacts with a large number of nonglycosylated membrane proteins, indicative of additional nonlectin binding modes. We find that calnexin preferentially bind misfolded membrane proteins and that it uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by calnexin, and identify sequence motifs within the calnexin TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of calnexin. Together, these data reveal a widespread role of calnexin client recognition in the lipid bilayer, which synergizes with its established lectin-based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome.
Assuntos
Chaperonas Moleculares , Proteoma , Humanos , Calnexina/metabolismo , Proteoma/metabolismo , Chaperonas Moleculares/metabolismo , Lectinas/metabolismo , Proteínas de Membrana/metabolismo , Dobramento de Proteína , Proteínas de Ligação ao Cálcio/metabolismoRESUMO
Self-assembled aggregation of peptides and proteins into regular amyloid fibrils is associated with several neurodegenerative diseases. In case of Alzheimer's disease proteolytic cleavage products of the amyloid precursor protein form pathological amyloid-beta fibrils in a nucleation and propagation phase. The molecular details and thermodynamic driving forces of amyloid formation are not well understood, but are of high relevance for potential pharmacological interference. We used atomistic binding free energy simulations to calculate the free energy of protofilament propagation by an additional Aß9-40 peptide binding to the protofilament tip. It requires sampling of relevant conformational transitions which is challenging since the monomeric Aß9-40 peptide is intrinsically disordered. However, the convergence of umbrella simulations can be enhanced by applying additional restraining potentials on the axial, orientational and conformational degrees of freedom. The improved convergence leads to a much closer agreement with experimental binding free energy data compared to unrestrained umbrella sampling. Moreover, the restraining approach results in a separation of contributions to the total binding free energy. The calculated contributions indicate that the free energy change associated with the restriction of conformational freedom upon propagation makes a large opposing contribution of higher magnitude than the total binding free energy. Finally, optimization of the approach leads to further significant reduction of the computational demand which is crucial for systematic studies on mutations, denaturants and inhibitors in the fibril propagation step.
RESUMO
The deposition of islet amyloid polypeptide (hIAPP) fibrils is a hallmark of ß-cell death in type II diabetes. In this study, we employ state-of-the-art MAS solid-state spectroscopy to investigate the previously elusive N-terminal region of hIAPP fibrils, uncovering both rigidity and heterogeneity. Comparative analysis between wild-type hIAPP and a disulfide-deficient variant (hIAPPC2S,C7S) unveils shared fibril core structures yet strikingly distinct dynamics in the N-terminus. Specifically, the variant fibrils exhibit extended ß-strand conformations, facilitating surface nucleation. Moreover, our findings illuminate the pivotal roles of specific residues in modulating secondary nucleation rates. These results deepen our understanding of hIAPP fibril assembly and provide critical insights into the molecular mechanisms underpinning type II diabetes, holding promise for future therapeutic strategies.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Humanos , Amiloide/química , Amiloide/metabolismo , Conformação ProteicaRESUMO
We investigated a cohort of 370 patients in Austria with hantavirus infections (7.8% ICU admission rate) and detected 2 cases (cumulative incidence 7%) of invasive pulmonary aspergillosis; 1 patient died. Hantavirus-associated pulmonary aspergillosis may complicate the course of critically ill patients who have hemorrhagic fever with renal syndrome.
Assuntos
Estado Terminal , Infecções por Hantavirus , Aspergilose Pulmonar Invasiva , Humanos , Áustria/epidemiologia , Infecções por Hantavirus/epidemiologia , Infecções por Hantavirus/complicações , Aspergilose Pulmonar Invasiva/epidemiologia , Aspergilose Pulmonar Invasiva/tratamento farmacológico , OrthohantavírusRESUMO
At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin (ß2m, encoded by B2M), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single-molecule co-tracking. We identify non-covalent MHC-I FHC dimers, with dimerization mediated by the α3 domain, as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single-molecule colocalization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the α3 domain of one FHC binds to another FHC in a manner similar to that seen for ß2m.
Assuntos
Antígenos de Histocompatibilidade Classe I , Microglobulina beta-2 , Animais , Antígenos de Histocompatibilidade Classe I/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Peptídeos/metabolismo , Ligação Proteica , Microglobulina beta-2/metabolismoRESUMO
AIMS: Mucormycosis is a fast-progressing disease with a high mortality rate. The most important factor determining survival of patients is early and accurate diagnosis. Although histopathology often recognises invasive mould infections at first, histomorphology alone is insufficient in providing an accurate diagnosis. Unbiased molecular methods to detect and identify fungi are promising, yet their role in complementing routine histopathological workflows has not been studied sufficiently. METHODS AND RESULTS: We performed a retrospective single-centre study examining the clinical value of complementing histopathology with internal transcribed spacer (ITS) sequencing of fungal DNA in the routine diagnosis of mucormycosis. At our academic centre, we identified 14 consecutive mucormycosis cases diagnosed by histopathology and subsequent ITS sequencing. Using histomorphological examination, fungal hyphae could be detected in all cases; however, morphological features were unreliable regarding specifying the taxa. Subsequent ITS sequencing identified a remarkable phylogenetic diversity among Mucorales: the most common species was Rhizopus microsporus (six of 14; 42.9%), followed by Lichtheimia corymbifera (three of 14, 21.4%) and single detections of Rhizopus oryzae, Actinomucor elegans, Mucor circinelloides, Rhizomucor pusillus and Rhizomucor miehei (one of 14; 7.1%, respectively). In one case, we additionally detected Pneumocystis jirovecii in the same lung tissue specimen, suggesting a clinically relevant co-infection. Fungal culture was performed in 10 cases but yielded positive results in only two of 10 (20%), revealing its limited value in the diagnosis of mucormycosis. CONCLUSIONS: Our study demonstrates that a combination of histopathology and ITS sequencing is a practically feasible approach that outperforms fungal culture in detecting Mucorales in tissue-associated infections. Therefore, pathologists might adapt diagnostic workflows accordingly when mucormycosis is suspected.
Assuntos
Mucormicose , Humanos , Mucormicose/diagnóstico , Mucormicose/microbiologia , Mucormicose/patologia , Estudos Retrospectivos , FilogeniaRESUMO
Compared to small molecules and antibodies, cyclic peptides exhibit unique biochemical and therapeutic attributes in the realm of pharmaceutical applications. The interaction between the inducible costimulator (ICOS) and its ligand (ICOSL) plays a key role in T-cell differentiation and activation. ICOS/ICOSL inhibition results in a reduction in the promotion of immunosuppressive regulatory T cells (Tregs) in both hematologic malignancies and solid tumors. Herein, we implement the computational cPEPmatch approach to design the first examples of cyclic peptides that inhibit ICOS/ICOSL interaction. The top cyclic peptide from our approach possessed an IC50 value of 1.87 ± 0.15 µM as an ICOS/ICOSL inhibitor and exhibited excellent in vitro pharmacokinetic properties as a drug candidate. Our work will lay the groundwork for future endeavors in cancer drug discovery, with the goal of developing cyclic peptides that target the ICOS/ICOSL interaction.
Assuntos
Antineoplásicos , Linfócitos T Reguladores , Anticorpos , Antineoplásicos/farmacologia , Proteína Coestimuladora de Linfócitos T Induzíveis , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologiaRESUMO
Many biological functions are mediated by large complexes formed by multiple proteins and other cellular macromolecules. Recent progress in experimental structure determination, as well as in integrative modeling and protein structure prediction using deep learning approaches, has resulted in a rapid increase in the number of solved multiprotein assemblies. However, the assembly process of large complexes from their components is much less well-studied. We introduce a rapid computational structure-based (SB) model, GoCa, that allows to follow the assembly process of large multiprotein complexes based on a known native structure. Beyond existing SB GoÌ -type models, it distinguishes between intra- and intersubunit interactions, allowing us to include coupled folding and binding. It accounts automatically for the permutation of identical subunits in a complex and allows the definition of multiple minima (native) structures in the case of proteins that undergo global transitions during assembly. The model is successfully tested on several multiprotein complexes. The source code of the GoCa program including a tutorial is publicly available on Github: https://github.com/ZachariasLab/GoCa. We also provide a web source that allows users to quickly generate the necessary input files for a GoCa simulation: https://goca.t38webservices.nat.tum.de.
Assuntos
Conformação Proteica , Proteínas , Proteínas/química , Proteínas/metabolismo , Sítios de Ligação , Modelos Moleculares , Software , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismoRESUMO
The sequence-dependent structure and deformability of DNA play a major role for binding of proteins and regulation of gene expression. So far, most efforts to model DNA flexibility are based on unimodal harmonic stiffness models at base-pair resolution. However, multimodal behavior due to distinct conformational substates also contributes significantly to the conformational flexibility of DNA. Moreover, these local substates are correlated to their nearest-neighbor substates. A description for DNA elasticity which includes both multimodality and nearest-neighbor coupling has remained a challenge, which we solve by combining our multivariate harmonic approximation with an Ising model for the substates. In a series of applications to DNA fluctuations and protein-DNA complexes, we demonstrate substantial improvements over the unimodal stiffness model. Furthermore, our multivariate Ising model reveals a mechanical destabilization for adenine (A)-tracts to undergo nucleosome formation. Our approach offers a wide range of applications to determine sequence-dependent deformation energies of DNA and to investigate indirect readout contributions to protein-DNA recognition.
Assuntos
DNA/química , Modelos Teóricos , Conformação de Ácido Nucleico , Animais , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Simulação de Dinâmica Molecular , Nucleossomos/química , Nucleossomos/metabolismo , Ligação ProteicaRESUMO
Novel biophysical tools allow the structural dynamics of proteins and the regulation of such dynamics by binding partners to be explored in unprecedented detail. Although this has provided critical insights into protein function, the means by which structural dynamics direct protein evolution remain poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the periplasmic-binding protein-like II domain family, have undergone divergent evolution, leading to adaptation of their structural dynamics. We performed a structural analysis on â¼600 bilobed proteins with a common primordial structural core, which we complemented with biophysical studies to explore the structural dynamics of selected examples by single-molecule Förster resonance energy transfer and Hydrogen-Deuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enable distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extracytoplasmic transport-related proteins. Structural embellishments of the core created interdomain interactions that stabilized structural states, reshaping the active site geometry, and ultimately altered substrate specificity. Our findings reveal an as-yet-unrecognized mechanism for the emergence of functional promiscuity during long periods of evolution and are applicable to a large number of domain architectures.
Assuntos
Proteínas/química , Proteínas/metabolismo , Escherichia coli/metabolismo , Evolução Molecular , Regulação da Expressão Gênica , Espectrometria de Massas , Modelos Moleculares , Filogenia , Conformação Proteica , Domínios Proteicos , Proteínas/genéticaRESUMO
Molecular dynamics simulations have strongly matured as a method to study biomolecular processes. Their validity, however, is determined by the accuracy of the underlying force fields that describe the forces between all atoms. In this article, we review the development of nucleic acids force fields. We describe the early attempts in the 1990s and emphasize their strong influence on recent force fields. State-of-the-art force fields still use the same Lennard-Jones parameters derived 25 years ago in spite of the fact that these parameters were in general not fitted for nucleic acids. In addition, electrostatic parameters also are deprecated, which may explain some of the current force field deficiencies. We compare different force fields for various systems and discuss new tests of the recently developed Tumuc1 force field. The OL-force fields and Tumuc1 are arguably the best force fields to describe the DNA double helix. However, no force field is flawless. In particular, the description of sugar-puckering remains a problem for nucleic acids force fields. Future refinements are required, so we review methods for force field refinement and give an outlook to the future of force fields.
Assuntos
Ácidos Nucleicos , Ácidos Nucleicos/química , Simulação de Dinâmica Molecular , DNA/química , CarboidratosRESUMO
γ-Secretase complexes (GSECs) are multimeric membrane proteases involved in a variety of physiological processes and linked to Alzheimer's disease (AD). Presenilin (PSEN, catalytic subunit), Nicastrin (NCT), Presenilin Enhancer 2 (PEN-2), and Anterior Pharynx Defective 1 (APH1) are the essential subunits of GSECs. Mutations in PSEN and the Amyloid Precursor Protein (APP) cause early-onset AD GSECs successively cut APP to generate amyloid-ß (Aß) peptides of various lengths. AD-causing mutations destabilize GSEC-APP/Aßn interactions and thus enhance the production of longer Aßs, which elicit neurotoxic events underlying pathogenesis. Here, we investigated the molecular strategies that anchor GSEC and APP/Aßn during the sequential proteolysis. Our studies reveal that a direct interaction between NCT ectodomain and APPC99 influences the stability of GSEC-Aßn assemblies and thereby modulates Aß length. The data suggest a potential link between single-nucleotide variants in NCSTN and AD risk. Furthermore, our work indicates that an extracellular interface between the protease (NCT, PSEN) and the substrate (APP) represents the target for compounds (GSMs) modulating Aß length. Our findings may guide future rationale-based drug discovery efforts.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Glicoproteínas de Membrana/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Secretases da Proteína Precursora do Amiloide/química , Precursor de Proteína beta-Amiloide/química , Animais , Células Cultivadas , Ativação Enzimática , Espaço Extracelular , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Proteólise , Relação Estrutura-AtividadeRESUMO
AzoChignolin is a photoswitchable variant of the mini-protein Chignolin with an azobenzene (AMPP) replacing the central loop. AzoChignolin is unfolded with AMPP in the trans-isomer. Transition to the cis-isomer causes ß-hairpin folding similar to Chignolin. The AzoChignolin system is excellently suited for comprehensive analysis of folding nucleation kinetics. Utilizing multiple long-time MD simulations of AzoChignolin and Chignolin in MeOH and water, we estimated Markov models to examine folding kinetics of both peptides. We show that while AzoChignolin mimics Chignolin's structure well, the folding kinetics are quite different. Not only folding times but also intermediate states differ, particularly Chignolin is able to fold in MeOH into an α-helical intermediate which is impossible to form in AzoChignolin. The Markov models demonstrate that AzoChignolin's kinetics are generally faster, specifically when comparing the two main microfolding processes of hydrophobic collapse and turn formation. Photoswitchable loops are used frequently to understand the kinetics of elementary protein folding nucleation. However, our results indicate that intermediates and folding kinetics may differ between natural loops and photoswitchable variants.
Assuntos
Oligopeptídeos , Peptídeos , Oligopeptídeos/química , Peptídeos/química , Dobramento de Proteína , CinéticaRESUMO
Zn2+ is one of the most versatile biologically available metal ions, but accurate modeling of Zn2+ -containing metalloproteins at the biomolecular force field level can be challenging. Since most Zn2+ models are parameterized in bulk solvent, in-depth knowledge about their performance in a protein environment is limited. Thus, we systematically investigate here the behavior of non-polarizable Zn2+ models for their ability to reproduce experimentally determined metal coordination and ligand binding in metalloproteins. The benchmarking is performed in challenging environments, including mono- (carbonic anhydrase II) and bimetallic (metallo-ß-lactamase VIM-2) ligand binding sites. We identify key differences in the performance between the Zn2+ models with regard to the preferred ligating atoms (charged/non-charged), attraction of water molecules, and the preferred coordination geometry. Based on these results, we suggest suitable simulation conditions for varying Zn2+ site geometries that could guide the further development of biomolecular Zn2+ models.
Assuntos
Metaloproteínas , Zinco , Zinco/química , Ligantes , Benchmarking , Sítios de Ligação , Metaloproteínas/químicaRESUMO
We present a case of bilateral cystic lung metastases originating from cutaneous angiosarcoma (cAS) of the scalp in a 73-year-old man. He presented with hemoptysis and recurrent bilateral pneumothorax. The clinical, radiological, and histological features and a potential pathophysiological mechanism of pulmonary changes in cutaneous angiosarcoma are discussed.
Assuntos
Cistos , Hemangiossarcoma , Pneumotórax , Neoplasias Cutâneas , Masculino , Humanos , Idoso , Pneumotórax/etiologia , Pneumotórax/patologia , Hemangiossarcoma/diagnóstico , Hemangiossarcoma/complicações , Hemangiossarcoma/patologia , Pulmão/patologia , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologia , Cistos/complicações , Cistos/patologiaRESUMO
The development of novel anti-infectives requires unprecedented strategies targeting pathways which are solely present in pathogens but absent in humans. Following this principle, we developed inhibitors of lipoic acid (LA) salvage, a crucial pathway for the survival of LA auxotrophic bacteria and parasites but non-essential in human cells. An LA-based probe was selectively transferred onto substrate proteins via lipoate protein ligase (LPL) in intact cells, and their binding sites were determined by mass spectrometry. Probe labeling served as a proxy of LPL activity, enabling in situ screenings for cell-permeable LPL inhibitors. Profiling a focused compound library revealed two substrate analogs (LAMe and C3) as inhibitors, which were further validated by binding studies and co-crystallography. Importantly, LAMe exhibited low toxicity in human cells and achieved killing of Plasmodium falciparum in erythrocytes with an EC50 value of 15â µM, making it the most effective LPL inhibitor reported to date.
Assuntos
Parasitos , Animais , Humanos , Proteômica , Plasmodium falciparum , Bactérias , EritrócitosRESUMO
In the past few decades, glycosaminoglycan (GAG) research has been crucial for gaining insights into various physiological, pathological, and therapeutic aspects mediated by the direct interactions between the GAG molecules and diverse proteins. The structural and functional heterogeneities of GAGs as well as their ability to bind specific proteins are determined by the sugar composition of the GAG, the size of the GAG chains, and the degree and pattern of sulfation. A deep understanding of the interactions in protein-GAG complexes is essential to explain their biological functions. In this study, the umbrella sampling (US) approach is used to pull away a GAG ligand from the binding site and then pull it back in. We analyze the binding interactions between GAGs of three types (heparin, desulfated heparan sulfate, and chondroitin sulfate) with three different proteins (basic fibroblast growth factor, acidic fibroblast growth factor, and cathepsin K). The main focus of our study was to evaluate whether the US approach is able to reproduce experimentally obtained structures, and how useful it can be for getting a deeper understanding of GAG properties, especially protein recognition specificity and multipose binding. We found that the binding free energy landscape in the proximity of the GAG native binding pose is complex and implies the co-existence of several binding poses. The sliding of a GAG chain along a protein surface could be a potential mechanism of GAG particular sequence recognition by proteins.