Biochemistry

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

CO dehydrogenases (CODH) are metalloenzymes that reversibly oxidize CO to CO2, at a buried NiFe4S4 active site. The substrates, CO and CO2, need therefore to be transported through the protein matrix to reach the active site. The most likely pathway for intra-protein diffusion is the hydrophobic channel identified in the crystal structures. Here, we use site-directed mutagenesis to study the highly conserved isoleucine 563 of Thermococcus sp. AM4 CODH2. Mutations at this position change the biochemical properties (KM for CO, product inhibition constant, catalytic bias...), and increase the resistance of the enzyme to the inhibitor O2, showing that isoleucine 563 indeed lines the gas channel. The I563F mutation decreases the bimolecular rate constant of inhibition by O2 15-fold, and increases the IC50 20-fold, which is the strongest improvement in O2 resistance reported so far. We show that the size of the introduced amino acids is less important than their flexibility - along with the size of the cavity formed near the active site in the channel. We also conclude that O2 access to the active site cannot be slowed down without also affecting CO diffusion. This tradeoff will have to be considered in further attempts to use site-directed mutagenesis to make CODHs more O2 tolerant.

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

Glutamate racemase (MurI) catalyzes the stereochemical interconversion of L-glutamate to D-glutamate, a key element of bacterial peptidoglycan biosynthesis. In this study, we present the crystal structure of Helicobacter pylori glutamate racemase at 1.43 [A] and in monoclinic symmetry, as previously reported models, but different unit-cell parameters. The present model contains a single dimer and retains the previously described head-to-head dimer arrangement. The differences between the models arise from variations in unit-cell parameters, which lead to altered crystal packing interactions rather than changes in the quaternary assembly. The monomeric fold and active-site architecture remain conserved and are consistent with the catalytic features described for bacterial glutamate racemases. This structure provides an updated, high-resolution structural model for H. pylori glutamate racemase and highlights the variability in crystal packing within the same space group.

Purified proteins are routinely flash frozen for use in functional and structural studies, providing a convenient way to reproduce results across complex experiments. Rho guanine-nucleotide exchange factors (RhoGEFs) are no exception to this practice, yet the effects of freezing on their activity and stability remain largely uncharacterized. This gap potentially affects the characterization of these important enzymes and how results are interpreted with respect to their prospective use as therapeutic targets. Here, we tested the isolated DH/PH tandems of P-Rex1, P-Rex2, and PRG under different cryoprotectant conditions and monitored activity and thermostability over time after flash freezing. Our results show a clear divergence between the activity of fresh and frozen purified RhoGEF protein samples in as little as one week for some conditions. Specifically, the variability in data collected on frozen samples was greatly increased. Despite these differences, thermostability seems to be preserved for much longer timepoints across RhoGEFs. Moreover, despite eventual changes in both activity and thermostability with respect to freezing, there are no obvious changes in global conformation between fresh and frozen samples of the isolated P-Rex2 DH/PH tandem. From our data, there are few generalizable trends between the different RhoGEFs and no single cryoprotective agent tested was a silver bullet to preserve both activity and thermostability across RhoGEFs. Overall, our findings emphasize the unpredictable effects of freezing RhoGEFs. As such, RhoGEF freezing should be carefully characterized for each protein and critically viewed when comparing analyses between different studies.

TBCK-related encephalopathy (TBCKE) is a neurodevelopmental disorder associated with biallelic mutations in TBCK. Despite the increasing number of reported cases worldwide, the biochemical and biophysical properties of TBCK remain unclear, hindering molecular understanding of its role in disease. Here, we present the successful expression, purification, and biochemical characterization of full-length human TBCK produced in Spodoptera frugiperda cells. Biochemical and biophysical analyses reveal that the catalytically inactive pseudokinase domain of TBCK lacks nucleotide binding, consistent with the absence of the canonical VAIK, HRD, and DFG motifs required for catalysis. These findings support that TBCK is a class I pseudokinase and provide a foundation for future structural and functional studies to elucidate its biological role.

Macrolides are an antibiotic class widely used in both human and veterinary medicine, and function by interfering with protein synthesis. Regrettably, numerous strategies for evading the antibiotic properties of macrolides have been found in bacteria, including enzyme-mediated inactivation. These mechanisms are now widely disseminated among pathogenic, animal-associated and environmental bacteria making them a One Health issue. Macrolide esterases, which hydrolyze the macrolactones ester bond, confer one such resistance mechanism. Two types of macrolide esterases have thus far been identified, the well-studied erythromycin esterases and the recently discovered Est-type enzymes that belong to the /{beta}-hydrolase superfamily. We present detailed structure-function studies for four diverse Est type esterases: which only share 44-66% sequence identity (EstTSf, EstTSt, EstTBc, and EstXEc). In addition to resistance profiling and substrate specificity studies, we present structures for all four enzymes, including structures for EstTBc and EstXEc in complex with tylosin and tylvalosin macrolides, post hydrolysis. Complementing the data with mutational and kinetic studies allowed for a detailed analysis of the structural basis for macrolide-enzyme interactions. Combined the data suggest that promiscuous binding and imprecise positioning, mediated by a water-cage, dictate substrate specificity for Est-type macrolide resistance enzymes. These insights may prove beneficial for next-generation antibiotic development.

Ferredoxins are central to cellular metabolism by mediating electron flow in energy conversion reactions. The focus of this study was to systematically examine twelve ferredoxin and ferredoxin-like proteins from Synechocystis sp. PCC 6803 to identify their properties, activities, and functions in electron transfer. Using electron paramagnetic resonance spectroscopy, we detected cluster types consistent with major ferredoxin families including plant-type [2Fe-2S], adrenodoxin, thioredoxin, and bacterial-type [4Fe- 4S] ferredoxins. In addition, we found that the ssr3184 ferredoxin-like protein exchanged between a [3Fe-4S] or a [4Fe-4S] cluster, pointing to a possible functional change in response to changes in oxygen or cellular redox poise. Electrochemical measurements demonstrated that these ferredoxins constitute a broad potential window, from -243 mV to -520 mV vs SHE. Investigations on their capacity to support electron-transfer focused on reactions with two major redox hubs: Photosystem I and pyruvate:ferredoxin oxidoreductase and included testing of binding interactions with nitrite reductase. Expression profiling under multiple environmental conditions was also used to predict function and revealed distinct regulatory patterns. Collectively, these findings identified a group of core ferredoxins that directly support photosynthetic electron transfer, and more specialized ones that may serve other functions. In summary, Synechocystis utilizes a suite of ferredoxins to maintain cellular redox homeostasis under dynamic environmental conditions.

One challenge to DNA replication is the presence of unrepaired damage on the template strand, which can stall the replication machinery. This stall can be resolved by the translesion synthesis (TLS) pathway, in which specialized translesion polymerases are recruited to copy damaged DNA. Because TLS polymerases are error-prone, their activity is regulated at multiple levels to minimize unnecessary mutagenesis. Although the molecular mechanisms of bacterial TLS have been extensively studied in Escherichia coli, less is known about this pathway in other species. In E. coli, the TLS polymerase Pol IV is minimally enriched at replication forks in the absence of DNA damage but is strongly recruited upon replication stalling, enabling TLS while minimizing mutagenesis. However, we recently showed that the Bacillus subtilis TLS polymerase Pol Y1, the homolog of Pol IV, is moderately enriched near replication sites even during normal growth and is not further enriched upon treatment with the DNA damaging agent 4-nitroquinoline 1-oxide (4-NQO). It is unknown whether this behavior is unique to 4-NQO or general to other types of DNA damage. In this study, we investigate the effects of four different DNA damaging agents (ultraviolet light, methyl methanesulfonate, nitrofurazone, and mitomycin C) in B. subtilis. We first characterize the contributions of the two TLS polymerases, Pol Y1 and Pol Y2, to DNA damage survival and damage-induced mutagenesis after treatment with these agents. We then use single-molecule fluorescence microscopy to measure the localization and dynamics of individual Pol Y1 molecules in live B. subtilis cells. We find that Pol Y1 and Pol Y2 have differing effects on survival and mutagenesis, but that under no circumstances is Pol Y1 strongly recruited to sites of replication upon DNA damage. This study broadens our understanding of TLS in B. subtilis, indicating that there are notable differences in TLS mechanisms across bacteria.

Proteins with intrinsically disordered regions (IDRs) migrate at a higher apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) complicating their analysis and identification. Here, we investigate the sequence determinants of the hypomobility of IDRs using a series of synthetic low complexity domains. We find that negative charge increases the apparent molecular weight, but neutral polar tracts also have abnormally slow migration. Positive charge and hydrophobic residues decrease the apparent molecular weight, although lysine residues show a biphasic effect with decreased migration at high fractional contents. Combinations of residues show that different sequence contributions to the apparent molecular weight are not additive. The results can be rationalized by the protein-decorated micelle model by considering both SDS binding and the compaction of protein SDS-complexes.

The core biopolymers (DNA, RNA and proteins) are assembled from their monomers under conditions that avoid water. RNA is crucial for the Origin of Life. When cleaved from its polymerized state, RNA first transitions to nucleoside 2,3-cyclic phosphates. In the reverse direction, RNA polymerizes from 2,3-cyclic monomers in dry states, creating short oligomers that then can ligate on a template under aqueous, alkaline conditions. We studied the role of the counterions in polymerization of 2,3-cyclic nucleotides under geologically plausible settings. Through experiments and simulations, we demonstrate that the presence of ammonium and alkylammonium counterions greatly improves RNA polymerization. The otherwise less reactive cytidine containing monomers formed polyC sequences of up to heptamers; copolymers of AU, GC, or GCAU were detected up to hexamers. Our findings suggest three reasons for this: (1) (Alkyl)ammonium cations form hydrogen bonds with phosphates, (2) their alkaline pKa value can trigger general base catalysis, and (3) (alkyl)ammonium salts naturally form dry, anhydrous materials. The findings indicate that pyrolyzed organic tars creating ammonia-rich gas pockets in subsurface rocks could have enhanced the early evolution of RNA. TOC image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/713775v1_ufig1.gif" ALT="Figure 1"> View larger version (112K): org.highwire.dtl.DTLVardef@1adc431org.highwire.dtl.DTLVardef@12b8da0org.highwire.dtl.DTLVardef@5f187dorg.highwire.dtl.DTLVardef@140ed1a_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

c-di-AMP is a bacterial second messenger with the crucial role of regulating turgor and osmotic adaptation. Due to the importance of intracellular K+ for osmotic balance, c-di-AMP controls the import and export of K+ by regulating the activity and transcription level of K+ transporters and channels. It has been postulated that c-di-AMP inactivates K+ import and activates K+ export. To gain a full understanding of the properties the K+ machinery in the Gram-positive model organism Bacillus subtilis and in particular, of how the machinery is regulated by c-di-AMP, we characterized the molecular properties of CpaA, a cation/H+ antiporter that has been shown to bind the dinucleotide. We determined the crystal structure of the cytosolic RCK domain with and without c-di-AMP and performed a functional characterization of full-length CpaA using a fluorescence-based flux assay. We found that c-di-AMP binds on the interface of the RCK-C subdomain but only small structural differences are detected between the apo- and holo-structure. We determined that CpaA is more active at high pH and that it slightly favors K+ over Na+ for exchange with H+. Unexpectedly, CpaA is inactivated by c-di-AMP with a K1/2 of inactivation around 1 {micro}M. Our results reinforce the emerging view that regulation of the bacterial K+ machinery by c-di-AMP is more complex than previously thought and that a detailed characterization of the molecular properties of the individual protein components and of how their activity is integrated is necessary for a complete view of the machinery physiological function.

The Ube2J1 enzyme that mediates the ubiquitination and proteasomal degradation of misfolded proteins at the ER is phosphorylated at serine S184. Following anisomycin treatment of HEK293T cells, we observed an inverse relationship between phosphorylation and dephosphorylation at this site. This suggested a dynamic interchange between the two forms, and we show that S184 is a target for protein phosphatase 2A. The S184-phosphorylated protein is known to exhibit increased sensitivity to proteasomal degradation, and we found that mutation at K186R increased the ratio of S184-phosphorylated to S184-dephosphorylated protein. Although the K186R mutant retained some sensitivity to proteasomal inhibition, our results show that Ube2J1 steady state expression can be exercised at multiple levels, and can involve dynamic phosphorylation and dephosphorylation at S184.

The conformational dynamics of a model cationic protein in water and in the presence of anionic sodium dodecyl sulphate (SDS) and cationic cetyltrimethylamonium bromide (CTAB) surfactants at different concentrations were investigated using all-atom molecular dynamics simulations. Free-energy landscapes constructed along principal components reveal a compact, well-defined native basin at 25 {degrees}C in water, whereas elevated temperature (100 {degrees}C) induces a broadening of the conformational space and the emergence of multiple metastable states. The presence of surfactants further modulates this behavior in a concentration-dependent manner. Cluster population analysis shows that SDS promotes a highly heterogeneous ensemble characterized by reduced dominance of the native-like cluster, while CTAB partially protects the protein from thermal denaturation at higher concentrations. Radial distribution functions demonstrate strong accumulation of SDS headgroups around the protein and pronounced insertion of SDS alkyl tails into hydrophobic protein regions, indicating direct hydrophobic destabilization and micelle-assisted unfolding. In contrast, CTAB exhibits weaker headgroup association owing to electrostatic repulsion and reduced tail-hydrophobic contacts, suggesting a less disruptive interaction mechanism. At high concentration, CTAB aggregates provide a structured hydrophobic environment that stabilizes the folded state and suppresses denaturation. Together, these results provide a molecular-level picture of how surfactant chemistry and concentration govern the conformational stability of a cationic protein, highlighting the dominant role of hydrophobic interactions in surfactant-induced denaturation at high temperature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/717321v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@f68004org.highwire.dtl.DTLVardef@14e9a98org.highwire.dtl.DTLVardef@18771d3org.highwire.dtl.DTLVardef@141fc6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

1Long QT syndrome Type 2 (LQT2) is a genetic disorder caused by missense mutations in the KCNH2 gene that encodes the potassium channel KV11.1. Previous studies have shown that most KV11.1 missense mutations with loss-of-function phenotypes result from impaired trafficking from the endoplasmic reticulum to the plasma membrane. To investigate the molecular basis of these defects, we used molecular dynamics simulations to analyze two sets of disease-associated missense mutations: those that suppress and those that maintain normal channel trafficking. We focused initially on the conformational and dynamics differences between wild-type and several mutants of KV11.1 via molecular dynamics simulations when two K+ were placed in the selectivity filter (SF). Our study reveals that missense mutations in the S4 helix allosterically disrupt the selectivity filter, a critical determinant for proper channel trafficking. Trafficking-competent variants largely retained a wild-type selectivity filter structure, whereas trafficking-deficient mutants exhibited pronounced structural perturbations in this region. These findings suggest that certain LQT2-associated missense mutations in KCNH2 impair channel trafficking by compromising the structural integrity of the selectivity filter. We additionally found that second-site variants Y652C in the drug binding vestibule can correct structural defects associated with some mistrafficking variants.

Staphylococcus aureus (S. aureus) is a clinically relevant pathogen capable of adapting its membrane composition in response to environmental stress. In this adaptive process, bacterial carotenoids play a crucial role. Although staphyloxanthin (STX) is the main carotenoid produced by the bacterium, S. aureus also synthesizes other pigmented intermediates that play an unknown role in regulating membrane biophysical properties. In this study, we purified 4,4-diaponeurosporenoic acid (4,4'-DNPA) from S. aureus carotenoid extracts and evaluated its effect on the thermotropic and biophysical properties of representative membrane models. The highly rigid triterpenoid 4,4'-DNPA is one of the last precursors in the biosynthesis of STX and is found in high concentrations in the stationary phase of S. aureus. Phase transition temperatures were determined using infrared spectroscopy, while interfacial hydration and hydrophobic core dynamics were investigated using fluorescence spectroscopy through Laurdan generalized polarization and DPH anisotropy. The results show that 4,4'-DNPA increases the main phase transition temperature of lipid bilayers in a concentration-dependent manner. This is in contrast to STX that decreases the transition temperature. This difference is consistent with the additional fatty acid present in STX that changes its effect on the phase behavior. Furthermore, 4,4'-DNPA reduced the interfacial hydration levels and restricted hydrophobic-core dynamics at higher concentrations, consistent with increased molecular order and stability. 4,4'-DNPA therefore complements STX in increasing membrane order and lipid packing. These findings support the notion that the production of bacterial carotenoids functions as a biophysical regulatory mechanism of lipid packing in S. aureus membranes.

Glutamate dehydrogenase (GDH) is a key mitochondrial enzyme that catalyzes the reversible oxidative deamination of glutamate to -ketoglutarate, thereby linking amino acid and carbohydrate metabolism. GDH forms catalytically active hexamers and is regulated by various allosteric modulators, including ADP and GTP. Here, we demonstrate that GDH undergoes auto-palmitoylation in the presence of palmitoyl-CoA, leading to a dose-dependent inhibition of enzymatic activity. Using acyl-PEG exchange assays and mass spectrometry, we show that GDH monomers are predominantly mono-palmitoylated, with modification detected at multiple cysteine residues, including Cys55, Cys115, and Cys197, among the six cysteines in the mature enzyme. Blue Native PAGE analysis revealed that palmitoylation disrupts the native hexameric assembly of mammalian GDH, which is organized as a dimer-of-trimers, promoting dissociation into dimers. Importantly, this modification is reversible, as incubation with mitochondrial acyl-protein thioesterases 1 (APT1) and, to a lesser extent, /{beta} hydrolase domain 10 (ABHD10) restores both the hexameric structure and enzymatic activity. The modified Cys55 residues are positioned near the trimer interface, providing a mechanism by which palmitoylation could prevent hexamer formation, whereas Cys115 and 197 may destabilize individual trimers. These findings establish S-palmitoylation as a novel regulatory mechanism for GDH, linking mitochondrial lipid metabolism to the reversible control of a central metabolic enzyme.

Inositols are a family of cyclic sugar alcohols comprising nine stereoisomers. Myo-inositol is the most abundant isomer found in humans and has been studied most extensively. It plays an important role in osmoregulation and is incorporated into membrane-anchored phosphatidylinositols. Scyllo-inositol is the second most abundant inositol isomer in the human brain and aberrant concentrations are associated with various diseases; however, its biological functions remain poorly understood. Here, the development and application of [13C6]scyllo-inositol as an isotopic tracer to study its metabolism is reported. A concise and robust synthetic route was established to obtain [13C6]scyllo-inositol from [13C6]myo-inositol in good yield. The uptake of [13C6]scyllo-inositol and responses of endogenous inositol isomers were measured in multiple cell lines by HILIC-MS/MS, showcasing the advantages of isotopic tracing. [13C6]scyllo-inositol proved to be a versatile isotopic tracer, when coupled with MS-based lipidomics and 2D NMR experiments. These experiments provide evidence that scyllo-inositol is incorporated into phosphatidylinositols in different cell lines. The results suggest a previously underappreciated role of scyllo-inositol in mammalian cells. The utilization of [13C6]scyllo-inositol will help to elucidate the role of scyllo-inositol metabolism in healthy and diseased states. SignificanceScyllo-inositol is a cyclic sugar alcohol found predominantly in the human brain. Changes in its concentration are associated with different diseases, and scyllo-inositol has been investigated as a potential drug against Alzheimers disease in clinical trials. However, its metabolic fate in mammalian cells is not well understood. We report here a synthetic strategy to obtain [13C6]scyllo-inositol and demonstrate, through isotopic tracing, its incorporation into phosphatidylinositols in different human-derived cell lines. This new stable isotopic tracer enables the investigation of the biological role of scyllo-inositol in mammals and beyond. HighlightsO_LIConcise synthesis of [13C6]scyllo-inositol C_LIO_LI[13C6]scyllo-inositol uptake and response of endogenous inositol isomers studied in multiple cell lines C_LIO_LIUse of [13C6]scyllo-inositol as an isotopic tracer in metabolomics and lipidomics experiments C_LIO_LIEvidence for scyllo-inositol incorporation into phosphatidylinositol in mammalian cells C_LI

Our current understanding of carbohydrate-binding module (CBM) function is limited by the fact that most CBM research has focused on single-binding-site modules. CBM family 92 (CBM92) is a recently characterized family of predominantly trivalent proteins that bind {beta}-1,3- and {beta}-1,6-glucans with high specificity. CpCBM92A from Chitinophaga pinensis stands out as the first trivalent member of the family to be structurally determined. Multivalent CBM families are rare, and the way in which the three binding sites cooperate in ligand recognition remains unclear. Here, we use NMR spectroscopy to demonstrate how each of the proteins binding sites plays distinct roles in ligand binding. One binding site, referred to as the {beta} site, can be identified as the primary attachment point because of its higher affinity for all tested ligands, consistent with previous biochemical data suggesting it is the strongest binding site on CpCBM92A. The other two binding sites, referred to as and {gamma}, preferentially bind longer segments of {beta}-1,3- and {beta}-1,6-glucan chains, respectively. We further show that the glycosidic bond position and anomeric configuration of the binding glucosyl unit strongly affects protein affinity due to a preferred ligand pose in the binding sites. Our results provide insight into how the trivalent architecture of CBM92 might enable cross-linking of scleroglucan chains, which may guide the development of new applications for CBMs in biotechnology.