JACS Au

The aggregation of -synuclein (Syn) is a molecular hallmark of Parkinson's disease (PD) and other synucleinopathies. Understanding the molecular mechanisms that determine the aggregation of this protein may thus facilitate the development of disease-modifying therapies. While Syn is most commonly expressed as a 140-residue protein (Syn-140), recent evidence suggests an involvement of alternatively spliced Syn isoforms in disease onset and progression. Here, we report and characterise the interaction between Syn-140 and the aggregation-prone Syn-112 variant, one of the most abundant Syn splice isoforms. We found that amounts as low as 1% of Syn-112 accelerate the nucleation and aggregation of Syn-140. To further investigate this phenomenon, we employed MALDI-MS and NMR spectroscopy, confirming that Syn-140 and Syn-112 monomers interact strongly with one another. Furthermore, to assess the association of Syn-112 with disease pathology, we performed immunohistochemical staining combined with confocal microscopy on PD brain samples. Thereby, we found an increase in the number as well as the area of Syn-112 immunoreactive aggregates compared to healthy controls. These results illustrate how low-abundance Syn splice isoforms can modulate the aggregation landscape of Syn-140 and in turn contribute to the molecular heterogeneity of synucleinopathies.

The inhibition of A{beta}42 ({beta}-amyloid) fibril formation is a key therapeutic strategy in Alzheimer's disease research. Paddlewheel diruthenium complexes have shown promising activity against A{beta}42 aggregation and preformed fibril disaggregation, yet their molecular mode of action remains poorly understood. In this work, we perform atomistic simulations to explore how charge modulation influences the interactions of three analogous paddlewheel diruthenium complexes, the parent neutral complex [Ru2Cl(D-p-FPhF)(O2CCH3)3], and its anionic [Ru2Cl2(D-p-FPhF)(O2CCH3)3]- and cationic [Ru2(D-p-FPhF)(O2CCH3)3]+ counterparts (D-p-FPhF- is the N,N' -bis(4-fluorophenyl)formamidinato ligand) with A{beta}42. Our results indicate that electrostatic tuning governs binding affinity and the extent of interaction across the A{beta}42 fibril surface. As the complexes' charge changes from -1 to +1, the interaction pattern shifts from localized contacts to widespread, multi-site engagement encompassing key charged, aromatic, and hydrophobic regions of A{beta}42. This enhanced binding correlates with longer-lived, thermodynamically stable interactions at the fibril interface, which effectively lower the free energy penalty for fibril disassembly. Overall, our findings propose a mechanism in which charge-dependent activation through ligand exchange enhances fibril recognition and promotes disruptive binding modes, demonstrating the potential of charge-tunable diruthenium complexes as therapeutic modulators of A{beta}42 fibril stability.

-Synuclein (S), a protein that plays a central role in Parkinsons disease and related synucleinopathies, is an intrinsically disordered protein (IDP) whose functional interactions and aggregation behavior can be strongly influenced by post-translational modifications (PTMs). Phosphorylation, acetylation, and other PTMs regulate Ss interactions with lipid membranes and binding partners, whereas their dysregulation is associated with aggregation and neuronal toxicity. Despite significant progress through chemical and semi-synthetic approaches, investigating the combinatorial effects of PTMs has remained challenging due to the lack of accessible, site-specific methods. Here, we present an integrated strategy combining genetic code expansion, enzymatic modification, and intein-mediated click chemistry to generate S variants bearing multiple defined PTMs and a C-terminal fluorescent label. The resulting constructs enable direct evaluation of how individual and combined PTMs influence S structure, lipid binding, and cellular internalization. Our approach expands the molecular toolkit for dissecting PTM crosstalk in S and other aggregation-prone IDPs, advancing mechanistic understanding and supporting the development of therapeutic strategies for neurodegenerative disease.

Chemical exchange saturation transfer (CEST) has become an indispensable NMR method to characterize slow exchange affecting biomacromolecules, especially for cases involving exchange between a major state and a minor state, the latter of which is often invisible in the spectrum. The CEST method is based on successive irradiation of selective regions of the NMR spectrum using a weak radiofrequency field, B1, while observing the effect on the visible major state when the B1 field saturates the invisible minor state. The need for selective saturation of narrow spectral regions has to date required acquisition of many tens of two-dimensional CEST spectra to sample the entire spectrum with sufficient resolution. Here we present the ACCEST method which measures an entire CEST profile from a single two-dimensional accordion-CEST spectrum plus a reference spectrum. ACCEST is based on the concept of accordion spectroscopy, where in the present implementation the carrier frequency of the weak saturating B1 field is stepped in synchrony with the dwell-time incrementation in the indirect dimension of the two-dimensional spectrum. We benchmarked ACCEST against conventional CEST, resulting in excellent agreement for both backbone 15N and methyl 13C CEST profiles. ACCEST offers substantial time savings that scale linearly with the number of spectra required in the corresponding conventional CEST experiment. Thus, ACCEST can dramatically speed up lengthy serial experiments, such as ligand titrations or temperature-dependent studies, and enable studies of non-equilibrium systems or samples with limited lifetimes.

Nuclear organization is dynamic and originates from the fundamental subunit of chromatin, the nucleosome. Post-translational modification of nucleosomal histones, particularly within intrinsically disordered histone tail regions, provides a dynamic regulatory mechanism of accessibility for chromatin-templated processes. While the epigenomic impacts of lysine acetylation and serine phosphorylation in the histone H3 tail are well-known, how these charge-altering post-translational modifications (PTMs) alter nucleosomal tail conformational dynamics remains incompletely characterized. Given that the functional implications of these PTMs are, at least in part, a consequence of modified nucleosome conformation, systematically cataloging the impact of histone PTMs on nucleosome dynamics provides crucial insight into both baseline cellular activity and epigenetic dysregulation that occurs in disease. Previously, our lab demonstrated that arginine citrullination mimetics lead to regional increases in H3 tail dynamics within nucleosome core particles. Here, we performed nuclear magnetic resonance spin relaxation experiments to investigate the effects of lysine acetylation and serine phosphorylation on H3 tail picosecond-nanosecond (ps-ns) dynamics. Using lysine-to-glutamine and serine-to-glutamate mutations as acetyllysine and phosphoserine mimetics, respectively, we found that these PTMs increase ps-ns conformational dynamics regionally around the PTM site, with a position-dependent effect. Additionally, we show that the type of PTM influences the extent of these increases: in general, the effect of mimetics trends in the order of phosphorylation [&le;] acetylation < citrullination, suggesting a tunable method for altering histone tail dynamics. Taken together, these results illustrate the role of nucleosome conformational dynamics in conveying the effects of epigenomic PTMs, elucidating a mechanism of the histone language.

The cellular uptake and propagation of tau are central features of tauopathies, including Alzheimers disease, and are mediated by the endocytic receptor low-density lipoprotein receptor-related protein 1 (LRP1). While prior studies have implicated LRP1 in tau binding and internalization, the biochemical features of this interaction and its suitability for therapeutic targeting remain incompletely defined. Here, we establish a quantitative and scalable framework to interrogate the tau-LRP1 interaction and identify small-molecule modulators. We engineered and purified the LRP1 ligand-binding domain 4 (BD4), a key region mediating tau interaction, and developed multiple orthogonal assays, including fluorescence polarization, split luciferase complementation, and time-resolved FRET, to measure LRP1-BD4 interactions with tau and a known peptide ligand. Across assay formats, we observe consistent binding affinities in the nanomolar range and demonstrate competitive displacement by tau, receptor-associated protein (RAP), and a peptide ligand, supporting overlapping binding interfaces. Leveraging these platforms, we performed small molecule high-throughput screening and identified a set of candidate inhibitors of the LRP1-BD4-tau interaction. Selected compounds reduced tau uptake in a cellular assay, phenocopying competitive inhibition by tau and a peptide ligand. Together, these studies define the LRP1-BD4-tau interaction as a biochemically tractable and druggable interface and establish an integrated discovery pipeline linking mechanistic characterization to functional cellular outcomes. This work provides a foundation for the development of therapeutic strategies targeting LRP1-mediated tau uptake.

Serotonin is a key neurotransmitter, and aptamer-based tools using the 44 nt Apt44 have been successfully developed for its in vitro and in vivo detection. Nevertheless, the structural basis of recognition by this aptamer remains unclear. Here we report high resolution NMR structures of Apt38, a 6-nt truncated variant in the third loop of Apt44, in free and serotonin-bound states. Both structures reveal a two layered antiparallel chair type G quadruplex core with three edgewise loops and a terminal duplex, forming a G quadruplex duplex hybrid structure. Serotonin binds at the G quadruplex duplex junction, stabilized by stacking, electrostatic attraction, hydrogen bonding, and hydrophobic contacts. Apt38 is preorganized for binding, whereas the longer third loop of Apt44 introduces conformational dynamics into the G quadruplex scaffold, which enables a pronounced binding triggered conformational switch in PBS buffer, explaining its sensing mechanism. Our work reveals the recognition and sensing mechanism of the serotonin aptamer and provides a framework for aptamer design in serotonin biosensing.

Recent advances in the discovery, characterisation, and engineering of urethanases provide new opportunities for the sustainable biocatalytic degradation of polyurethane waste. A mechanistic understanding of enzyme-plastic interactions is essential for structure-based engineering to enhance urethanase activity. However, the extremely complex and hydrophobic nature of polyurethane makes it challenging to elucidate the structural basis of enzyme-plastic interactions. Here, we used a fragment-based approach to characterise the active sites of two novel urethanases with different catalytic scaffolds, employing both a crystallographic fragment-screening (FASE) campaign and soluble fragments of plastic-like analogues that mimic the substrate, transition state, or product. FASE identified new substrate-binding subpockets while interactions of plastic mimetics in the active site provided a mechanistic understanding of the recognition and binding of polyurethane fragments by these subpockets. These results highlight a diversity of binding modes among urethanases toward different polyurethane fragments.

Precisely modifying proteins at multiple sites in their native, folded structures offers unique opportunities to answer molecular and cellular-level biological questions. Here, we present a genetic code expansion strategy for site-specific integration of a fluorophore-quencher pair comprising two non-canonical amino acids--acridonylalanine (Acd) and methyltetrazinyl phenylalanine (Tet) -- into a protein expressed in E. coli. The Acd and Tet pair requires no post-translational labeling, and quenching can be switched off by biorthogonal or photochemical reactions of Tet for convenient internal control experiments. Mechanistic studies based on Stern-Volmer quenching, fluorescence lifetime measurements, and "proline ruler" peptides established the distance dependence of quenching. As proof-of-concept, we applied this strategy to study: 1) calmodulin, a calcium-sensing protein, 2) RecA, a DNA damage sensor in bacteria, and 3) LexA, a transcriptional repressor whose activation by RecA governs acquired antibiotic resistance in bacteria. Using these proteins, we demonstrate that dual Acd/Tet labeling provides molecular-level insights into protein dynamics, enables high-throughput drug screening, and advances tools for studying protein structure-function relationships.

Thermodynamic characterization of biomolecular interactions is essential for understanding the enthalpic and entropic driving forces of molecular recognition, but established label-free techniques are limited either by bulk refractive-index sensitivity or by the lengthy thermal equilibration required to suppress it. Here, we used focal molography to investigate the temperature-dependent binding of the protein kinase A regulatory subunit (PKA-R) to cyclic AMP (cAMP) derivatives and to derive apparent thermodynamic signatures from kinetic measurements. We first validated the diffractometric readout under conditions that challenge refractometric sensors: the coherent mass density channel strongly suppressed temperature-induced bulk refractive-index effects and resolved binding in 50% human serum despite measurable non-specific adsorption, reducing the need for lengthy equilibration and buffer matching. We then combined focal molography with DNA-directed immobilization (DDI), allowing five cAMP derivatives to be presented in parallel on the same multiplexed chip and followed across five temperatures. This format yielded distinct, internally consistent apparent thermodynamic fingerprints for each derivative, separating ligands with similar affinities by their enthalpic and entropic contributions. Together, these results establish focal molography with DDI as a multiplexed workflow for comparative thermodynamic fingerprinting of biomolecular interactions at higher throughput.

Cryptic binding sites generated by local conformational dynamics have become an important concept in protein-targeted ligand discovery, yet their energetic accessibility and relevance to RNA recognition remain less well understood. Here, we use the env8 cobalamin (Cbl) riboswitch as a model system to investigate the energetic consequences of cryptic-site formation through base displacement. Structural analysis revealed that binding of {beta}-axial substituted Cbl derivatives displaces a conserved adenosine (A20) from the RNA core, exposing a previously hidden binding site that is subsequently occupied by the {beta}-axial substituent. Using selective abasic substitution at this position, we quantified the energetic contributions associated with A20 in the native RNA core and with base displacement. Isothermal titration calorimetry and fluorescence measurements revealed that cryptic-site formation incurs a modest energetic penalty of ~1.4 kcal mol-1. Guided by this experimentally derived framework, computational conformational sampling recapitulated cryptic-site formation in the Cbl riboswitch and identified analogous cryptic sites in structurally unrelated RNAs from HIV-1 and HCV. These cryptic-site conformers were identified within low-energy conformational windows and exposed ligand-accessible surfaces through local base displacement. Finally, a ligand previously identified to target the env8 cryptic site bound both RNAs and yielded docking poses consistent with engagement of the newly exposed binding surfaces. Together, these results indicate that cryptic RNA binding sites can be both energetically accessible and chemically addressable, expanding the range of conformational states that may contribute to RNA ligandability.

I.Rapid developments in magic-angle-spinning (MAS) hardware over the past two decades have made possible the acquisition of high-resolution spectra of protons in solids, fuelling studies of small and large molecules alike. Nevertheless, proton resolution, limited by the strong dipole-dipole coupling network, remains a bottleneck even at MAS frequencies exceeding 100 kHz. We present here techniques based on phase-modulated homonuclear decoupling that dramatically improve proton coherence times and resolution compared to 60-95 kHz MAS alone using low average radio-frequency amplitudes (< 100 kHz). A relatively high sensitivity (40- 70%) and a straightforward optimization procedure directly on the sample being studied allows these gains to be realised in large biomolecules, as demonstrated here on a 326-residue cytoskeletal protein in its filamentous state. These techniques enable experiments with improved resolution on biomolecules while simultaneously taking advantage of the higher sensitivity available on probes with relatively large rotor volumes that cannot reach higher MAS frequencies.

Non-canonical amino acids (ncAAs) are valuable tools in chemical biology and biochemistry for labeling, probing, and tracking biomolecules. ncAAs that can be recombinantly incorporated using native E. coli machinery are particularly useful because they allow for global protein incorporation and avoid complex genetic code expansion. Here, we demonstrate successful incorporation of a methionine analog, L-cyanohomoalanine (Cha), by the methionyl-tRNA synthetase of E. coli into mutant superfolder GFP (sfGFP) expressed in methionine auxotroph bacterial cultures. We compare to methionine auxotroph bacterial cultures supplemented with L-methionine (Met) or L-azidohomoalanine (Aha). In control prototrophic E. coli, bacterial growth rates are inhibited with high concentrations of Aha but not Cha. However, less sfGFP is produced in auxotrophic cells supplemented with Cha compared to Aha and Met. Thus, while Cha is non-toxic to E. coli it is incorporated less efficiently into proteins than Aha or Met. Mass spectrometry confirmed that N-terminal Cha, Aha, and Met are cleaved, as expected for the sfGFP mutants. Other sites of Cha and Aha incorporation were confirmed by mass spectrometry, with labeling efficiency varying by position. Thermal melts of purified sfGFPs demonstrate that Cha and Aha labeling does not significantly perturb the protein stability. In the future, Cha may be useful for proteome labeling by wild-type methionyl-tRNA synthetase and could be implemented in metabolic pulse-labeling of newly synthesized proteins with other methionine analogs. Additionally, the nitrile moiety of Cha may be used to perform reactions orthogonal to azide/alkyne click chemistry or could serve as a vibrational reporter of the environment.

Biomolecular condensates are increasingly implicated in protein aggregation, yet their contribution is often reduced to that of concentrating reactants. Whether the phase state of a protein itself changes how it aggregates remains unclear. Using complex coacervates of a tau repeat-domain construct (K12) with heparin, we show that phase separation does not simply accelerate tau aggregation but redirects it along a distinct kinetic regime. Amyloid nucleation and growth are largely associated with the condensed phase, where intact phase-separated mixtures nucleate with a markedly shortened lag phase, whereas the corresponding dilute phase contributes little to overall amyloid formation. Aggregation kinetics in this regime become largely decoupled from total protein concentration. Because phase equilibrium pins the composition of the dense phase, additional tau partitions largely into the coexisting dilute phase without substantially altering the reacting population. This weak concentration dependence provides a kinetic signature of compartmentalized aggregation, and it recurs across chemically distinct coacervates formed with heparin, RNA, and polyglutamate, pointing to a general feature of coacervate-mediated tau assembly rather than a heparin-specific effect. Aggregation within coacervates also yields fibrils with altered morphology and secondary structure, suggesting access to alternative regions of the assembly landscape, and shows reduced sensitivity to bulk pH perturbations. Together, these results show that condensation changes tau aggregation by defining the local reaction environment: phase equilibrium buffers the dense-phase composition, in turn altering aggregation kinetics and the properties of the amyloid formed.

Fluorescent dyes are critical to visualizing nucleic acids in many applications. SYTOX Orange and SYTOX Green are cyanine dyes, used in dead cell staining and increasingly in single-molecule assays to probe DNA supercoiling and processing. However, their structures and effects on DNA mechanics are not or only partially known. We determine the structure of SYTOX Orange to be (E)-2-((2-(4 ((diethyl(methyl)ammonio)methyl)phenyl)-6-methoxy-1-methylquinolin-4(1H)-ylidene)methyl)-4-methyloxazolo[4,5-b]pyridin-4-ium, identical to SYBR Gold except for an aza-benzoxazol core that is fundamentally different from other dyes in the SYTOX and SYBR families. We report SYTOX Green to be (Z)-2-(bis(3-(trimethylammonio)propyl)amino)-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-1-phenylquinolin-1-ium, similar to PicoGreen. Using magnetic tweezers, we characterize the effect of SYTOX Orange and SYTOX Green on DNA mechanics. They lengthen and unwind DNA consistent with intercalation and the DNA unwinding angles per dye are 21.1(1) degree and 20.5(1) degree for SYTOX Orange and Green, respectively. Both dyes leave the DNA bending persistence length and plectoneme size almost unaltered (<10% change up to 1 uM), which is advantageous in assays probing DNA supercoiling. Their photophysical properties reveal close agreement between single-molecule manipulation and optical absorbance and fluorescence spectroscopy. Our comprehensive set of complementary measurements relates mechanical and optical properties to the molecular structures and provides recommendations for their use in applications.

Biomolecular condensates exhibit spontaneous electrochemical microenvironments characterized by asymmetric ion distributions and pH gradients that emerge from protein-sequence-dependent charge regulation. Despite their biological importance, mechanistic understanding of these microenvironments has been constrained by the absence of computationally tractable frameworks capable of treating proton exchange, counterion partitioning, and buffer equilibria on consistent thermodynamic footing. Here, we introduce the buffered Charge-Regulation Monte Carlo (b-CR-MC) framework, which couples grand-canonical exchange of ions and buffer species with explicit charge regulation of titratable residues. By extending the CR-MC ion-merging strategy to multicomponent reservoirs and employing the Restricted Primitive Model, b-CR-MC achieves computational efficiency while maintaining thermodynamic rigor, with quantitative agreement to the more expensive generalized G-RxMC approach. Applied to full-length FUS (net positive) and PGL-3 (net negative) under physiological conditions, the framework reveals sequence-dependent pH gradients: the dense phase of FUS exhibits an alkaline shift, while PGL-3 exhibits an acidic shift, in both cases driving the condensate interior toward the protein's isoelectric point. Slab-geometry simulations further resolve the Donnan potential and continuous ion profiles across the condensate interface, confirming the direction and magnitude of these electrochemical shifts. Additionally, we identify spatially resolved buffer depletion within dense phases, establishing that dynamic charge regulation is a primary determinant rather than a secondary correction to condensate electrochemistry. By establishing a sequence-resolved, thermodynamically consistent computational platform, b-CR-MC enables quantitative prediction of how mutations and post-translational modifications reprogram condensate microenvironments across biological and pathophysiological contexts.

Proximity labeling has transformed spatial proteomics by enabling compartment-resolved mapping of protein environments in living cells, yet its extension to small-molecule metabolites has not been demonstrated, probably due to limitations in labeling chemistry and identification of labeled metabolites. Here, we introduce DESTNI, an engineered desthiobiotin (DTB) ligase derived from TurboID through directed evolution, and establish a platform for spatially resolved profiling of amine-containing metabolites. A directed evolution strategy based on a yeast display system yielded DESTNI with an efficient DTB-dependent reactivity, enabling robust and compartment-specific proximity labeling across diverse subcellular environments. To identify the DTB-modified amino metabolome, we developed an integrated analytical framework combining DTB-modified amino metabolite standards, in vitro DESTNI profiling, and in silico MS/MS prediction, enabling systematic annotation of DTB-modified amino metabolites. To extend this chemistry to metabolites, we combined synthetic DTB-conjugated metabolite reference standards, in vitro DESTNI-reactive metabolite discovery, and machine-learning prediction of DTB-derivatized metabolites and oligopeptides. Organelle-targeted DESTNI recovered reproducible compartment-enriched amino metabolite signatures, including mitochondrial matrix-enriched glycine, 5-aminolevulinic acid, ornithine and spermidine adducts, as well as nuclear-enriched {gamma}-aminobutyric acid and 5-aminovaleric acid adducts. Together, this work establishes DESTNI as a proximity labeling platform that bridges spatial proteomics and metabolomics and provides a general strategy for mapping subcellular biochemical environments in living cells.

Enzyme adaptation toward novel substrates involves the rewiring of intramolecular residue networks, yet how this rewiring differs across multiple substrates, and how it underpins functional trade-offs and promiscuity, remains poorly understood. Here, we profile all 64 combinations of six key mutations in a phosphotriesterase across nine structurally diverse substrates spanning three chemical classes (organophosphates, esters, and lactones), thus generating a multi-dimensional map of epistasis and promiscuity within the phosphotriesterase's active site. We developed a statistically robust reference-based analysis pipeline incorporating error propagation and significance testing to move beyond global epistatic trends and resolve idiosyncratic, substrate-dependent intramolecular wiring in specific genetic backgrounds. Simulations confirm that this pipeline reliably identifies genuine higher-order epistatic interactions while minimizing false positives. We reveal that intramolecular network wiring varies substantially between substrates, even within the same chemical class, with notable divergences between the adaptive target substrate 2-naphthyl hexanoate and its shorter-chain ester analogs. Key higher-order networks, including d233E/h254R/l271F and l271F/f306I/i313F, exhibit substrate-specific epistatic signatures that discriminate between subtle structural features such as acyl chain length, leaving group identity, and heteroatom substitution. These substrate-dependent rewiring events account for observed functional trade-offs, particularly the strong anti-correlation between the adaptive and native substrates. Collectively, these findings demonstrate that comprehensive cross-substrate epistatic profiling, paired with rigorous statistical analysis, provides a powerful framework for dissecting the molecular basis of enzyme promiscuity and the trade-offs that define adaptive evolution.

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of proteins in aqueous solution is often limited by water absorption and other optical artifacts. To overcome these limitations, we evaluated the structural features and hydrogen-deuterium exchange (HDX) kinetics of the -helical protein GCN4 in both hydrated (wet) and vacuum-dried (dry) states. While solvent heavily mask the second-derivative spectra of wet samples, vacuum drying yielded a thin, protein-rich film on the ATR crystal, significantly enhancing the signal-to-noise ratio and resolving the protein features without altering the native structure. Dry-state analysis clearly resolved the Amide I, Amide II, and deuterium-shifted Amide II' (1450 cm-1) bands. Notably, second-derivative analysis of the dry spectra of the HDX samples revealed a bimodal Amide I distribution consisting of a stationary band at 1653 cm-1 from the solvent-inaccessible regions and an isotopically sensitive band shifting from 1648 cm-1 to 1644 cm-1 from solvent-accessible regions. These results demonstrate that vacuum-dried ATR-FTIR spectroscopy effectively eliminates solvent masking, providing the spectral clarity required to resolve discrete -helical sub-populations after deuteration.

The cryptococcal polysaccharide capsule is a unique eukaryotic virulence factor that is a target for the immune system and the development of therapeutic antibodies. Our understanding of capsular architecture is limited to a few studies suggesting that metal dications play a role. In this work we explore a mechanism of cryptococcal aggregation that depends on calcium phosphate precipitation. We describe the chemical and biophysical properties of calcium interaction with the predominant cryptococcal polysaccharide, glucuronoxylomannan (GXM). We show that cell aggregation is a pH-dependent and occurs in a calcium dose-dependent manner. Furthermore, this cellular aggregation phenomenon as well as interpolymer capsular polysaccharide interactions are unique to calcium dications and do not occur with other mono- or dications as shown by size exclusion chromatography and circular dichroism. Diffusion ordered spectroscopy nuclear magnetic resonance and ab-initio calculations support complexation of calcium with glucuronic acid (GlcA). The ab-initio calculations also suggest that calcium ions can complex up to four GlcA monomers. Not only does calcium act as a scaffold for the cryptococcal capsule, interacting with up to four glucuronic acid residues of GXM, but calcium phosphate treatment of cells reduces the anti-phagocytic properties of the capsule, promoting ingestion by macrophages and altering antibody interactions with the capsule. This work advances our understanding of the cryptococcal capsule, its biophysical properties, by providing a model for the critical role of calcium interactions with capsular polymers of Cryptococcus neoformans including important impacts at the host-cell interface.