Oligomerization Dynamics Research Articles

Advanced multiparametric image spectroscopy and super-resolution microscopy reveal a minimal model of CD95 signal initiation.

Unraveling the concentration-dependent spatiotemporal organization of receptors in the plasma membrane is crucial to understand cell signal initiation. A paradigm of this process is the oligomerization of CD95 during apoptosis signaling, with different oligomerization models being discussed. Here, we establish the molecular-sensitive approach cell lifetime Förster resonance energy transfer image spectroscopy to determine CD95 configurations in live cells. These data are corroborated by stimulated emission depletion microscopy, confocal photobleaching step analysis, and fluorescence correlation spectroscopy. We probed CD95 interactions for concentrations of ~10 to 1000 molecules per square micrometer, over nanoseconds to hours, and molecular to cellular scales. Quantitative benchmarking was achieved establishing high-fidelity monomer and dimer controls. While CD95 alone is primarily monomeric (~96%) and dimeric (4%), the addition of ligand induces oligomerization to dimers/trimers (~15%) leading to cell death. This study highlights molecular concentration effects and oligomerization dynamics. It reveals a minimal model, where small CD95 oligomers suffice to efficiently initiate signaling.

Carbon dots fluorescence can be used to distinguish isobaric peptide and to monitor protein oligomerization dynamics

Carbon dots (CD) are widely investigated particles with interesting fluorescent properties which are reported to be used for various purposes, as they are biocompatible, resistant to photobleaching and with tuneable properties depending on the specific CD surface chemistry. In this work, we report on the possibility to use opportunely designed CD to distinguish among isobaric peptides almost undistinguishable by mass spectrometry, as well as to monitor protein aggregation phenomena. Particularly, cell-penetrating peptides containing the carnosine moiety at different positions in the peptide chain produce sequence specific fluorescent signals. Analogously, different insulin oligomerization states can also be distinguished by the newly proposed experimental approach. The latter is here described in details and can be potentially applied to any kind of peptide or protein.

Dissecting the effect of ALS mutation S375G on the conformational properties and aggregation dynamics of TDP-43370-375 fragment

The aggregation of transactive response deoxyribonucleic acid (DNA) binding protein of 43 kDa (TDP-43) into ubiquitin-positive inclusions is closely associated with amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration, and chronic traumatic encephalopathy. The 370–375 fragment of TDP-43 (370GNNSYS375, TDP-43370-375), the amyloidogenic hexapeptides, can be prone to forming pathogenic amyloid fibrils with the characteristic of steric zippers. Previous experiments reported the ALS-associated mutation, serine 375 substituted by glycine (S375G) is linked to early onset disease and protein aggregation of TDP-43. Based on this, it is necessary to explore the underlying molecular mechanisms. By utilizing all-atom molecular dynamics (MD) simulations of 102 μs in total, we investigated the impact of S375G mutation on the conformational ensembles and oligomerization dynamics of TDP-43370-375 peptides. Our replica exchange MD simulations show that S375G mutation could promote the unstructured conformation formation and induce peptides to form a loose packed oligomer, thus inhibiting the aggregation of TDP-43370-375. Further analyses suggest that S375G mutation displays a reduction effect on the number of total hydrogen bonds and contacts among TDP-43370-375 peptides. Hydrogen bonding and polar interactions among TDP-43370-375 peptides, as well as Y374-Y374 π-π stacking interaction, are attenuated by S375G mutation. Additional microsecond MD simulations demonstrate that S375G mutation could prohibit the conformational conversion to β-structure-rich aggregates and possess an inhibitory effect on the oligomerization dynamics of TDP-43370-375. This study offers for the first time of molecular insights into the S375G mutation affecting the aggregation of TDP-43370-375 at the atomic level, and may open new avenues in the development of future site-specific mutation therapeutics.

Exploring the significance of potassium homeostasis in copper ion binding to human αB-Crystallin

αB-Crystallin (αB-Cry) is a small heat shock protein known for its protective role, with an adaptable structure that responds to environmental changes through oligomeric dynamics. Cu(II) ions are crucial for cellular processes but excessive amounts are linked to diseases like cataracts and neurodegeneration. This study investigated how optimal and detrimental Cu(II) concentrations affect αB-Cry oligomers and their chaperone activity, within the potassium-regulated ionic-strength environment. Techniques including isothermal titration calorimetry, differential scanning calorimetry, fluorescence spectroscopy, inductively coupled plasma atomic emission spectroscopy, cyclic voltammetry, dynamic light scattering, circular dichroism, and MTT assay were employed and complemented by computational methods. Results showed that potassium ions affected αB-Cry's structure, promoting Cu(II) binding at multiple sites and scavenging ability, and inhibiting ion redox reactions. Low concentrations of Cu(II), through modifications of oligomeric interfaces, induce regulation of surface charge and hydrophobicity, resulting in an increase in chaperone activity. Subunit dynamics were regulated, maintaining stable interfaces, thereby inhibiting further aggregation and allowing the functional reversion to oligomers after stress. High Cu(II) disrupted charge/hydrophobicity balance, sewing sizable oligomers together through subunit-subunit interactions, suppressing oligomer dissociation, and reducing chaperone efficiency. This study offers insights into how Cu(II) and potassium ions influence αB-Cry, advancing our understanding of Cu(II)-related diseases.

Osmolyte influence on stability and oligomerization dynamics of the Saccharomyces cerevisiae PCNA sliding clamp

Osmolyte influence on stability and oligomerization dynamics of the Saccharomyces cerevisiae PCNA sliding clamp

Overoxidation and Oligomerization of Trypanosoma cruzi Cytosolic and Mitochondrial Peroxiredoxins.

Peroxiredoxins (Prxs) have been shown to be important enzymes for trypanosomatids, counteracting oxidative stress and promoting cell infection and intracellular survival. In this work, we investigate the in vitro sensitivity to overoxidation and the overoxidation dynamics of Trypanosoma cruzi Prxs in parasites in culture and in the infection context. We showed that recombinant m-TXNPx, in contrast to what was observed for c-TXNPx, exists as low molecular mass forms in the overoxidized state. We observed that T. cruzi Prxs were overoxidized in epimastigotes treated with oxidants, and a significant proportion of the overoxidized forms were still present at least 24 h after treatment suggesting that these forms are not actively reversed. In in vitro infection experiments, we observed that Prxs are overoxidized in amastigotes residing in infected macrophages, demonstrating that inactivation of at least part of the Prxs by overoxidation occurs in a physiological context. We have shown that m-TXNPx has a redox-state-dependent chaperone activity. This function may be related to the increased thermotolerance observed in m-TXNPx-overexpressing parasites. This study suggests that despite the similarity between protozoan and mammalian Prxs, T. cruzi Prxs have different oligomerization dynamics and sensitivities to overoxidation, which may have implications for their function in the parasite life cycle and infection process.

Deciphering the Inhibitory Mechanism of Naphthoquinone-Dopamine on the Aggregation of Tau Core Fragments PHF6* and PHF6.

The formation of neurofibrillary tangles by abnormal aggregation of tau protein is considered to be an important pathological characteristic of tauopathies, including Alzheimer's disease and chronic traumatic encephalopathy. Two hexapeptides 275VQIINK280 and 306VQIVYK311 in the microtubule binding region, named PHF6* and PHF6, are known to be aggregation-prone and responsible for tau fibrillization. Previous experiments reported that naphthoquinone-dopamine (NQDA) could effectively inhibit the aggregation of PHF6* and PHF6 and disrupt the fibrillar aggregates into nontoxic species, displaying a dual effect on the amyloid aggregation. However, the underlying molecular mechanism remains mostly elusive. Herein, we performed all-atom molecular dynamics (MD) simulations for 114 μs in total to systematically investigate the impacts of NQDA on the oligomerization of PHF6* and PHF6. The conformational ensembles of PHF6* and PHF6 peptides generated by replica exchange MD simulations show that NQDA could effectively prevent the hydrogen bond formation, reduce the ability of peptides to self-assemble into long β-strand and large β-sheets, and induce peptides to form a loosely packed and coil-rich oligomer. The interaction analysis shows that the binding of NQDA to PHF6* is mainly through hydrophobic interactions with residue I277 and hydrogen bonding interactions with Q276; for the PHF6 peptides, NQDA displays a strong π-π stacking interaction with residue Y310, thus impeding the Y310-Y310 π-π stacking and I308-Y310 CH-π interactions. The DA group of NQDA displays a stronger cation-π interaction than the NQ group, while the NQ group exhibits a stronger π-π stacking interaction. MD simulations demonstrate that NQDA prevents the conformational conversion to β-sheet-rich aggregates and displays an inhibitory effect on the oligomerization dynamics of PHF6* and PHF6. Our results provide a complete picture of inhibitory mechanisms of NQDA on PHF6* and PHF6 oligomerization, which may pave the way for designing drug candidates for the treatment of tauopathies.

Biophysical tools to study the oligomerization dynamics of Prx1-class peroxiredoxins.

Peroxiredoxins (Prx) are ubiquitous, highly conserved peroxidases whose activity depends on catalytic cysteine residues. The Prx1-class of the peroxiredoxin family, also called typical 2-Cys Prx, organize as head-to-tail homodimers containing two active sites. The peroxidatic cysteine CP of one monomer reacts with the peroxide substrate to form sulfenic acid that reacts with the resolving cysteine (CR) of the adjacent subunit to form an intermolecular disulfide, that is reduced back by the thioredoxin/thioredoxin reductase/NADPH system. Although the minimal catalytic unit is the dimer, these Prx oligomerize into (do)decamers. In addition, these ring-shaped decamers can pile-up into high molecular weight structures. Prx not only display peroxidase activity reducing H2O2, peroxynitrous acid and lipid hydroperoxides (antioxidant enzymes), but also exhibit holdase activity protecting other proteins from unfolding (molecular chaperones). Highly relevant is their participation in redox cellular signaling that is currently under active investigation. The different activities attributed to Prx are strongly ligated to their quaternary structure. In this review, we will describe different biophysical approaches used to characterize the oligomerization dynamics of Prx that include the classical size-exclusion chromatography, analytical ultracentrifugation, calorimetry, and also fluorescence anisotropy and lifetime measurements, as well as mass photometry.

Dissecting diffusion and oligomerization dynamics of GPCRs in live cells using single-particle tracking.

Crucial insights about the signaling and trafficking mechanisms of G Protein Coupled Receptors (GPCRs) can be derived from their membrane diffusion properties. Previous studies in our lab (Shivnaraine et al., JACS 2016, Li at al., Biophys. J. 2018) have shown that the M2 muscarinic receptor (M2R) can be purified as oligomers, yet the oligomerization dynamics and the implications for signalling in live cells remain elusive. Here we used single-molecule fluorescence techniques, such as single-particle tracking (SPT) and single-molecule photobleaching (smPB), to characterize the dynamic distribution of M2R in HEK293 cells. To that end, receptors have been co-expressed at their N-terminus either with a green fluorescent protein (eGFP), or with a HaloTag for labelling with HaloTag ligand (HTL) dyes, such as JF549 HTL. For comparison, we applied the same techniques to μ-opioid receptors (MORs) in live U2OS cells. For both cases, measured diffusion maps are spatially and temporally heterogeneous, with receptors transitioning between normal and anomalous diffusion regimes. The analysis of experimental data was reinforced by simulations and by control measurements on monomeric (CD86) and dimeric (CD28) membrane proteins. Intensity traces of immobile, single receptor complexes and of monomeric/dimeric controls in the membrane of fixed cells were analyzed using an in-house smPB code based on a Bayesian algorithm (Garry et al., J. Chem. Phys. 2020). The results confirm the presence, correlated with expression levels, of higher order M2R oligomers in cells, and lay out the foundation for subsequent studies of their functional role.

Focus in numbers: Characterizing protein oligomerization dynamics at DNA double strand breaks.

During radiotherapy, ionizing radiation (IR) is used to kill cancer cells by directly inducing high levels of genotoxic DNA double-strand breaks (DSB). These breaks are quickly detected and bound by DNA damage sensor proteins that activate a cascade of events leading to the recruitment and accumulation of repair factors (e.g. 53BP1) at sites of damage in dot-like structures called foci. The balance and interplay between the plethora of proteins recruited at the break site determine the choice of the repairing pathway, which can have paramount consequences on cell fate and ultimately in the response to cancer radiotherapy. Indeed, substantial evidence indicates that enhanced DSB repair (DSBR) capacity in individual patients is a major determinant in tumour radioresistance, while both radiosensitivity and the occurrence of radiotherapy-induced secondary cancers, are presumably associated with reduced DSBR function. Therefore, there is a pressing need to better understand the fundamental mechanisms of DSBR, towards improving the clinical management of patients undergoing radiotherapy. While it is clear that DNA repair relies on the balanced contribution of various repair factors, the oligomerization dynamics governing their organization into foci remain largely unknown. Here we combine Spatial Intensity Distribution Analysis (SpIDA) with an automated high-content foci detection algorithm to quantify the number of 53BP1 molecules at DSBR foci in cells challenged with increasing doses of the radiomimetic drug zeocin. Consistently with published data, 53BP1 foci number increases linearly at lower zeocin doses while plateauing at higher DNA damage load (zeocin concentration above 500 μg/mL). At this plateau, despite the availability of a nucleoplasmic protein pool, the number of 53BP1 molecules per focus decreases, suggesting a further not yet clarified layer of dynamic regulation potentially leverageable as therapeutical target.

A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranes.

Membrane proteins are often observed as higher-order oligomers, and in some cases in multiple stoichiometric forms, raising the question of whether dynamic oligomerization can be linked to modulation of function. To better understand this potential regulatory mechanism, there is an ongoing effort to quantify equilibrium reactions of membrane protein oligomerization directly in membranes. Single-molecule photobleaching analysis is particularly useful for this as it provides a binary readout of fluorophores attached to protein subunits at dilute conditions. However, any quantification of stoichiometry also critically requires knowing the probability that a subunit is fluorescently labeled. Since labeling uncertainty is often unavoidable, we developed an approach to estimate labeling yields using the photobleaching probability distribution of an intrinsic dimeric control. By iterative fitting of an experimental dimeric photobleaching probability distribution to an expected dimer model, we estimate the fluorophore labeling yields and find agreement with direct measurements of labeling of the purified protein by UV-VIS absorbance before reconstitution. Using this labeling prediction, similar estimation methods are applied to determine the dissociation constant of reactive CLC-ec1 dimerization constructs without prior knowledge of the fluorophore labeling yield. Finally, we estimate the operational range of subunit labeling yields that allows for discrimination of monomer and dimer populations across the reactive range of mole fraction densities. Thus, our study maps out a practical method for quantifying fluorophore labeling directly from single-molecule photobleaching data, improving the ability to quantify reactive membrane protein stoichiometry in membranes.

Atomistic Insights into the Inhibitory Mechanism of Tyrosine Phosphorylation against the Aggregation of Human Tau Fragment PHF6.

Abnormal aggregation of the microtubule-associated protein tau into intracellular fibrillary inclusions is characterized as the hallmark of tauopathies, including Alzheimer's disease and chronic traumatic encephalopathy. The hexapeptide 306VQIVYK311 (PHF6) of R3 plays an important role in the aggregation of tau. Recent experimental studies reported that phosphorylation of residue tyrosine 310 (Y310) could decrease the propensity of PHF6 to form fibrils and inhibit tau aggregation. However, the underlying inhibitory mechanism is not well understood. In this work, we systematically investigated the influences of phosphorylation on the conformational ensembles and oligomerization dynamics of PHF6 by performing extensive all-atom molecular dynamics (MD) simulations. Our replica exchange MD simulations demonstrate that Y310 phosphorylation could effectively suppress the formation of β-structure and shift PHF6 oligomers toward coil-rich aggregates. The interaction analyses show that hydrogen bonding and hydrophobic interactions among PHF6 peptides, as well as Y310-Y310 π-π stacking and I308-Y310 CH-π interactions, are weakened by phosphorylation. Additional microsecond MD simulations show that Y310 phosphorylation could inhibit the oligomerization of PHF6 by preventing the formation of large β-sheet oligomers and multi-layer β-sheet aggregates. This study provides mechanistic insights into the phosphorylation-inhibited tau aggregation, which may be helpful for the in-depth understanding of the pathogenesis of tauopathies.

Investigating Diffusion Dynamics and Interactions with Scanning Fluorescence Correlation Spectroscopy (sFCS).

Activation of immune cells and formation of immunological synapses (IS) rely critically on the reorganization of the plasma membrane. These highly orchestrated processes are driven by diffusion and oligomerization dynamics, as well as by single molecule interactions. While slow macro- and meso-scale changes in organization can be observed with conventional imaging, fast nano-scale dynamics are often missed with traditional approaches, but resolving them is, nonetheless, essential to understand the underlying biological mechanisms at play. Here, we describe the use of scanning fluorescence correlation spectroscopy (sFCS) and scanning fluorescence cross-correlation spectroscopy (sFCCS) to study reorganization and changes in molecular diffusion dynamics and interactions during IS formation and in other biological settings. We focus on the practical aspects of the measurements including calibration and alignment of the optical setup, present a comprehensive protocol to perform the measurements, and provide data analysis pipelines and strategies. Finally, we show an exemplary application of the technology to studying Lck diffusion during T-cell signaling.

Is the lipochaperone activity of sHSP a key to the stress response encoded in its primary sequence?

Several strategies have been put in place by organisms to adapt to their environment. One of these strategies is the production of stress proteins such as sHSPs, which have been widely described over the last 30years for their role as molecular chaperones. Some sHSPs have, in addition, the particularity to exert a lipochaperone role by interacting with membrane lipids to maintain an optimal membrane fluidity. However, the mechanisms involved in this sHSP-lipid interaction remain poorly understood and described rather sporadically in the literature. This review gathers the information concerning the structure and function of these proteins available in the literature in order to highlight the mechanism involved in this interaction. In addition, analysis of primary sequence data of sHSPs available in database shows that sHSPs can interact with lipids via certain amino acid residues present on some β sheets of these proteins. These residues could have a key role in the structure and/or oligomerization dynamics of sHPSs, which is certainly essential for interaction with membrane lipids and consequently for maintaining optimal cell membrane fluidity.

Real-time detection of response regulator phosphorylation dynamics in live bacteria

Bacteria utilize two-component system (TCS) signal transduction pathways to sense and adapt to changing environments. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and activates a transcriptional response. Here, we demonstrate that oligomerization-dependent depolarization of excitation light by fused mNeonGreen fluorescent protein probes enables real-time monitoring of RR dimerization dynamics in live bacteria. Using inducible promoters to independently express SHKs and RRs, we detect RR dimerization within seconds of stimulus addition in several model pathways. We go on to combine experiments with mathematical modeling to reveal that TCS phosphosignaling accelerates with SHK expression but decelerates with RR expression and SHK phosphatase activity. We further observe pulsatile activation of the SHK NarX in response to addition and depletion of the extracellular electron acceptor nitrate when the corresponding TCS is expressed from both inducible systems and the native chromosomal operon. Finally, we combine our method with polarized light microscopy to enable single-cell measurements of RR dimerization under changing stimulus conditions. Direct invivo characterization of RR oligomerization dynamics should enable insights into the regulation of bacterial physiology.

Molecular insights into the oligomerization dynamics and conformations of amyloidogenic and non-amyloidogenic amylin from discrete molecular dynamics simulations.

The amyloid aggregation of human islet amyloid polypeptide (hIAPP) is associated with pancreatic β-cell death in type 2 diabetes. The S20G substitution of hIAPP (hIAPP(S20G)), found in Japanese and Chinese people, is more amyloidogenic and cytotoxic than wild-type hIAPP. Rat amylin (rIAPP) does not have aggregation propensity or cytotoxicity. Mounting evidence suggests that soluble low-molecular-weight amyloid oligomers formed during early aggregation are more cytotoxic than mature fibrils. The self-assembly dynamics and oligomeric conformations remain unknown because the oligomers are heterogeneous and transient. The molecular mechanism of sequence-variation rendering dramatically different aggregation propensity and cytotoxicity is also elusive. Here, we investigate the oligomerization dynamics and conformations of amyloidogenic hIAPP, hIAPP(S20G), and non-amyloidogenic rIAPP using atomistic discrete molecular dynamics (DMD) simulations. Our simulation results demonstrated that all three monomeric amylin peptides mainly adopted an unstructured formation with partial dynamical helices near the N-terminus. Relatively transient β-hairpins were more abundant in hIAPP and hIAPP(S20G) than in rIAPP. The S20G-substituting mutant of hIAPP altered the turn region of the β-hairpin motif, resulting in more hydrophobic residue-pairwise contacts within the β-hairpin. Oligomerization dynamic investigation revealed that all three peptides spontaneously accumulated into helix-populated oligomers. The conformational conversion to form β-sheet-rich oligomers was only observed in hIAPP and hIAPP(S20G). The population of high-β-sheet-content oligomers was enhanced by S20G substitution. Interestingly, both hIAPP and hIAPP(S20G) could form β-barrel formations, and the β-barrel propensity of hIAPP(S20G) was three times larger than that of hIAPP. No β-sheet-rich or β-barrel formations were observed in rIAPP. Our direct observation of the correlation between β-barrel oligomer formation and cytotoxicity suggests that β-barrels might play a critically important role in the cytotoxicity of amyloidosis.

Single molecule mass photometry reveals the dynamic oligomerization of human and plant peroxiredoxins

SummaryProtein oligomerization is central to biological function and regulation, yet its experimental quantification and measurement of dynamic transitions in solution remain challenging. Here, we show that single molecule mass photometry quantifies affinity and polydispersity of heterogeneous protein complexes in solution. We demonstrate these capabilities by studying the functionally relevant oligomeric equilibria of 2-cysteine peroxiredoxins (2CPs). Comparison of the polydispersity of plant and human 2CPs as a function of concentration and redox state revealed features conserved among all 2CPs. In addition, we also find species-specific differences in oligomeric transitions, the occurrence of intermediates and the formation of high molecular weight complexes, which are associated with chaperone activity or act as a storage pool for more efficient dimers outlining the functional differentiation of human 2CPs. Our results point to a diversified functionality of oligomerization for 2CPs and illustrate the power of mass photometry for characterizing heterogeneous oligomeric protein distributions in near native conditions.

Physical tuning of galectin-3 signaling

Galectin-3 (Gal3) exhibits dynamic oligomerization and promiscuous binding, which can lead to concomitant activation of synergistic, antagonistic, or noncooperative signaling pathways that alter cell behavior. Conferring signaling pathway selectivity through mutations in the Gal3-glycan binding interface is challenged by the abundance of common carbohydrate types found on many membrane glycoproteins. Here, employing alpha-helical coiled-coils as scaffolds to create synthetic Gal3 constructs with defined valency, we demonstrate that oligomerization can physically regulate extracellular signaling activity of Gal3. Constructs with 2 to 6 Gal3 subunits ("Dimer," "Trimer," "Tetramer," "Pentamer," "Hexamer") demonstrated glycan-binding properties and cell death-inducing potency that scaled with valency. Dimer was the minimum functional valency. Unlike wild-type Gal3, which signals apoptosis and mediates agglutination, synthetic Gal3 constructs induced cell death without agglutination. In the presence of CD45, Hexamer was distributed on the cell membrane, whereas it clustered in absence of CD45 via membrane glycans other than those found on CD7. Wild-type Gal3, Pentamer, and Hexamer required CD45 and CD7 to signal apoptosis, and the involvement of caspases in apoptogenic signaling was increased in absence of CD45. However, wild-type Gal3 depended on caspases to signal apoptosis to a greater extent than Hexamer, which had greater caspase dependence than Pentamer. Diminished caspase activation downstream of Hexamer signaling led to decreased pannexin-1 hemichannel opening and interleukin-2 secretion, events facilitated by the increased caspase activation downstream of wild-type Gal3 signaling. Thus, synthetic fixation of Gal3 multivalency can impart physical control of its outside-in signaling activity by governing membrane glycoprotein engagement and, in turn, intracellular pathway activation.

Tuning Multivalent Signaling of Extracellular Galectin‐3

Promiscuous glycan-binding properties of galectin-3 coupled with its dynamic oligomerization can concomitantly activate various signal transduction pathways and, in turn, modulate cell behavior. Modifying the lectin-glycan binding interface to confer signaling pathway selectivity is challenged by the abundance of common carbohydrates that are present on a wide variety of membrane glycoproteins. Alternatively, controlling the multivalent assembly of galectin-3 could provide a physical means to direct its complex extracellular functions. Here, we use a library of α-helical coiled-coils as scaffolds to construct synthetic galectin-3 oligomers with specified valency, and we demonstrate that synthetic galectin-3 assembly can physically regulate membrane glycoprotein recognition to bias the activation of intracellular signaling pathways. Constructs displaying 2-6 galectin-3 subunits (i.e., “Dimer” - “Hexamer”) demonstrated increasing apparent glycan-binding affinity and cell death signaling potency. Though, whereas wild-type galectin-3 signaled caspase-dependent death via CD45 and CD7, Hexamer preferentially signaled death via CD45 due to its abundance and length protruding from the membrane. Preferential engagement of CD45 by Hexamer signaled caspase-independent cell death and diminished caspase-dependent cell activities, such as pannexin-1 hemichannel opening and interleukin-2 secretion, which were more closely associated with wild-type galectin-3 signaling. Therefore, synthetic galectin-3 oligomerization can impart physical control of its carbohydrate-binding promiscuity and, in turn, emergent extracellular functions.

A computational study of co-inhibitory immune complex assembly at the interface between T cells and antigen presenting cells.

The activation and differentiation of T-cells are mainly directly by their co-regulatory receptors. T lymphocyte-associated protein-4 (CTLA-4) and programed cell death-1 (PD-1) are two of the most important co-regulatory receptors. Binding of PD-1 and CTLA-4 with their corresponding ligands programed cell death-ligand 1 (PD-L1) and B7 on the antigen presenting cells (APC) activates two central co-inhibitory signaling pathways to suppress T cell functions. Interestingly, recent experiments have identified a new cis-interaction between PD-L1 and B7, suggesting that a crosstalk exists between two co-inhibitory receptors and the two pairs of ligand-receptor complexes can undergo dynamic oligomerization. Inspired by these experimental evidences, we developed a coarse-grained model to characterize the assembling of an immune complex consisting of CLTA-4, B7, PD-L1 and PD-1. These four proteins and their interactions form a small network motif. The temporal dynamics and spatial pattern formation of this network was simulated by a diffusion-reaction algorithm. Our simulation method incorporates the membrane confinement of cell surface proteins and geometric arrangement of different binding interfaces between these proteins. A wide range of binding constants was tested for the interactions involved in the network. Interestingly, we show that the CTLA-4/B7 ligand-receptor complexes can first form linear oligomers, while these oligomers further align together into two-dimensional clusters. Similar phenomenon has also been observed in other systems of cell surface proteins. Our test results further indicate that both co-inhibitory signaling pathways activated by B7 and PD-L1 can be down-regulated by the new cis-interaction between these two ligands, consistent with previous experimental evidences. Finally, the simulations also suggest that the dynamic and the spatial properties of the immune complex assembly are highly determined by the energetics of molecular interactions in the network. Our study, therefore, brings new insights to the co-regulatory mechanisms of T cell activation.