Heat of supersaturation-limited amyloid burst directly monitored by isothermal titration calorimetry

Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. Although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. Furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. Here, with β₂-microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (ITC). The spontaneous fibrillation after a lag phase was accompanied by exothermic heat. The thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. We also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. These results indicate that ITC will become a promising approach for clarifying comprehensively the thermodynamics of protein folding and misfolding.Aggregation has often been an obstacle to studying the structure, function, and physical properties of proteins. However, a large number of aggregates associated with serious diseases, including Alzheimer’s, Parkinson, and prion diseases (1, 2) promoted the challenge of studying protein misfolding and aggregation. Researchers succeeded in distinguishing amyloid fibrils and oligomers from other amorphous aggregates and characterized the ordered structures present in amyloid fibrils or oligomers, which led to the development of the field of amyloid structural biology (3–8). These advances have been attributed to various methodologies that are also useful for studying the structural properties of globular proteins. Even X-ray crystallography has become a powerful approach for studying amyloid microcrystals (5) or oligomers (9). The atomic details of amyloid fibrils are becoming increasingly clearer, and a cross-β structure was shown to be the main structural component of fibrils (5, 6, 8). Although tightly packed core regions of amyloid fibrils have been reported, the overall structures were shown to be dominated by common cross-β structures, which supported the argument for the main-chain dominated architecture in contrast to the side-chain dominated architecture of globular native states (10–12).These structural studies have been complemented by a series of efforts to clarify the mechanism for the formation of amyloid fibrils (i.e., amyloid fibrillation). The presence of a long lag time in spontaneous fibrillation and rapid fibrillation by the addition of preformed fibrils represent a similarity with the supersaturation-limited crystallization of substances (13–18). We have revisited “supersaturation” and argued its critical role for amyloid fibrillation (17–19). The role of supersaturation in neurodegenerative diseases at the proteome level has been reported recently (20).However, calorimetry, one of the most powerful methods used to study the thermodynamic properties of globular proteins (21–24), has not played a significant role in understanding protein aggregation. The aggregation of proteins following heat denaturation as monitored by differential scanning calorimetry is an infamous example demonstrating how aggregation can prevent exact analyses (25, 26). To date, few studies have investigated protein aggregation including amyloid fibrils with calorimetry (27–32). Our previous study on the exothermic heat effects accompanying fibril growth was achieved by monitoring the seed-dependent elongation of fibrils formed by β₂-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, using isothermal titration calorimetry (ITC) (28).In the present study using β2m, we succeeded in characterizing the total heat of spontaneous fibrillation and amorphous aggregation. An analysis of the heat burst associated with fibrillation or amorphous aggregation under various temperatures clarified their thermodynamic properties. The results obtained enabled the calorimetric characterization of amyloid fibrils and amorphous aggregates relative to that of the native globular structures, which opens a new field for the calorimetric study of protein aggregates.     

Cross-talk between amyloidogenic proteins in type-2 diabetes and Parkinson’s disease

In type-2 diabetes (T2D) and Parkinson’s disease (PD), polypeptide assembly into amyloid fibers plays central roles: in PD, α-synuclein (aS) forms amyloids and in T2D, amylin [islet amyloid polypeptide (IAPP)] forms amyloids. Using a combination of biophysical methods in vitro we have investigated whether aS, IAPP, and unprocessed IAPP, pro-IAPP, polypeptides can cross-react. Whereas IAPP forms amyloids within minutes, aS takes many hours to assemble into amyloids and pro-IAPP aggregates even slower under the same conditions. We discovered that preformed amyloids of pro-IAPP inhibit, whereas IAPP amyloids promote, aS amyloid formation. Amyloids of aS promote pro-IAPP amyloid formation, whereas they inhibit IAPP amyloid formation. In contrast, mixing of IAPP and aS monomers results in coaggregation that is faster than either protein alone; moreover, pro-IAPP can incorporate aS monomers into its amyloid fibers. From this intricate network of cross-reactivity, it is clear that the presence of IAPP can accelerate aS amyloid formation. This observation may explain why T2D patients are susceptible to developing PD.Parkinson’s disease (PD) is the second most common neurological disorder and the most common movement disorder. It is characterized by widespread degeneration of subcortical structures of the brain, especially dopaminergic neurons in the substantia nigra. These changes are coupled with bradykinesia, rigidity, and tremor, resulting in difficulties in walking and abnormal gait in patients (1). The assembly process of the intrinsically unstructured 140-residue protein α-synuclein (aS) into amyloid fibers has been linked to the molecular basis of PD. aS is a major component of amyloid aggregates found in Lewy body inclusions, which are the pathological hallmark of PD, and duplications, triplications, and point mutations in the aS gene are related to familial PD cases (2, 3). The exact function of aS is unknown, but it is suggested to be involved in synaptic vesicle release and trafficking, regulation of enzymes and transporters, and control of the neuronal apoptotic response (4, 5). aS is present at presynaptic nerve terminals (6–8) and, intriguingly, also in many cells outside the brain (e.g., red blood cells and pancreatic β-cells). aS can assemble via oligomeric intermediates to amyloid fibrils under pathological conditions (9). Although soluble aS oligomers have been proposed to be toxic (10, 11), work with preformed aS fibrils has demonstrated that the amyloid fibrils themselves are toxic and can be transmitted from cell to cell and are also able to cross the blood–brain barrier (12–14).Type-2 diabetes (T2D) is another disease involving amyloid formation. Here, the primary pathological characteristic is islet amyloid of the hormone amylin, also known as islet amyloid polypeptide (IAPP), in pancreatic β-cells (15–18). The process of islet amyloid formation (19–21) leads to pancreatic β-cell dysfunction, cell death, and development of diabetes. IAPP (37 residues, natively unfolded) is cosecreted with insulin after enzymatic maturation of prohormones pro-IAPP (67 residues) and proinsulin in secretory granules. IAPP and insulin play roles in controlling gastric emptying, glucose homeostasis, and in the suppression of glucagon release. Although not understood on a mechanistic level, impairment of prohormone processing has been thought to play a role in initiation and progression of T2D (22, 23). Insulin and pro-IAPP (22, 24–26), but not proinsulin, can inhibit IAPP amyloid formation in vitro and in mice, suggesting that accumulation of unprocessed proinsulin may promote IAPP amyloid formation (22, 24). Insulin-degrading enzyme (IDE) is a conserved metallopeptidase that can degrade insulin and a variety of other small peptides including IAPP in the pancreas (27, 28). Genome-wide association studies have linked IDE to T2D (29, 30) and Ide mutant mice were found to have impaired glucose-stimulated insulin secretion as well as increased levels of IAPP, insulin, and, surprisingly, aS in pancreatic islets (31, 32). Here, aS may be associated with insulin biogenesis and exocytic release, as it was found to localize with insulin-secretory granules in pancreatic β-cells (33). We recently demonstrated in vitro that IDE readily inhibits aS amyloid formation via C-terminal binding and, in parallel, IDE activity toward insulin and other small substrates increases (34, 35).Together, the key role of aS in PD and the inverse correlation of impaired insulin secretion and increased aS levels in the pancreatic β-cells, imply that PD and T2D may be connected. In support, reports have suggested that patients with T2D are predisposed toward PD (36, 37). For Alzheimer’s disease (AD), a direct link with T2D was found (15, 38). Amyloid fiber seeds of the AD peptide, amyloid-β, were shown to efficiently accelerate amyloid formation of IAPP in vitro (39, 40) and IAPP was part of amyloid-β plaque found in mice brains (41). To address the unexplored question of cross-reactivity between the amyloidogenic peptides in PD and T2D, we here investigated cross-reactivity among aS, IAPP, and pro-IAPP using biophysical methods in vitro.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Rational design of antibodies targeting specific epitopes within intrinsically disordered proteins

Antibodies are powerful tools in life sciences research, as well as in diagnostic and therapeutic applications, because of their ability to bind given molecules with high affinity and specificity. Using current methods, however, it is laborious and sometimes difficult to generate antibodies to target specific epitopes within a protein, in particular if these epitopes are not effective antigens. Here we present a method to rationally design antibodies to enable them to bind virtually any chosen disordered epitope in a protein. The procedure consists in the sequence-based design of one or more complementary peptides targeting a selected disordered epitope and the subsequent grafting of such peptides on an antibody scaffold. We illustrate the method by designing six single-domain antibodies to bind different epitopes within three disease-related intrinsically disordered proteins and peptides (α-synuclein, Aβ42, and IAPP). Our results show that all these designed antibodies bind their targets with good affinity and specificity. As an example of an application, we show that one of these antibodies inhibits the aggregation of α-synuclein at substoichiometric concentrations and that binding occurs at the selected epitope. Taken together, these results indicate that the design strategy that we propose makes it possible to obtain antibodies targeting given epitopes in disordered proteins or protein regions.Antibodies are versatile molecules that are increasingly used in therapeutic and diagnostic applications, as they can be used to treat a wide range of diseases, including cancer and autoimmune disorders (1–5). These molecules can be obtained with well-established methods, such as immunization or phage and associated display methods, against a wide variety of targets (6–11). In some cases, however, these procedures may require significant amounts of time and resources, in particular if one is interested in targeting weakly immunogenic epitopes in protein molecules. In this work, we introduce a computational method of rational design of complementarity determining regions (CDRs) that makes it possible to obtain antibody against virtually any target epitope within intrinsically disordered peptides and proteins or within disordered regions in structured proteins.Intrinsically disordered proteins, in particular, play major roles in a wide range of biochemical processes in living organisms. A range of recent studies has revealed that the functional diversity provided by disordered regions complements that of ordered regions of proteins, in particular in terms of key cellular functions such as signaling and regulation (12–18). The high flexibility and lack of stable secondary and tertiary structures allow intrinsically disordered proteins to have multiple interactions with multiple partners, often placing them at the hubs of protein–protein interaction networks (19–21). It has also been realized that the failure of the regulatory processes responsible for the correct behavior of intrinsically disordered proteins is associated with a variety of different pathological conditions (22–24). Indeed, intrinsic disorder is often observed in peptides and proteins implicated in a series of human conditions, including cancer, cardiovascular diseases, and neurodegenerative disorders (22–24). It would therefore be very helpful to develop methods to facilitate the generation of antibodies against disordered proteins, a goal that has a great therapeutic potential (25, 26).Here, we address this problem by introducing a rational design procedure that enables one to obtain antibodies that bind specifically target disordered regions. This procedure is based on the identification of a peptide complementary to a target region and on its grafting on to the CDR of an antibody scaffold. Related methods of altering rationally antibodies have been discussed in the literature, which include the exploration of specificity-enhancing mutations (27, 28), the design of CDRs to bind structured epitopes (28, 29), and the grafting of peptides extracted from aggregation prone proteins (30–32) or from other antibodies (33) in the CDR of an antibody scaffold. Here we show that designed antibodies can be obtained by the method that we present for essentially any disordered epitope. We illustrate the method for the Aβ peptide, α-synuclein, and the islet amyloid polypeptide (IAPP, or amylin peptide), which are respectively involved in Alzheimer’s and Parkinson’s diseases and type II diabetes (24).     

C-terminal sequence of amyloid-resistant type F apolipoprotein A-II inhibits amyloid fibril formation of apolipoprotein A-II in mice

In murine senile amyloidosis, misfolded serum apolipoprotein (apo) A-II deposits as amyloid fibrils (AApoAII) in a process associated with aging. Mouse strains carrying type C apoA-II (APOA2C) protein exhibit a high incidence of severe systemic amyloidosis. Previously, we showed that N- and C-terminal sequences of apoA-II protein are critical for polymerization into amyloid fibrils in vitro. Here, we demonstrate that congenic mouse strains carrying type F apoA-II (APOA2F) protein, which contains four amino acid substitutions in the amyloidogenic regions of APOA2C, were absolutely resistant to amyloidosis, even after induction of amyloidosis by injection of AApoAII. In vitro fibril formation tests showed that N- and C-terminal APOA2F peptides did not polymerize into amyloid fibrils. Moreover, a C-terminal APOA2F peptide was a strong inhibitor of nucleation and extension of amyloid fibrils during polymerization. Importantly, after the induction of amyloidosis, we succeeded in suppressing amyloid deposition in senile amyloidosis-susceptible mice by treatment with the C-terminal APOA2F peptide. We suggest that the C-terminal APOA2F peptide might inhibit further extension of amyloid fibrils by blocking the active ends of nuclei (seeds). We present a previously unidentified model system for investigating inhibitory mechanisms against amyloidosis in vivo and in vitro and believe that this system will be useful for the development of novel therapies.Amyloidosis refers to a group of protein structural disorders characterized by the extracellular deposits of insoluble amyloid fibrils resulting from abnormal conformational changes (1–5). Amyloid fibrils have a characteristic ultrastructural appearance and a β-pleated sheet core structure that consists of full-length proteins and/or fragments of either WT or mutant proteins found in familial diseases (2, 6–8). In humans, 28 amyloidogenic proteins have been identified. They are associated with prominent diseases such as Alzheimer’s disease, hemodialysis-associated amyloidosis, and familial amyloid polyneuropathy (2, 9, 10). To develop a therapeutic strategy for these disorders, it is essential to understand the mechanisms of amyloid fibril formation. Currently, the molecular and biological mechanisms that convert proteins into amyloid fibrils in vivo and in vitro remain largely unknown.Apolipoprotein (apo) A-II is the second most abundant apolipoprotein in human and mouse plasma high-density lipoproteins (HDLs) (11) and the most important protein associated with murine senile amyloidosis because it is the precursor of amyloid fibrils (AApoAII) (12–15). Seven alleles of the apoA-II gene have been found among inbred strains of mice, with polymorphisms in 15 nucleotide positions comprising eight amino acid positions (16). Each inbred laboratory mouse strain has a single type apoA-II protein, and the pathological findings of senile amyloidosis in strains with type A, B, or C apoA-II (APOA2A, APOA2B, or APOA2C, respectively) have been investigated (13, 17, 18). C57BL/6J, ICR, and DBA/2 strains have APOA2A and exhibit a moderate incidence of mild amyloid deposits with aging (19, 20). BALB/c, C3H/He, N2B, 129/SV, and SAMR1 strains have APOA2B and exhibit a low incidence of slight amyloid deposits with aging. In contrast, the SAMP1 strain has APOA2C and spontaneously exhibits a high incidence of severe systemic amyloid deposits with aging (20–22). We previously reported a unique mechanism in which N- and C-terminal peptides of apoA-II protein associated into amyloid fibrils in vitro (23) according to the nucleation-dependent polymerization model, which can explain the general mechanisms of amyloid fibril formation (24–28). The 11-residue amino acid sequence from positions 6–16 in the N terminus of apoA-II protein is critical for polymerization into amyloid fibrils. The 18-residue amino acid sequence from positions 48–65 in the C terminus of apoA-II is also necessary for nucleation, but not for the extension phase. Both sequences are common, and there is no substitution among APOA2A, APOA2B, and APOA2C (Fig. 1).Open in a separate windowFig. 1.Amino acid sequences of mouse apoA-II and synthetic partial peptides. For types A, B, and C apoA-II proteins (APOA2A, APOA2B, and APOA2C, respectively), the two amino acid sequences indicated in the red-colored boxes at positions 6–16 at the N terminus and 48–65 at the C terminus are the essential and common sequences required for amyloid fibril formation (23). Synthetic partial peptides were used to evaluate polymerization into amyloid fibrils in vitro and suppression against amyloid deposition in mice. The bold and blue-colored letters at positions 9, 16, 54, and 62 indicate the four variant amino acids in the core sequences for types A/B/C and F apoA-II proteins. Peptides containing orange letters represent substitutions of the a48/65(N62K) peptide.We hypothesized that some amino acid substitutions in these N- and C-terminal amyloidogenic sequences of apoA-II might inhibit the polymerization of apoA-II into amyloid fibrils. In that regard, type F apoA-II (APOA2F) contains four substitutions in the N- and C-terminal peptides relative to APOA2C (16) (Fig. 1). In this study, we evaluated the in vivo incidence of amyloidosis in mice having APOA2F and compared it with those in mice having APOA2A or APOA2C. We also analyzed the ability of APOA2F peptides to polymerize into amyloid fibrils in vitro. In previous studies, we found that injection of a very small amount of AApoAII amyloid fibrils markedly accelerated amyloid deposition (13–15). We demonstrated that mice with APOA2F were absolutely resistant against senile amyloidosis, even after induction of amyloidosis by injection with type C AApoAII fibrils. Thus, we have succeeded in suppressing amyloid deposits in amyloidosis-susceptible mice by treatment with the C-terminal APOA2F peptide. We thus demonstrate that the C-terminal sequence of APOA2F is an important inhibitor of polymerization into amyloid fibrils in vitro and in vivo. These findings provide a previously unidentified model system for investigating inhibitory mechanisms against amyloidosis in vivo and in vitro.     

Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg²⁺ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand.Argonaute (Ago) proteins, critical components of the RNA-induced silencing complex, play a key role in guide strand-mediated target RNA recognition, cleavage, and product release (reviewed in refs. 1–3). Ago proteins adopt a bilobal scaffold composed of an amino terminal PAZ-containing lobe (N and PAZ domains), a carboxyl-terminal PIWI-containing lobe (Mid and PIWI domains), and connecting linkers L1 and L2. Ago proteins bind guide strands whose 5′-phosphorylated and 3′-hydroxyl ends are anchored within Mid and PAZ pockets, respectively (4–7), with the anchored guide strand then serving as a template for pairing with the target strand (8, 9). The cleavage activity of Ago resides in the RNase H fold adopted by the PIWI domain (10, 11), whereby the enzyme’s Asp-Asp-Asp/His catalytic triad (12–15) initially processes loaded double-stranded siRNAs by cleaving the passenger strand and subsequently processes guide-target RNA duplexes by cleaving the target strand (reviewed in refs. 16–18). Such Mg²⁺ cation-mediated endonucleolytic cleavage of the target RNA strand (19, 20) resulting in 3′-OH and 5′-phosphate ends (21) requires Watson–Crick pairing of the guide and target strands spanning the seed segment (positions 2–2′ to 8–8′) and the cleavage site (10′–11′ step on the target strand) (9). Insights into target RNA recognition and cleavage have emerged from structural (9), chemical (22), and biophysical (23) experiments.Notably, bacterial and archaeal Ago proteins have recently been shown to preferentially bind 5′-phosphoryated guide DNA (14, 15) and use an activated water molecule as the nucleophile (reviewed in ref. 24) to cleave both RNA and DNA target strands (9). Structural studies have been undertaken on bacterial and archaeal Ago proteins in the free state (10, 15) and bound to a 5′-phosphorylated guide DNA strand (4) and added target RNA strand (8, 9). The structural studies of Thermus thermophilus Ago (TtAgo) ternary complexes have provided insights into the nucleation, propagation, and cleavage steps of target RNA silencing in a bacterial system (9). These studies have highlighted the conformational transitions on proceeding from Ago in the free state to the binary complex (4) to the ternary complexes (8, 9) and have emphasized the requirement for a precisely aligned Asp-Asp-Asp triad and a pair of Mg²⁺ cations for cleavage chemistry (9), typical of RNase H fold-mediated enzymes (24, 25). Structural studies have also been extended to binary complexes of both human (5, 6) and yeast (7) Agos bound to 5′-phosphorylated guide RNA strands.Despite these singular advances in the structural biology of RNA silencing, further progress was hampered by the modest resolution (2.8- to 3.0-Å resolution) of TtAgo ternary complexes with guide DNA (4) and added target RNAs (8, 9). This precluded identification of water molecules coordinated with the pair of Mg²⁺ cations, including the key water that acts as a nucleophile and targets the cleavable phosphate between positions 10′-11′ on the target strand. We have now extended our research to TtAgo ternary complexes with guide DNA and target DNA strands, which has permitted us to grow crystals of ternary complexes that diffract to higher (2.2–2.3 Å) resolution in the cleavage-incompatible, cleavage-compatible, and postcleavage steps. These high-resolution structures of TtAgo ternary complexes provide snapshots of distinct key steps in the catalytic cleavage pathway, opening opportunities for experimental probing into DNA target cleavage as a defense mechanism against plasmids and possibly other mobile elements (26, 27).     

Conformational dynamics of a crystalline protein from microsecond-scale molecular dynamics simulations and diffuse X-ray scattering

X-ray diffraction from protein crystals includes both sharply peaked Bragg reflections and diffuse intensity between the peaks. The information in Bragg scattering is limited to what is available in the mean electron density. The diffuse scattering arises from correlations in the electron density variations and therefore contains information about collective motions in proteins. Previous studies using molecular-dynamics (MD) simulations to model diffuse scattering have been hindered by insufficient sampling of the conformational ensemble. To overcome this issue, we have performed a 1.1-μs MD simulation of crystalline staphylococcal nuclease, providing 100-fold more sampling than previous studies. This simulation enables reproducible calculations of the diffuse intensity and predicts functionally important motions, including transitions among at least eight metastable states with different active-site geometries. The total diffuse intensity calculated using the MD model is highly correlated with the experimental data. In particular, there is excellent agreement for the isotropic component of the diffuse intensity, and substantial but weaker agreement for the anisotropic component. Decomposition of the MD model into protein and solvent components indicates that protein–solvent interactions contribute substantially to the overall diffuse intensity. We conclude that diffuse scattering can be used to validate predictions from MD simulations and can provide information to improve MD models of protein motions.Proteins explore many conformations while carrying out their functions in biological systems (1–3). X-ray crystallography is the dominant source of information about protein structure; however, crystal structure models usually consist of just a single major conformation and at most a small portion of the model as alternate conformations. Crystal structures therefore are missing many details about the underlying conformational ensemble (4).Proteins assembled in crystalline arrays, like proteins in solution, exhibit rich conformational diversity (4) and often can perform their native functions (5). Many methods have emerged for using Bragg data to model conformational diversity in protein crystals (6–17). The development of these methods has been important as conformational diversity can lead to inaccuracies in protein structure models (9, 18–20). A key limitation of using the Bragg data, however, is that different models of conformational diversity can yield the same mean electron density.Whereas the Bragg scattering only contains information about the mean electron density, diffuse scattering (diffraction resulting in intensity between the Bragg peaks) is sensitive to spatial correlations in electron density variations (21–28) and therefore contains information about the way that atomic positions vary together in protein crystals. Because models that yield the same mean electron density can yield different correlations in electron density variations, diffuse scattering provides a means to increase the accuracy of crystallography for determining protein conformational variations (29). Peter Moore (30) and Mark Wilson (31) have argued that diffuse scattering should be used to test models of conformational diversity in X-ray crystallography.Several pioneering studies used diffuse scattering to reveal insights into correlated motions in proteins (17, 30, 32–49). Some of these studies used diffuse scattering to experimentally validate predictions of correlated motions from molecular-dynamics (MD) simulations (35–37, 40, 42–44). These studies revealed important insights but were limited by inadequate sampling of the conformational ensemble, leading to lack of convergence of the diffuse scattering calculations (35). Microsecond-scale simulations of staphylococcal nuclease were predicted to be adequate for convergence of diffuse scattering calculations (42). Modern simulation algorithms and computer hardware now enable microsecond or longer MD simulations of protein crystals (50).Here, we present calculations of diffuse X-ray scattering using a 1.1-μs MD simulation of crystalline staphylococcal nuclease. The results demonstrate that we have overcome the past limitation of inadequate sampling. We chose staphylococcal nuclease because the experiments of Wall et al. (49) still represent the only complete, high-quality, 3D diffuse scattering data set from a protein crystal. The calculated diffuse intensity is very similar using two independent halves of the trajectory; the results therefore are reproducible and can be meaningfully compared with the experimental data. The MD simulation provides a rich picture of conformational diversity in the energy landscape of a protein crystal, consisting of at least eight metastable states. Like previous MD studies of crystalline staphylococcal nuclease (42–44), the agreement of the simulation with the total experimental diffuse intensity is excellent, supporting the use of MD simulations to model diffuse scattering data. Unlike previous MD studies, we separately compared the more finely structured, anisotropic component of the diffuse intensity with experimental data. The agreement is substantial but weaker than for the isotropic component, indicating there are inaccuracies in the MD models. Our results therefore point toward using diffuse scattering to improve MD models of protein motions.     

Contingency and entrenchment in protein evolution under purifying selection

The phenotypic effect of an allele at one genetic site may depend on alleles at other sites, a phenomenon known as epistasis. Epistasis can profoundly influence the process of evolution in populations and shape the patterns of protein divergence across species. Whereas epistasis between adaptive substitutions has been studied extensively, relatively little is known about epistasis under purifying selection. Here we use computational models of thermodynamic stability in a ligand-binding protein to explore the structure of epistasis in simulations of protein sequence evolution. Even though the predicted effects on stability of random mutations are almost completely additive, the mutations that fix under purifying selection are enriched for epistasis. In particular, the mutations that fix are contingent on previous substitutions: Although nearly neutral at their time of fixation, these mutations would be deleterious in the absence of preceding substitutions. Conversely, substitutions under purifying selection are subsequently entrenched by epistasis with later substitutions: They become increasingly deleterious to revert over time. Our results imply that, even under purifying selection, protein sequence evolution is often contingent on history and so it cannot be predicted by the phenotypic effects of mutations assayed in the ancestral background.Whether a heritable mutation is advantageous or deleterious to an organism often depends on the evolutionary history of the population. A mutation that is beneficial at the time of its introduction may confer its beneficial effect only in the presence of other potentiating or permissive mutations (1–9). Thus, the fate of a mutation arising in a population may be contingent on previous mutations (10–13). Conversely, once a mutation has fixed in a population, the mutation becomes part of the genetic background onto which subsequent modifications are introduced. Because the beneficial effects of the subsequent modifications may depend on the focal mutation, as time passes reversion of the focal mutation may become increasingly deleterious, leading to a type of evolutionary conservatism, or entrenchment (14–18).In the context of protein evolution, the effects of contingency and entrenchment are most easily studied by considering a sequence of single amino acid changes (19) that extends both forward and backward in time from some focal substitution. To assess the roles of contingency and entrenchment we can study the degree to which each focal substitution was facilitated by previous substitutions, and the degree to which the focal substitution influences the subsequent course of evolution (Fig. 1A).Open in a separate windowFig. 1.(A) A schematic model indicating how a focal substitution may be contingent on prior substitutions and may constrain future substitutions along an evolutionary trajectory, owing to epistasis. (B) A model of protein evolution under weak mutation and purifying selection for thermodynamic stability. Starting from the wild-type sequence of argT we propose 10 random 1-aa point mutations. For each of the proposed mutants we compute its predicted stability (ΔG) using FoldX, and its associated fitness. The fitness function is assumed to be either Gaussian or semi-Gaussian, with a maximum at the wild-type stability. One of the proposed mutants fixes in the population, based on its relative fixation probability under the Moran model with effective population size N_e. This process is iterated for 30 consecutive substitutions to produce an evolutionary trajectory. We simulate 100 replicate trajectories, each initiated at the wild-type argT sequence.Dependencies within a sequence of substitutions are closely connected to the concept of epistasis—that is, the idea that the phenotypic effect of a mutation at a particular genetic site may depend on the genetic background in which it arises (20–24). In the absence of epistasis, a mutation has the same effect regardless of its context and therefore regardless of any prior history or subsequent evolution. By contrast, in the presence of epistasis, each substitution may be contingent on the entire prior history of the protein, and it may constrain all subsequent evolution.The potential for epistasis to play an important role in evolution, including protein evolution, has not been overlooked by researchers (1, 8, 25–34), nor have the concepts of contingency (3, 4, 9, 12, 35–38) and, more recently, entrenchment (18, 39, 40). However, most studies have addressed the role of epistasis in the context of adaptive evolution (1–9, 27, 30, 31, 36, 38), whereas the consequences of epistasis under purifying selection have received less attention (18, 41–44). Indeed, although some more sophisticated models have been proposed (e.g., refs. 45–50), all commonly used phylogenetic models of long-term protein evolution assume that epistasis is absent so that sites evolve independently (51–56).Here we explore the relationships between epistasis, contingency, and entrenchment under long-term purifying selection on protein stability. Our analysis combines computational models for protein structures with population-genetic models for evolutionary dynamics. We use a force-field-based model, FoldX (57), to characterize the effects of point mutations on a protein’s stability and fitness. This approach allows us to simulate evolutionary trajectories of protein sequences under purifying selection, by the sequential fixation of nearly neutral mutations. We can then dissect the epistatic relationships between these substitutions by systematically inserting or reverting particular substitutions at various time points along the evolutionary trajectory.Our analysis considers epistasis both at the level of protein stability and at the level of fitness. Whereas empirical studies in diverse proteins have demonstrated that the stability effects of point mutations are typically additive across sites (58, 59), in this study we are specifically interested in epistasis for stability among the mutations that fix during evolution. Even if most random mutations are virtually additive in their effects on stability, the mutations that fix under purifying selection are highly nonrandom, and so there is reason to suspect that epistasis for stability may be enriched among such mutations. Moreover, because the mapping from stability to fitness is itself nonlinear (18, 26, 60, 61) and because selection is sensitive to selection coefficients as small as the inverse of the population size (62), even slight variation in the stability effects of mutations across different genetic backgrounds may be sufficient to influence the course of evolution.Using the computational approach summarized above, we will demonstrate that the nearly neutral mutations that fix under purifying selection are, indeed, often epistatic with each other for both stability and fitness. In particular, we find that each mutation that fixes is typically permitted to fix by the presence of preceding substitutions—that is, most substitutions would be too deleterious to fix were it not for epistasis with preceding substitutions. Conversely, we also find that mutations that fix typically become entrenched over time by epistasis—so that a substitution that was nearly neutral when it fixed becomes increasingly deleterious to revert as subsequent substitutions accumulate (18, 39). These results imply an important role for epistasis in shaping the course of sequence evolution in a protein under selection to maintain thermodynamic stability.     

Dopamine release from transplanted neural stem cells in Parkinsonian rat striatum in vivo

Embryonic stem cell-based therapies exhibit great potential for the treatment of Parkinson’s disease (PD) because they can significantly rescue PD-like behaviors. However, whether the transplanted cells themselves release dopamine in vivo remains elusive. We and others have recently induced human embryonic stem cells into primitive neural stem cells (pNSCs) that are self-renewable for massive/transplantable production and can efficiently differentiate into dopamine-like neurons (pNSC–DAn) in culture. Here, we showed that after the striatal transplantation of pNSC–DAn, (i) pNSC–DAn retained tyrosine hydroxylase expression and reduced PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in the striatum both in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC–DAn (and not from injured original cells). Thus, pNSC–DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proof-of-concept for human clinical translation.Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder characterized by the specific loss of dopaminergic neurons in the substantia nigra pars compacta and their projecting axons, resulting in loss of dopamine (DA) release in the striatum (1). During the last two decades, cell-replacement therapy has proven, at least experimentally, to be a potential treatment for PD patients (2–7) and in animal models (8–15). The basic principle of cell therapy is to restore the DA release by transplanting new DA-like cells. Until recently, obtaining enough transplantable cells was a major bottleneck in the practicability of cell therapy for PD. One possible source is embryonic stem cells (ESCs), which can develop infinitely into self-renewable pluripotent cells with the potential to generate any type of cell, including DA neurons (DAns) (16, 17).Recently, several groups including us have introduced rapid and efficient ways to generate primitive neural stem cells (pNSCs) from human ESCs using small-molecule inhibitors under chemically defined conditions (12, 18, 19). These cells are nonpolarized neuroepithelia and retain plasticity upon treatment with neuronal developmental morphogens. Importantly, pNSCs differentiate into DAns (pNSC–DAn) with high efficiency (∼65%) after patterning by sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) in vitro, providing an immediate and renewable source of DAns for PD treatment. Importantly, the striatal transplantation of human ESC-derived DA-like neurons, including pNSC–DAn, are able to relieve the motor defects in a PD rat model (11–13, 15, 19–23). Before attempting clinical translation of pNSC–DAn, however, there are two fundamental open questions. (i) Can pNSC–DAn functionally restore the striatal DA levels in vivo? (ii) What cells release the restored DA, pNSC–DAn themselves or resident neurons/cells repaired by the transplants?Regarding question 1, a recent study using nafion-coated carbon fiber electrodes (CFEs) reported that the amperometric current is rescued in vivo by ESC (pNSC–DAn-like) therapy (19). Both norepinephrine (NE) and serotonin are present in the striatum (24, 25). However, CFE amperometry/chronoamperometry alone cannot distinguish DA from other monoamines in vivo, such as NE and serotonin (Fig. S1) (see also refs. 26–28). Considering that the compounds released from grafted ESC-derived cells are unknown, the work of Kirkeby et al. was unable to determine whether DA or other monoamines are responsible for the restored amperometric signal. Thus, the key question of whether pNSC–DAn can rescue DA release needs to be reexamined for the identity of the restored amperometric signal in vivo.Regarding question 2, many studies have proposed that DA is probably released from the grafted cells (8, 12, 13, 20), whereas others have proposed that the grafted stem cells might restore striatal DA levels by rescuing injured original cells (29, 30). Thus, whether the grafted cells are actually capable of synthesizing and releasing DA in vivo must be investigated to determine the future cellular targets (residual cells versus pNSC–DAn) of treatment.To address these two mechanistic questions, advanced in vivo methods of DA identification and DA recording at high spatiotemporal resolution are required. Currently, microdialysis-based HPLC (HPLC) (31–33) and CFE amperometric recordings (34, 35) have been used independently by different laboratories to assess evoked DA release from the striatum in vivo. The major advantage of microdialysis-based HPLC is to identify the substances secreted in the cell-grafted striatum (33), but its spatiotemporal resolution is too low to distinguish the DA release site (residual cells or pNSC–DAn). In contrast, the major advantage of CFE-based amperometry is its very high temporal (ms) and spatial (μm) resolution, making it possible to distinguish the DA release site (residual cells or pNSC–DAn) in cultured cells, brain slices, and in vivo (34–39), but it is unable to distinguish between low-level endogenous oxidizable substances (DA versus serotonin and NE) in vivo.In the present study, we developed a challenging experimental paradigm of combining the two in vivo methods, microdialysis-based HPLC and CFE amperometry, to identify the evoked substance as DA and its release site as pNSC–DAn in the striatum of PD rats.     

Residue-level resolution of alphavirus envelope protein interactions in pH-dependent fusion

Alphavirus envelope proteins, organized as trimers of E2–E1 heterodimers on the surface of the pathogenic alphavirus, mediate the low pH-triggered fusion of viral and endosomal membranes in human cells. The lack of specific treatment for alphaviral infections motivates our exploration of potential antiviral approaches by inhibiting one or more fusion steps in the common endocytic viral entry pathway. In this work, we performed constant pH molecular dynamics based on an atomic model of the alphavirus envelope with icosahedral symmetry. We have identified pH-sensitive residues that cause the largest shifts in thermodynamic driving forces under neutral and acidic pH conditions for various fusion steps. A series of conserved interdomain His residues is identified to be responsible for the pH-dependent conformational changes in the fusion process, and ligand binding sites in their vicinity are anticipated to be potential drug targets aimed at inhibiting viral infections.Alphaviruses, mosquito-borne human pathogens causing severe inflammations and fatal fevers, have infected many millions of people in recent outbreaks worldwide since 2005 (1–3). The lack of a vaccine or specific treatment prompts investigations of the fundamental mechanisms of the alphaviral lifecycle to facilitate the development of effective antiviral therapies (4). Alphaviruses have been reported to enter the cell through receptor-mediated endocytosis. Here, alphaviruses are ferried toward the perinuclear space of the host cell inside vesicles towed by molecular motors and delivered to specific locations for productive replication (5–11). Even when direct entry into the cytoplasm is possible (11–15), the endocytic entry pathway facilitates the transportation of viruses across the crowded cytoplasmic space and delays detection by the immune system without leaving empty capsid or envelope as obvious evidence of the viral infection exposed outside the host cell (10, 11). Before the delivery of its viral genome into the cytoplasm of a host cell, the alphavirus must undergo a critical step of low pH-triggered membrane fusion, which is a common mechanism in the endocytic viral entry pathway among many different viruses. Understanding the mechanism of the low pH-triggered alphaviral membrane fusion is essential for the development of therapies against alphavirus as well as other viruses using similar endocytic entry mechanisms.Recent studies of the lifecycle of alphavirus reveal that a precursor, p62, is first synthesized as a chaperon forming a heterodimer with E1, which is essential for viral budding (16); p62 protects the E1 protein in the low-pH environment of the secretory pathway before being cleaved by cellular furin to produce mature E2-E1 and a smaller fragment, E3 (17–21). After the virus buds from the cytoplasmic membrane, E3 is released from the virus particle under neutral pH conditions outside the host cell (13, 22–24).On the surface of a mature alphavirus, 80 (E2–E1)₃ viral spikes, organized in T = 4 icosahedral symmetry on the viral lipid membrane, enclose the viral capsid and genome (25–43). On internalization of the mature virus in the endosome of the host cell in a new round of infection cycle, the increasingly acidified endosomal environment triggers a series of conformational changes in the alphaviral spike (E2–E1)₃ (38), including the dissociation of E2 (42, 44, 45), release of a fusion loop on E1 (46, 47), and trimerization of E1 (48). The fusion loop, roughly residues 83–100 on the cd loop of each E1 protein (13, 49, 50), in the newly formed E1 homotrimer (HT), inserts into the endosomal membrane. Then, the E1 proteins fold back, pulling the viral and endosomal membranes together and thus, promoting membrane fusion (13, 24).Recently solved high-resolution structures of the alphavirus envelope proteins E2–E1 fitted into cryo-EM data representing the intact virus under both acidic and neutral pH conditions (43, 51, 52) provide excellent atomic models for studies of the low pH-triggered fusion process. The structure of Chikungunya virus (CHIKV) obtained at pH 8.0 represents the initial mature state (M state) of the (E2–E1)₃ viral spike before the fusion process (51). Under pH 5.6, domain B (DB) of E2, which protects the E1 fusion loop, is observed to be disordered in Sindbis virus (52). The rest of the domains of the (E2–E1)₃ spike show moderate conformational differences with an rmsd = 4.0 Å among C_α atoms compared with the structures obtained at pH 8.0 for CHIKV (43, 51). The structure of the envelope proteins in acidic conditions most likely depicts a fusion intermediate (FI) state (52) before E2 dissociation during the low pH-triggered fusion process. In addition, the crystal structure of the folded-back E1 HT (53) is a good model to describe the postfusion state.Based on these atomic models of the E2 and E1 envelope proteins and our previously developed constant pH molecular dynamics (CPHMD) method (54–58), we simulated the envelope proteins with icosahedral symmetry under various pH conditions covering pH 2.0–9.0. We used pH replica exchange in CPHMD and calculated pK_a values using pH titration fitting, which has been shown as a reliable and accurate approach to capture pK_a values of protein residues in various systems (59–64). Through the CPHMD modeling, we calculated the pK_a of the possible pH-sensitive residues (Asp, Glu, and His) in the M, FI, dissociated E2 (Dis), and HT states. We, therefore, derive the shifts in the thermodynamic stabilities originating from each titrating residue for the steps from the M to the FI state (M→FI) of (E2–E1)₃, from the FI to the Dis state (FI→Dis) of E2 proteins, and from the FI to the HT state (FI→HT) of E1 proteins as shown in Fig. 1D. For these processes, we assume that the virus is in the endosomal environment, and we do not consider possible receptor-induced conformational changes. Our residue-level resolution simulations and analyses allow us to identify the critical functional residues with significant pK_a shifts and changes in thermodynamic stability in the low pH-triggered fusion activation. Our results suggest that the most pH-sensitive residues are highly conserved among different alphaviral species and that these critical residues control the pH threshold of fusion activities, provide guidance to further mutagenesis experiments, and lead to more fundamental understanding of low pH-triggered alphaviral membrane fusion.Open in a separate windowFig. 1.Structure and organization of alphaviral envelope proteins. (A) The alphaviral envelope modeled in our simulations. (B) The alphaviral envelope proteins in an MAU. (C) The heterodimer of E2 (DA–DB–DC) and E1 (DI–DII–DIII). (D) Structures of a viral spike in different conformational states simulated for shifts in pK_a values and thermodynamic stabilities. E1 proteins are shown in blue, cyan, and light blue. E2 proteins are shown in red, magenta, and pink.     

Enhanced receptor–clathrin interactions induced by N-glycan–mediated membrane micropatterning

Glycan–protein interactions are emerging as important modulators of membrane protein organization and dynamics, regulating multiple cellular functions. In particular, it has been postulated that glycan-mediated interactions regulate surface residence time of glycoproteins and endocytosis. How this precisely occurs is poorly understood. Here we applied single-molecule-based approaches to directly visualize the impact of glycan-based interactions on the spatiotemporal organization and interaction with clathrin of the glycosylated pathogen recognition receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). We find that cell surface glycan-mediated interactions do not influence the nanoscale lateral organization of DC-SIGN but restrict the mobility of the receptor to distinct micrometer-size membrane regions. Remarkably, these regions are enriched in clathrin, thereby increasing the probability of DC-SIGN–clathrin interactions beyond random encountering. N-glycan removal or neutralization leads to larger membrane exploration and reduced interaction with clathrin, compromising clathrin-dependent internalization of virus-like particles by DC-SIGN. Therefore, our data reveal that cell surface glycan-mediated interactions add another organization layer to the cell membrane at the microscale and establish a novel mechanism of extracellular membrane organization based on the compartments of the membrane that a receptor is able to explore. Our work underscores the important and complex role of surface glycans regulating cell membrane organization and interaction with downstream partners.Glycans are fundamental cellular components ubiquitously present in the extracellular matrix and cell membrane as glycoproteins or glycolipids. Glycan-binding proteins such as galectins, siglecs, and selectins are mostly multivalent and thus thought to cross-link glycoproteins into higher-order aggregates, creating a cell surface glycan-based connectivity also called glycan lattice or network (1–3). By concentrating specific glycoproteins or glycolipids while excluding other cell surface molecules, surface glycan-based connectivity can organize the plasma membrane into specialized domains that perform unique functions (1, 3–6). Nevertheless, direct observation of glycan-mediated ligand cross-linking in living cells remains challenging (7). Notwithstanding, there is no doubt that surface glycan-based connectivity is essential in the control of multiple biological processes including immune cell activation and homeostasis, cell proliferation and differentiation, and receptor turnover and endocytosis (1, 5, 6, 8).Clathrin-mediated endocytosis (CME) constitutes the primary pathway of cargo internalization in mammalian cells regulating the surface expression of receptors (9). Formation of clathrin-coated pits (CCPs) starts by nucleation of coat assembly at distributed positions in the inner surface of the plasma membrane, where it continues to grow or dissolve rapidly unless coat stabilization occurs (10, 11). One event that clearly correlates with successful CCP stabilization is cargo loading (11). Recent studies show that cargo molecules diffuse randomly on the cell membrane until they meet growing CCPs, with the extent of cargo interactions regulating CCP maturation (12). As such, factors that affect cargo mobility within/at the cell surface will inevitably impact on CCP maturation and successful internalization. In the context of surface glycan–protein interactions, it has been shown that glycoproteins with an intact glycan-based connectivity exhibit reduced lateral mobility and this correlates with compromised endocytosis (3, 13–17). How this precisely occurs is poorly defined, although fluorescence recovery after photobleaching on the EGF receptor (EGFR) suggested that cell surface glycan-based interactions restrict EGFR dynamics and localization into membrane regions away from endocytic platforms (14, 17). Whether this is a general mechanism for glycosylated proteins or specific to EGFR is not known. Moreover, visualization of receptor interactions with the endocytic machinery under the influence of the glycan network has not yet been attained.In this work we applied superresolution nanoscopy and developed a dedicated dual-color single-molecule spatio-dynamic exploration approach to visualize the impact of glycan-based interactions on the spatiotemporal organization and clathrin interaction of a glycosylated membrane receptor involved in pathogen recognition and uptake. We focused on the transmembrane glycoprotein dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) given its importance in supporting primary immune responses such as pathogen recognition and uptake on immature dendritic cells (imDCs), signaling, and cell adhesion (6, 18–20). Moreover, DC-SIGN contains a single N-glycosylation site, organizes in nanoclusters at the cell membrane (19, 21–23), and internalizes bound antigens via CPPs for subsequent processing and presentation to T cells (20, 24–26). Our work provides insights on how surface glycan-mediated interactions tune spatiotemporal micropatterning of receptors on the cell membrane, potentially regulating interactions with the endocytic machinery and underscoring the importance and complex role of surface glycans on cell membrane organization and function.