Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor ß (TGF-ß), is reduced; that then leads to the activation of the TGF-ß pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins.

Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Mass photometry: A powerful tool for carbohydrates-proteins conjugation monitoring and glycoconjugates molecular mass determination.

Glycoconjugate vaccines are important additions to the existing means for prevention of diseases caused by bacterial and viral pathogens. Conjugating carbohydrates to proteins is a crucial step in the development of these vaccines. Traditional mass spectrometry techniques, such as MALDI-TOF and SELDI-TOF, have difficulties in detecting glycoconjugates with high molecular masses. Mass photometry (MP) is a single-molecule technique that has been recently developed, which allows mass measurements of individual molecules and generates mass distributions based on hundreds to thousands of these measurements. In this study, we evaluated the performance of MP in monitoring carbohydrate-protein conjugation reactions and characterization of conjugates. Three different glycoconjugates were prepared from carrier protein BSA, and one from a large protein complex, a virus capsid with 3.74 MDa molecular mass. The masses measured by MP were consistent with those obtained by SELDI-TOF-MS and SEC-MALS. The conjugation of BSA dimer to carbohydrate antigen was also successfully characterized. This study shows that the MP technique is a promising alternative to methods developed earlier for monitoring glycoconjugation reactions and characterization of glycoconjugates. It measures intact molecules in solution and it is highly accurate over a wide mass range. MP requires only a very small amount of sample and has no specific buffer constraints. Other MP advantages include minimal cost of consumables and rapid data collection and analysis. Its advantages over other methods make it a valuable tool for researchers in the glycoconjugation field.

Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry.

Recombinant adeno-associated viruses (rAAV) are used extensively as gene delivery vectors in clinical studies, and several rAAV based treatments have already been approved. Significant progress has been made in rAAV manufacturing; however, better and more precise capsid characterization techniques are still needed to guarantee the purity and safety of rAAV preparations. Current analytical techniques used to characterize rAAV preparations are susceptible to background signals, have limited accuracy, or require a large amount of time and material. A recently developed single-molecule technique, mass photometry (MP), measures mass distributions of biomolecules with high-resolution and sensitivity. Here we explore applications of MP for the characterization of capsid fractions. We demonstrate that MP is able to resolve and quantify not only empty and full-genome containing capsid populations but also identify partially packaged capsid impurities. MP data accurately measures full and empty capsid ratios, and can be used to estimate the size of the encapsidated genome. MP distributions provide information on sample heterogeneity and on the presence of aggregates. Sub-picomole quantities of sample are sufficient for MP analysis, and data can be obtained and analyzed within minutes. This method provides a simple, robust, and effective tool to monitor the physical attributes of rAAV vectors.

A New Structural Model of Apolipoprotein B100 Based on Computational Modeling and Cross Linking.

ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the "divide and conquer" algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 Å threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 Å constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions.

Chlorpromazine binding to the PAS domains uncovers the effect of ligand modulation on EAG channel activity.

Ether-a-go-go (EAG) potassium selective channels are major regulators of neuronal excitability and cancer progression. EAG channels contain a Per-Arnt-Sim (PAS) domain in their intracellular N-terminal region. The PAS domain is structurally similar to the PAS domains in non-ion channel proteins, where these domains frequently function as ligand-binding domains. Despite the structural similarity, it is not known whether the PAS domain can regulate EAG channel function via ligand binding. Here, using surface plasmon resonance, tryptophan fluorescence, and analysis of EAG currents recorded in Xenopus laevis oocytes, we show that a small molecule chlorpromazine (CH), widely used as an antipsychotic medication, binds to the isolated PAS domain of EAG channels and inhibits currents from these channels. Mutant EAG channels that lack the PAS domain show significantly lower inhibition by CH, suggesting that CH affects currents from EAG channels directly through the binding to the PAS domain. Our study lends support to the hypothesis that there are previously unaccounted steps in EAG channel gating that could be activated by ligand binding to the PAS domain. This has broad implications for understanding gating mechanisms of EAG and related ERG and ELK K+ channels and places the PAS domain as a new target for drug discovery in EAG and related channels. Up-regulation of EAG channel activity is linked to cancer and neurological disorders. Our study raises the possibility of repurposing the antipsychotic drug chlorpromazine for treatment of neurological disorders and cancer.

Standard protocol for mass photometry experiments.

Mass photometry (MP) is a relatively new experimental technique with a quickly expanding list of applications. Using optical detection, MP measures the mass of individual molecules to obtain molecular mass distributions of proteins and other biomolecules in solution. The combination of speed, sensitivity, and very low sample consumption with label- and immobilization-free detection sets MP apart from other analytical methods. An increasing number of laboratories incorporates mass photometry as a routine sample analysis technique. However, MP measurements can sometimes be challenging, especially for users without previous experience with single-molecule techniques. Here, we present a protocol for the determination of protein molecular mass distributions by MP. It describes the sample and materials preparation as well as data collection and analysis. The advantages and limitations of this technique and the potential sources of artifacts are also given. This protocol can be used by new MP users and serve as a checklist for laboratories routinely performing MP experiments to guide consistent data collection and documentation.

A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA.

A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called "stoichiometry" (n), a concentration-correction factor.

Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

Measuring the affinity of protein-protein interactions on a single-molecule level by mass photometry.

Measurements of biomolecular interactions are crucial to understand the mechanisms of the biological processes they facilitate. Bulk-based methods such as ITC and SPR provide important information on binding affinities, stoichiometry, and kinetics of interactions. However, the ensemble averaging approaches are not able to probe the intrinsic heterogeneity often displayed by biological systems. Interactions that involve cooperativity or result in the formation of multicomponent complexes pose additional experimental challenges. Single-molecule techniques have previously been applied to solve these problems. However, single-molecule experiments are often technically demanding and require labeling or immobilization of the molecules under study. A recently developed single-molecule method, mass photometry (MP), overcomes these limitations. Here we applied MP to measure the affinities of biomolecular interactions. We have demonstrated how MP allows the user to study multivalent complexes and quantify the affinities of different binding sites in a single measurement. Results obtained from this single-molecule technique have been validated by ITC and BLI. The quality and information content of the MP data, combined with simple and fast measurements and low sample consumption makes MP a new preferred method for measuring strong protein-protein interactions.

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II.

Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLC-phosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.

The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts.

Protein trafficking across membranes is an essential function in cells; however, the exact mechanism for how this occurs is not well understood. In the endosymbionts, mitochondria and chloroplasts, the vast majority of proteins are synthesized in the cytoplasm as preproteins and then imported into the organelles via specialized machineries. In chloroplasts, protein import is accomplished by the TOC (translocon on the outer chloroplast membrane) and TIC (translocon on the inner chloroplast membrane) machineries in the outer and inner envelope membranes, respectively. TOC mediates initial recognition of preproteins at the outer membrane and includes a core membrane channel, Toc75, and two receptor proteins, Toc33/34 and Toc159, each containing GTPase domains that control preprotein binding and translocation. Toc75 is predicted to have a ß-barrel fold consisting of an N-terminal intermembrane space (IMS) domain and a C-terminal 16-stranded ß-barrel domain. Here we report the crystal structure of the N-terminal IMS domain of Toc75 from Arabidopsis thaliana, revealing three tandem polypeptide transport-associated (POTRA) domains, with POTRA2 containing an additional elongated helix not observed previously in other POTRA domains. Functional studies show an interaction with the preprotein, preSSU, which is mediated through POTRA2-3. POTRA2-3 also was found to have chaperone-like activity in an insulin aggregation assay, which we propose facilitates preprotein import. Our data suggest a model in which the POTRA domains serve as a binding site for the preprotein as it emerges from the Toc75 channel and provide a chaperone-like activity to prevent misfolding or aggregation as the preprotein traverses the intermembrane space.

V-1 regulates capping protein activity in vivo.

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."

Fixation-resistant photoactivatable fluorescent proteins for CLEM.

Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.

Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges.

Although capping protein (CP) terminates actin filament elongation, it promotes Arp2/3-dependent actin network assembly and accelerates actin-based motility both in vitro and in vivo. In vitro, capping protein Arp2/3 myosin I linker (CARMIL) antagonizes CP by reducing its affinity for the barbed end and by uncapping CP-capped filaments, whereas the protein V-1/myotrophin sequesters CP in an inactive complex. Previous work showed that CARMIL can readily retrieve CP from the CP:V-1 complex, thereby converting inactive CP into a version with moderate affinity for the barbed end. Here we further clarify the mechanism of this exchange reaction, and we demonstrate that the CP:CARMIL complex created by complex exchange slows the rate of barbed-end elongation by rapidly associating with, and dissociating from, the barbed end. Importantly, the cellular concentrations of V-1 and CP determined here argue that most CP is sequestered by V-1 at steady state in vivo. Finally, we show that CARMIL is recruited to the plasma membrane and only at cell edges undergoing active protrusion. Assuming that CARMIL is active only at this location, our data argue that a large pool of freely diffusing, inactive CP (CP:V-1) feeds, via CARMIL-driven complex exchange, the formation of weak-capping complexes (CP:CARMIL) at the plasma membrane of protruding edges. In vivo, therefore, CARMIL should promote Arp2/3-dependent actin network assembly at the leading edge by promoting barbed-end capping there.

Nonenzymatic conversion of ADP-ribosylated arginines to ornithine alters the biological activities of human neutrophil peptide-1.

Activated neutrophils, recruited to the airway of diseased lung, release human neutrophil peptides (HNP1-4) that are cytotoxic to airway cells as well as microbes. Airway epithelial cells express arginine-specific ADP ribosyltransferase (ART)-1, a GPI-anchored ART that transfers ADP-ribose from NAD to arginines 14 and 24 of HNP-1. We previously reported that ADP-ribosyl-arginine is converted nonenzymatically to ornithine and that ADP-ribosylated HNP-1 and ADP-ribosyl-HNP-(ornithine) were isolated from bronchoalveolar lavage fluid of a patient with idiopathic pulmonary fibrosis, indicating that these reactions occur in vivo. To determine effects of HNP-ornithine on the airway, three analogs of HNP-1, HNP-(R14orn), HNP-(R24orn), and HNP-(R14,24orn), were tested for their activity against Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus; their cytotoxic effects on A549, NCI-H441, small airway epithelial-like cells, and normal human lung fibroblasts; and their ability to stimulate IL-8 and TGF-ß1 release from A549 cells, and to serve as ART1 substrates. HNP and the three analogs had similar effects on IL-8 and TGF-ß1 release from A549 cells and were all cytotoxic for small airway epithelial cells, NCI-H441, and normal human lung fibroblasts. HNP-(R14,24orn), when compared with HNP-1 and HNP-1 with a single ornithine substitution for arginine 14 or 24, exhibited reduced cytotoxicity, but it enhanced proliferation of A549 cells and had antibacterial activity. Thus, arginines 14 and 24, which can be ADP ribosylated by ART1, are critical to the regulation of the cytotoxic and antibacterial effects of HNP-1. The HNP analog, HNP-(R14,24orn), lacks the epithelial cell cytotoxicity of HNP-1, but partially retains its antibacterial activity and thus may have clinical applications in airway disease.

SEDPHAT--a platform for global ITC analysis and global multi-method analysis of molecular interactions.

Isothermal titration calorimetry experiments can provide significantly more detailed information about molecular interactions when combined in global analysis. For example, global analysis can improve the precision of binding affinity and enthalpy, and of possible linkage parameters, even for simple bimolecular interactions, and greatly facilitate the study of multi-site and multi-component systems with competition or cooperativity. A pre-requisite for global analysis is the departure from the traditional binding model, including an 'n'-value describing unphysical, non-integral numbers of sites. Instead, concentration correction factors can be introduced to account for either errors in the concentration determination or for the presence of inactive fractions of material. SEDPHAT is a computer program that embeds these ideas and provides a graphical user interface for the seamless combination of biophysical experiments to be globally modeled with a large number of different binding models. It offers statistical tools for the rigorous determination of parameter errors, correlations, as well as advanced statistical functions for global ITC (gITC) and global multi-method analysis (GMMA). SEDPHAT will also take full advantage of error bars of individual titration data points determined with the unbiased integration software NITPIC. The present communication reviews principles and strategies of global analysis for ITC and its extension to GMMA in SEDPHAT. We will also introduce a new graphical tool for aiding experimental design by surveying the concentration space and generating simulated data sets, which can be subsequently statistically examined for their information content. This procedure can replace the 'c'-value as an experimental design parameter, which ceases to be helpful for multi-site systems and in the context of gITC.

Structure of transmembrane domain of lysosome-associated membrane protein type 2a (LAMP-2A) reveals key features for substrate specificity in chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.

Dissociation of glucocerebrosidase dimer in solution by its co-factor, saposin C.

Mutations in the gene for the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease and are the most common risk factor for Parkinson disease (PD). Analytical ultracentrifugation of 8 µM GCase shows equilibrium between monomer and dimer forms. However, in the presence of its co-factor saposin C (Sap C), only monomer GCase is seen. Isothermal calorimetry confirms that Sap C associates with GCase in solution in a 1:1 complex (Kd = 2.1 ± 1.1 µM). Saturation cross-transfer NMR determined that the region of Sap C contacting GCase includes residues 63-66 and 74-76, which is distinct from the region known to enhance GCase activity. Because α-synuclein (α-syn), a protein closely associated with PD etiology, competes with Sap C for GCase binding, its interaction with GCase was also measured by ultracentrifugation and saturation cross-transfer. Unlike Sap C, binding of α-syn to GCase does not affect multimerization. However, adding α-syn reduces saturation cross-transfer from Sap C to GCase, confirming displacement. To explore where Sap C might disrupt multimeric GCase, GCase x-ray structures were analyzed using the program PISA, which predicted stable dimer and tetramer forms. For the most frequently predicted multimer interface, the GCase active sites are partially buried, suggesting that Sap C might disrupt the multimer by binding near the active site.