Amino-terminally elongated Aß peptides are generated by the secreted metalloprotease ADAMTS4 and deposit in a subset of Alzheimer's disease brains.

AIMS: The aggregation and deposition of amyloid-ß (Aß) peptides in the brain is thought to be the initial driver in the pathogenesis of Alzheimer's disease (AD). Aside from full-length Aß peptides starting with an aspartate residue in position 1, both N-terminally truncated and elongated Aß peptides are produced by various proteases from the amyloid precursor protein (APP) and have been detected in brain tissues and body fluids. Recently, we demonstrated that the particularly abundant N-terminally truncated Aß4-x peptides are generated by ADAMTS4, a secreted metalloprotease that is exclusively expressed in the oligodendrocyte cell population. In this study, we investigated whether ADAMTS4 might also be involved in the generation of N-terminally elongated Aß peptides. METHODS: We used cell-free and cell-based assays in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) and electrochemiluminescence sandwich immunoassays to identify and quantify N-terminally elongated Aß peptide variants. Antibodies against these Aß variants were characterised by peptide microarrays and employed for the immunohistochemical analyses of human brain samples. RESULTS: In this study, we discovered additional ADAMTS4 cleavage sites in APP. These were located N-terminal to Asp-(1) in the Aß peptide sequence between residues Glu-(-7) and Ile-(-6) as well as Glu-(-4) and Val-(-3), resulting in the release of N-terminally elongated Aß-6-x and Aß-3-x peptides, of which the latter serve as a component in a promising Aß-based plasma biomarker. Aß-6/-3-40 peptides were detected in supernatants of various cell lines and in the cerebrospinal fluid (CSF), and ADAMTS4 enzyme activity promoted the release of Aß-6/-3-x peptides. Furthermore, by immunohistochemistry, a subset of AD cases displayed evidence of extracellular and vascular localization of N-terminally elongated Aß-6/-3-x peptides. DISCUSSION: The current findings implicate ADAMTS4 in both the pathological process of Aß peptide aggregation and in the early detection of amyloid pathology in AD.

The MYCN oncoprotein is an RNA-binding accessory factor of the nuclear exosome targeting complex.

The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA in vitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN's ability to activate cell growth-related genes but is required for MYCN's ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.

Molecular dissection of an immunodominant epitope in Kv1.2-exclusive autoimmunity.

Introduction: Subgroups of autoantibodies directed against voltage-gated potassium channel (Kv) complex components have been associated with immunotherapy-responsive clinical syndromes. The high prevalence and the role of autoantibodies directly binding Kv remain, however, controversial. Our objective was to determine Kv autoantibody binding requirements and to clarify their contribution to the observed immune response. Methods: Binding epitopes were studied in sera (n = 36) and cerebrospinal fluid (CSF) (n = 12) from a patient cohort positive for Kv1.2 but negative for 32 common neurological autoantigens and controls (sera n = 18 and CSF n = 5) by phospho and deep mutational scans. Autoantibody specificity and contribution to the observed immune response were resolved on recombinant cells, cerebellum slices, and nerve fibers. Results: 83% of the patients (30/36) within the studied cohort shared one out of the two major binding epitopes with Kv1.2-3 reactivity. Eleven percent (4/36) of the serum samples showed no binding. Fingerprinting resolved close to identical sequence requirements for both shared epitopes. Kv autoantibody response is directed against juxtaparanodal regions in peripheral nerves and the axon initial segment in central nervous system neurons and exclusively mediated by the shared epitopes. Discussion: Systematic mapping revealed two shared autoimmune responses, with one dominant Kv1.2-3 autoantibody epitope being unexpectedly prevalent. The conservation of the molecular binding requirements among these patients indicates a uniform autoantibody repertoire with monospecific reactivity. The enhanced sensitivity of the epitope-based (10/12) compared with that of the cell-based detection (7/12) highlights its use for detection. The determined immunodominant epitope is also the primary immune response visible in tissue, suggesting a diagnostic significance and a specific value for routine screening.

CD200+ fibroblasts form a pro-resolving mesenchymal network in arthritis.

Fibroblasts are important regulators of inflammation, but whether fibroblasts change phenotype during resolution of inflammation is not clear. Here we use positron emission tomography to detect fibroblast activation protein (FAP) as a means to visualize fibroblast activation in vivo during inflammation in humans. While tracer accumulation is high in active arthritis, it decreases after tumor necrosis factor and interleukin-17A inhibition. Biopsy-based single-cell RNA-sequencing analyses in experimental arthritis show that FAP signal reduction reflects a phenotypic switch from pro-inflammatory MMP3+/IL6+ fibroblasts (high FAP internalization) to pro-resolving CD200+DKK3+ fibroblasts (low FAP internalization). Spatial transcriptomics of human joints indicates that pro-resolving niches of CD200+DKK3+ fibroblasts cluster with type 2 innate lymphoid cells, whereas MMP3+/IL6+ fibroblasts colocalize with inflammatory immune cells. CD200+DKK3+ fibroblasts stabilized the type 2 innate lymphoid cell phenotype and induced resolution of arthritis via CD200-CD200R1 signaling. Taken together, these data suggest a dynamic molecular regulation of the mesenchymal compartment during resolution of inflammation.

KCNA2 IgG autoimmunity in neuropsychiatric diseases.

BACKGROUND: Autoantibodies against the potassium voltage-gated channel subfamily A member 2 (KCNA2) have been described in a few cases of neuropsychiatric disorders, but their diagnostic and pathophysiological role is currently unknown, imposing challenges to medical practice. DESIGN / METHODS: We retrospectively collected comprehensive clinical and paraclinical data of 35 patients with KCNA2 IgG autoantibodies detected in cell-based and tissue-based assays. Patients' sera and cerebrospinal fluid (CSF) were used for characterization of the antigen, clinical-serological correlations, and determination of IgG subclasses. RESULTS: KCNA2 autoantibody-positive patients (n = 35, median age at disease onset of 65 years, range of 16-83 years, 74 % male) mostly presented with cognitive impairment and/or epileptic seizures but also ataxia, gait disorder and personality changes. Serum autoantibodies belonged to IgG3 and IgG1 subclasses and titers ranged from 1:32 to 1:10,000. KCNA2 IgG was found in the CSF of 8/21 (38 %) patients and in the serum of 4/96 (4.2 %) healthy blood donors. KCNA2 autoantibodies bound to characteristic anatomical areas in the cerebellum and hippocampus of mammalian brain and juxtaparanodal regions of peripheral nerves but reacted exclusively with intracellular epitopes. A subset of four KCNA2 autoantibody-positive patients responded markedly to immunotherapy alongside with conversion to seronegativity, in particular those presenting an autoimmune encephalitis phenotype and receiving early immunotherapy. An available brain biopsy showed strong immune cell invasion. KCNA2 autoantibodies occurred in less than 10 % in association with an underlying tumor. CONCLUSION: Our data suggest that KCNA2 autoimmunity is clinically heterogeneous. Future studies should determine whether KCNA2 autoantibodies are directly pathogenic or develop secondarily. Early immunotherapy should be considered, in particular if autoantibodies occur in CSF or if clinical or diagnostic findings suggest ongoing inflammation. Suspicious clinical phenotypes include autoimmune encephalitis, atypical dementia, new-onset epilepsy and unexplained epileptic seizures.

MARTin-an open-source platform for microarray analysis.

Background: Microarray technology has brought significant advancements to high-throughput analysis, particularly in the comprehensive study of biomolecular interactions involving proteins, peptides, and antibodies, as well as in the fields of gene expression and genotyping. With the ever-increasing volume and intricacy of microarray data, an accurate, reliable and reproducible analysis is essential. Furthermore, there is a high level of variation in the format of microarrays. This not only holds true between different sample types but is also due to differences in the hardware used during the production of the arrays, as well as the personal preferences of the individual users. Therefore, there is a need for transparent, broadly applicable and user-friendly image quantification techniques to extract meaningful information from these complex datasets, while also addressing the challenges posed by specific microarray and imager formats, which can flaw analysis and interpretation. Results: Here we introduce MicroArray Rastering Tool (MARTin), as a versatile tool developed primarily for the analysis of protein and peptide microarrays. Our software provides state-of-the-art methodologies, offering researchers a comprehensive tool for microarray image quantification. MARTin is independent of the microarray platform used and supports various configurations including high-density formats and printed arrays with significant x and y offsets. This is made possible by granting the user the ability to freely customize parts of the application to their specific microarray format. Thanks to built-in features like adaptive filtering and autofit, measurements can be done very efficiently and are highly reproducible. Furthermore, our tool integrates metadata management and integrity check features, providing a straightforward quality control method, along with a ready-to-use interface for in-depth data analysis. This not only promotes good scientific practice in the field of microarray analysis but also enhances the ability to explore and examine the generated data. Conclusion: MARTin has been developed to empower its users with a reliable, efficient, and intuitive tool for peptidomic and proteomic array analysis, thereby facilitating data-driven discovery across disciplines. Our software is an open-source project freely available via the GNU Affero General Public License licence on GitHub.

Inhibition of the YAP-MMB interaction and targeting NEK2 as potential therapeutic strategies for YAP-driven cancers.

YAP activation in cancer is linked to poor outcomes, making it an attractive therapeutic target. Previous research focused on blocking the interaction of YAP with TEAD transcription factors. Here, we took a different approach by disrupting YAP's binding to the transcription factor B-MYB using MY-COMP, a fragment of B-MYB containing the YAP binding domain fused to a nuclear localization signal. MY-COMP induced cell cycle defects, nuclear abnormalities, and polyploidization. In an AKT and YAP-driven liver cancer model, MY-COMP significantly reduced liver tumorigenesis, highlighting the importance of the YAP-B-MYB interaction in tumor development. MY-COMP also perturbed the cell cycle progression of YAP-dependent uveal melanoma cells but not of YAP-independent cutaneous melanoma cell lines. It counteracted YAP-dependent expression of MMB-regulated cell cycle genes, explaining the observed effects. We also identified NIMA-related kinase (NEK2) as a downstream target of YAP and B-MYB, promoting YAP-driven transformation by facilitating centrosome clustering and inhibiting multipolar mitosis.

Glycine Receptor ß-Targeting Autoantibodies Contribute to the Pathology of Autoimmune Diseases.

BACKGROUND AND OBJECTIVES: Stiff-person syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM) are rare neurologic disorders of the CNS. Until now, exclusive GlyRα subunit-binding autoantibodies with subsequent changes in function and surface numbers were reported. GlyR autoantibodies have also been described in patients with focal epilepsy. Autoimmune reactivity against the GlyRß subunits has not yet been shown. Autoantibodies against GlyRα1 target the large extracellular N-terminal domain. This domain shares a high degree of sequence homology with GlyRß making it not unlikely that GlyRß-specific autoantibody (aAb) exist and contribute to the disease pathology. METHODS: In this study, we investigated serum samples from 58 patients for aAb specifically detecting GlyRß. Studies in microarray format, cell-based assays, and primary spinal cord neurons and spinal cord tissue immunohistochemistry were performed to determine specific GlyRß binding and define aAb binding to distinct protein regions. Preadsorption approaches of aAbs using living cells and the purified extracellular receptor domain were further used. Finally, functional consequences for inhibitory neurotransmission upon GlyRß aAb binding were resolved by whole-cell patch-clamp recordings. RESULTS: Among 58 samples investigated, cell-based assays, tissue analysis, and preadsorption approaches revealed 2 patients with high specificity for GlyRß aAb. Quantitative protein cluster analysis demonstrated aAb binding to synaptic GlyRß colocalized with the scaffold protein gephyrin independent of the presence of GlyRα1. At the functional level, binding of GlyRß aAb from both patients to its target impair glycine efficacy. DISCUSSION: Our study establishes GlyRß as novel target of aAb in patients with SPS/PERM. In contrast to exclusively GlyRα1-positive sera, which alter glycine potency, aAbs against GlyRß impair receptor efficacy for the neurotransmitter glycine. Imaging and functional analyses showed that GlyRß aAbs antagonize inhibitory neurotransmission by affecting receptor function rather than localization.

Synapsin autoantibodies during pregnancy are associated with fetal abnormalities.

Anti-neuronal autoantibodies can be transplacentally transferred during pregnancy and may cause detrimental effects on fetal development. It is unclear whether autoantibodies against synapsin-I, one of the most abundant synaptic proteins, are associated with developmental abnormalities in humans. We recruited a cohort of 263 pregnant women and detected serum synapsin-I IgG autoantibodies in 13.3% using cell-based assays. Seropositivity was strongly associated with abnormalities of fetal development including structural defects, intrauterine growth retardation, amniotic fluid disorders and neuropsychiatric developmental diseases in previous children (odds ratios of 3-6.5). Autoantibodies reached the fetal circulation and were mainly of IgG1/IgG3 subclasses. They bound to conformational and linear synapsin-I epitopes, five distinct epitopes were identified using peptide microarrays. The findings indicate that synapsin-I autoantibodies may be clinically useful biomarkers or even directly participate in the disease process of neurodevelopmental disorders, thus being potentially amenable to antibody-targeting interventional strategies in the future.

Molecular characterization of AIFM2/FSP1 inhibition by iFSP1-like molecules.

Ferroptosis is a form of cell death characterized by phospholipid peroxidation, where numerous studies have suggested that the induction of ferroptosis is a therapeutic strategy to target therapy refractory cancer entities. Ferroptosis suppressor protein 1 (FSP1), an NAD(P)H-ubiquinone reductase, is a key determinant of ferroptosis vulnerability, and its pharmacological inhibition was shown to strongly sensitize cancer cells to ferroptosis. A first generation of FSP1 inhibitors, exemplified by the small molecule iFSP1, has been reported; however, the molecular mechanisms underlying inhibition have not been characterized in detail. In this study, we explore the species-specific inhibition of iFSP1 on the human isoform to gain insights into its mechanism of action. Using a combination of cellular, biochemical, and computational methods, we establish a critical contribution of a species-specific aromatic architecture that is essential for target engagement. The results described here provide valuable insights for the rational development of second-generation FSP1 inhibitors combined with a tracer for screening the druggable pocket. In addition, we pose a cautionary notice for using iFSP1 in animal models, specifically murine models.

Filamin A organizes γ­aminobutyric acid type B receptors at the plasma membrane.

The γ-aminobutyric acid type B (GABAB) receptor is a prototypical family C G protein-coupled receptor (GPCR) that plays a key role in the regulation of synaptic transmission. Although growing evidence suggests that GPCR signaling in neurons might be highly organized in time and space, limited information is available about the mechanisms controlling the nanoscale organization of GABAB receptors and other GPCRs on the neuronal plasma membrane. Using a combination of biochemical assays in vitro, single-particle tracking, and super-resolution microscopy, we provide evidence that the spatial organization and diffusion of GABAB receptors on the plasma membrane are governed by dynamic interactions with filamin A, which tethers the receptors to sub-cortical actin filaments. We further show that GABAB receptors are located together with filamin A in small nanodomains in hippocampal neurons. These interactions are mediated by the first intracellular loop of the GABAB1 subunit and modulate the kinetics of Gαi protein activation in response to GABA stimulation.

Peptide Microarrays for Studying Autoantibodies in Neurological Disease.

Antibody-mediated neurological diseases constitute an emerging clinical entity that remains to be fully explored. Recent studies identified autoantibodies that directly confer pathogenicity, and it was shown that in these cases immunotherapies can result in profound positive patient responses. These advances highlight the urgent need for improved means to effectively screen patient samples for novel autoantibodies (aAbs) and their subsequent characterization. Here, we discuss challenges and opportunities for peptide microarrays to contribute to the identification, mapping, and characterization of the underlying monospecific disease-defining binding surfaces. We outline control experiments, workflow modifications and bioinformatic filtering methods that enhance the predictive power of array-based studies. Further, we highlight experimental and computer-based display approaches that have the potential to expand the use of synthetic microarrays over the detection of discontinuous epitopes. Knowledge over the autoantibody epitopes in neurological disease will enhance our understanding of the pathological mechanisms and thereby potentially contribute to novel diagnostic approaches or even innovative antigen-specific treatments that avoid the serious adverse effects seen with currently used immunosuppressive therapies.

Peptide Microarray-Based Protein Interaction Studies Across Affinity Ranges: Enzyme Stalling, Cross-Linking, Depletion, and Neutralization.

While an ever-increasing number of protein-protein interactions were studied by peptide microarrays with great success, array-based investigations of transiently binding proteins, such as HDACs, and precise binding quantification, remained challenging. Here, we present an updated protocol for the preparation and use of peptide microarrays including the necessary adjustments for simple semi-quantitative and precise measurements across affinity ranges. This procedure describes the mass spectrometric controlled preparation of peptide microarrays in µSPOT format, and their application in binding profiling of recombinant, as well as endogenous, native proteins. We further highlight how cross-linking, blocking, and enzyme stalling can be leveraged to enhance sensitivity and describe how in situ on-chip binding neutralization can enhance the predictive value and robustness of the binding readout. Finally, we included examples for the integration of precise biophysical binding readouts that complement the traditional array-based binding assays.

MYC multimers shield stalled replication forks from RNA polymerase.

Oncoproteins of the MYC family drive the development of numerous human tumours1. In unperturbed cells, MYC proteins bind to nearly all active promoters and control transcription by RNA polymerase II2,3. MYC proteins can also coordinate transcription with DNA replication4,5 and promote the repair of transcription-associated DNA damage6, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks7,8. MYC multimerization is triggered in a HUWE16 and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double strand break formation during S-phase, suggesting that the multimerization of MYC enables tumour cells to proliferate under stressful conditions.

A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening.

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.

Multivalent binding kinetics resolved by fluorescence proximity sensing.

Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors.

Complex regulation of Gephyrin splicing is a determinant of inhibitory postsynaptic diversity.

Gephyrin (GPHN) regulates the clustering of postsynaptic components at inhibitory synapses and is involved in pathophysiology of neuropsychiatric disorders. Here, we uncover an extensive diversity of GPHN transcripts that are tightly controlled by splicing during mouse and human brain development. Proteomic analysis reveals at least a hundred isoforms of GPHN incorporated at inhibitory Glycine and gamma-aminobutyric acid A receptors containing synapses. They exhibit different localization and postsynaptic clustering properties, and altering the expression level of one isoform is sufficient to affect the number, size, and density of inhibitory synapses in cerebellar Purkinje cells. Furthermore, we discovered that splicing defects reported in neuropsychiatric disorders are carried by multiple alternative GPHN transcripts, demonstrating the need for a thorough analysis of the GPHN transcriptome in patients. Overall, we show that alternative splicing of GPHN is an important genetic variation to consider in neurological diseases and a determinant of the diversity of postsynaptic inhibitory synapses.

A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity.

Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding "Sylite"-a probe with outstanding signal-to-background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super-resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.

Low-cost synthesis of peptide libraries and their use for binding studies via temperature-related intensity change.

Protein-peptide interactions are involved in many fundamental cellular functions and constitute promising drug targets. Here, we provide a detailed protocol for the cost-effective preparation of a cellulose-based solid support for synthesis of nanoscale to micromolar-scale peptide libraries. Their subsequent use for high-throughput protein interaction screening as well as affinity determination in solution provides binding data for thousands of unique peptides with a turnover of 1 to 2 weeks, thereby facilitating in vitro assessment and development of high-affinity binders. For complete details on the use and execution of this protocol, please refer to Schulte et al., (2020).

Conformational Plasticity of Hepatitis B Core Protein Spikes Promotes Peptide Binding Independent of the Secretion Phenotype.

Hepatitis B virus is a major human pathogen, which forms enveloped virus particles. During viral maturation, membrane-bound hepatitis B surface proteins package hepatitis B core protein capsids. This process is intercepted by certain peptides with an "LLGRMKG" motif that binds to the capsids at the tips of dimeric spikes. With microcalorimetry, electron cryo microscopy and peptide microarray-based screens, we have characterized the structural and thermodynamic properties of peptide binding to hepatitis B core protein capsids with different secretion phenotypes. The peptide "GSLLGRMKGA" binds weakly to hepatitis B core protein capsids and mutant capsids with a premature (F97L) or low-secretion phenotype (L60V and P5T). With electron cryo microscopy, we provide novel structures for L60V and P5T and demonstrate that binding occurs at the tips of the spikes at the dimer interface, splaying the helices apart independent of the secretion phenotype. Peptide array screening identifies "SLLGRM" as the core binding motif. This shortened motif binds only to one of the two spikes in the asymmetric unit of the capsid and induces a much smaller conformational change. Altogether, these comprehensive studies suggest that the tips of the spikes act as an autonomous binding platform that is unaffected by mutations that affect secretion phenotypes.