Structure of Merkel Cell Polyomavirus Capsid and Interaction with Its Glycosaminoglycan Attachment Receptor.

Merkel cell polyomavirus (MCPyV) is a human double-stranded DNA tumor virus. MCPyV cell entry is unique among members of the polyomavirus family as it requires the engagement of two types of glycans, sialylated oligosaccharides and sulfated glycosaminoglycans (GAGs). Here, we present crystallographic and cryo-electron microscopic structures of the icosahedral MCPyV capsid and analysis of its glycan interactions via nuclear magnetic resonance (NMR) spectroscopy. While sialic acid binding is specific for α2-3-linked sialic acid and mediated by the exposed apical loops of the major capsid protein VP1, a broad range of GAG oligosaccharides bind to recessed regions between VP1 capsomers. Individual VP1 capsomers are tethered to one another by an extensive disulfide network that differs in architecture from previously described interactions for other PyVs. An unusual C-terminal extension in MCPyV VP1 projects from the recessed capsid regions. Mutagenesis experiments show that this extension is dispensable for receptor interactions.IMPORTANCE The MCPyV genome was found to be clonally integrated in 80% of cases of Merkel cell carcinoma (MCC), a rare but aggressive form of human skin cancer, strongly suggesting that this virus is tumorigenic. In the metastasizing state, the course of the disease is often fatal, especially in immunocompromised individuals, as reflected by the high mortality rate of 33 to 46% and the low 5-year survival rate (<45%). The high seroprevalence of about 60% makes MCPyV a serious health care burden and illustrates the need for targeted treatments. In this study, we present the first high-resolution structural data for this human tumor virus and demonstrate that the full capsid is required for the essential interaction with its GAG receptor(s). Together, these data can be used as a basis for future strategies in drug development.

Structural Basis and Evolution of Glycan Receptor Specificities within the Polyomavirus Family.

Asymptomatic infections with polyomaviruses in humans are common, but these small viruses can cause severe diseases in immunocompromised hosts. New Jersey polyomavirus (NJPyV) was identified via a muscle biopsy in an organ transplant recipient with systemic vasculitis, myositis, and retinal blindness, and human polyomavirus 12 (HPyV12) was detected in human liver tissue. The evolutionary origins and potential diseases are not well understood for either virus. In order to define their receptor engagement strategies, we first used nuclear magnetic resonance (NMR) spectroscopy to establish that the major capsid proteins (VP1) of both viruses bind to sialic acid in solution. We then solved crystal structures of NJPyV and HPyV12 VP1 alone and in complex with sialylated glycans. NJPyV employs a novel binding site for a α2,3-linked sialic acid, whereas HPyV12 engages terminal α2,3- or α2,6-linked sialic acids in an exposed site similar to that found in Trichodysplasia spinulosa-associated polyomavirus (TSPyV). Gangliosides or glycoproteins, featuring in mammals usually terminal sialic acids, are therefore receptor candidates for both viruses. Structural analyses show that the sialic acid-binding site of NJPyV is conserved in chimpanzee polyomavirus (ChPyV) and that the sialic acid-binding site of HPyV12 is widely used across the entire polyomavirus family, including mammalian and avian polyomaviruses. A comparison with other polyomavirus-receptor complex structures shows that their capsids have evolved to generate several physically distinct virus-specific receptor-binding sites that can all specifically engage sialylated glycans through a limited number of contacts. Small changes in each site may have enabled host-switching events during the evolution of polyomaviruses.IMPORTANCE Virus attachment to cell surface receptors is critical for productive infection. In this study, we have used a structure-based approach to investigate the cell surface recognition event for New Jersey polyomavirus (NJPyV) and human polyomavirus 12 (HPyV12). These viruses belong to the polyomavirus family, whose members target different tissues and hosts, including mammals, birds, fish, and invertebrates. Polyomaviruses are nonenveloped viruses, and the receptor-binding site is located in their capsid protein VP1. The NJPyV capsid features a novel sialic acid-binding site that is shifted in comparison to other structurally characterized polyomaviruses but shared with a closely related simian virus. In contrast, HPyV12 VP1 engages terminal sialic acids in a manner similar to the human Trichodysplasia spinulosa-associated polyomavirus. Our structure-based phylogenetic analysis highlights that even distantly related avian polyomaviruses possess the same exposed sialic acid-binding site. These findings complement phylogenetic models of host-virus codivergence and may also reflect past host-switching events.

A post-translational modification of human Norovirus capsid protein attenuates glycan binding.

Attachment of human noroviruses to histo blood group antigens (HBGAs) is essential for infection, but how this binding event promotes the infection of host cells is unknown. Here, we employ protein NMR experiments supported by mass spectrometry and crystallography to study HBGA binding to the P-domain of a prevalent virus strain (GII.4). We report a highly selective transformation of asparagine 373, located in an antigenic loop adjoining the HBGA binding site, into an iso-aspartate residue. This spontaneous post-translational modification (PTM) proceeds with an estimated half-life of a few days at physiological temperatures, independent of the presence of HBGAs but dramatically affecting HBGA recognition. Sequence conservation and the surface-exposed position of this PTM suggest an important role in infection and immune recognition for many norovirus strains.

Divalent Sialylated Precision Glycooligomers Binding to Polyomaviruses and the Effect of Different Linkers.

Divalent precision glycooligomers terminating in N-acetylneuraminic acid (Neu5Ac) or 3'-sialyllactose (3'-SL) with varying linkers between scaffold and the glycan portions are synthesized via solid phase synthesis for co-crystallization studies with the sialic acid-binding major capsid protein VP1 of human Trichodysplasia spinulosa-associated Polyomavirus. High-resolution crystal structures of complexes demonstrate that the compounds bind to VP1 depending on the favorable combination of carbohydrate ligand and linker. It is found that artificial linkers can replace portions of natural carbohydrate linkers as long as they meet certain requirements such as size or flexibility to optimize contact area between ligand and receptor binding sites. The obtained results will influence the design of future high affinity ligands based on the structures presented here, and they can serve as a blueprint to develop multivalent glycooligomers as inhibitors of viral adhesion.

Sialic Acids in Nonenveloped Virus Infections.

Sialic acid-based glycoconjugates cover the surfaces of many different cell types, defining key properties of the cell surface such as overall charge or likely interaction partners. Because of this prominence, sialic acids play prominent roles in mediating attachment and entry to viruses belonging to many different families. In this review, we first describe how interactions between viruses and sialic acid-based glycan structures can be identified and characterized using a range of techniques. We then highlight interactions between sialic acids and virus capsid proteins in four different viruses, and discuss what these interactions have taught us about sialic acid engagement and opportunities to interfere with binding.

Infectious Entry of Merkel Cell Polyomavirus.

Merkel cell polyomavirus (MCPyV) is a small, nonenveloped tumor virus associated with an aggressive form of skin cancer, Merkel cell carcinoma (MCC). MCPyV infections are highly prevalent in the human population, with MCPyV virions being continuously shed from human skin. However, the precise host cell tropism(s) of MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. However, MCPyV appears different from other polyomaviruses, as it requires sulfated polysaccharides, such as heparan sulfates and/or chondroitin sulfates, for initial attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co)receptor. To explore the infectious entry process of MCPyV, we analyzed the cell biological determinants of MCPyV entry into A549 cells, a highly transducible lung carcinoma cell line, in comparison to well-studied simian virus 40 and a number of other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis, or glycosphingolipid-enriched carriers. The viruses were internalized in small endocytic pits that led the virus to endosomes and from there to the endoplasmic reticulum (ER). Similar to other polyomaviruses, trafficking required microtubular transport, acidification of endosomes, and a functional redox environment. To our surprise, the virus was found to acquire a membrane envelope within endosomes, a phenomenon not reported for other viruses. Only minor amounts of viruses reached the ER, while the majority was retained in endosomal compartments, suggesting that endosome-to-ER trafficking is a bottleneck during infectious entry.IMPORTANCE MCPyV is the first polyomavirus directly implicated in the development of an aggressive human cancer, Merkel cell carcinoma (MCC). Although MCPyV is constantly shed from healthy skin, the MCC incidence increases among aging and immunocompromised individuals. To date, the events connecting initial MCPyV infection and subsequent transformation still remain elusive. MCPyV differs from other known polyomaviruses concerning its cell tropism, entry receptor requirements, and infection kinetics. In this study, we examined the cellular requirements for endocytic entry as well as the subcellular localization of incoming virus particles. A thorough understanding of the determinants of the infectious entry pathway and the specific biological niche will benefit prevention of virus-derived cancers such as MCC.

Structural Optimization of a Pyridinylimidazole Scaffold: Shifting the Selectivity from p38α Mitogen-Activated Protein Kinase to c-Jun N-Terminal Kinase 3.

Starting from known p38α mitogen-activated protein kinase (MAPK) inhibitors, a series of inhibitors of the c-Jun N-terminal kinase (JNK) 3 was obtained. Altering the substitution pattern of the pyridinylimidazole scaffold proved to be effective in shifting the inhibitory activity from the original target p38α MAPK to the closely related JNK3. In particular, a significant improvement for JNK3 selectivity could be achieved by addressing the hydrophobic region I with a small methyl group. Furthermore, additional structural modifications permitted to explore structure-activity relationships. The most potent inhibitor 4-(4-methyl-2-(methylthio)-1H-imidazol-5-yl)-N-(4-morpholinophenyl)pyridin-2-amine showed an IC50 value for the JNK3 in the low triple digit nanomolar range and its binding mode was confirmed by X-ray crystallography.

Spin ballet for sweet encounters: saturation-transfer difference NMR and X-ray crystallography complement each other in the elucidation of protein-glycan interactions.

Biomolecular NMR spectroscopy has limitations in the determination of protein structures: an inherent size limit and the requirement for expensive and potentially difficult isotope labelling pose considerable hurdles. Therefore, structural analysis of larger proteins is almost exclusively performed by crystallography. However, the diversity of biological NMR applications outperforms that of any other structural biology technique. For the characterization of transient complexes formed by proteins and small ligands, notably oligosaccharides, one NMR technique has recently proven to be particularly powerful: saturation-transfer difference NMR (STD-NMR) spectroscopy. STD-NMR experiments are fast and simple to set up, with no general protein size limit and no requirement for isotope labelling. The method performs best in the moderate-to-low affinity range that is of interest in most of glycobiology. With small amounts of unlabelled protein, STD-NMR experiments can identify hits from mixtures of potential ligands, characterize mutant proteins and pinpoint binding epitopes on the ligand side. STD-NMR can thus be employed to complement and improve protein-ligand complex models obtained by other structural biology techniques or by purely computational means. With a set of protein-glycan interactions from our own work, this review provides an introduction to the technique for structural biologists. It exemplifies how crystallography and STD-NMR can be combined to elucidate protein-glycan (and other protein-ligand) interactions in atomic detail, and how the technique can extend structural biology from simplified systems amenable to crystallization to more complex biological entities such as membranes, live viruses or entire cells.

Synthesis of highly controlled carbohydrate-polymer based hybrid structures by combining heparin fragments and sialic acid derivatives, and solid phase polymer synthesis.

Heparin is a polymeric carbohydrate with a variety of biomedical applications that is particularly challenging from a synthetic point of view. Here, we present the synthesis of carbohydrate-polymer based hybrid structures by combining defined heparin fragments with monodisperse, sequence-controlled glycooligo(amidoamines) suitable as glycan mimetic model compounds of heparin as demonstrated by STD-NMR binding studies with viral capsids.

Biophysical analysis of sialic acid recognition by the complement regulator Factor H.

Complement factor H (FH), an elongated and substantially glycosylated 20-domain protein, is a soluble regulator of the complement alternative pathway (AP). It contains several glycan binding sites which mediate recognition of α2-3-linked sialic acid (FH domain 20) and glycosaminoglycans (domains 6-8 and 19-20). FH also binds the complement C3-activation product C3b, a powerful opsonin and focal point for the formation of C3-convertases of the AP feedback loop. In freely circulating FH the C3b binding site in domains 19-20 is occluded, a phenomenon that is not fully understood and could be mediated by an intramolecular interaction between FH's intrinsic sialylated glycosylation and its own sialic acid binding site. In order to assess this possibility, we characterized FH's sialylation with respect to glycosidic linkage type and searched for further potential, not yet characterized sialic acid binding sites in FH and its seven-domain spanning splice variant and fellow complement regulator FH like-1 (FHL-1). We also probed FH binding to the sialic acid variant Neu5Gc which is not expressed in humans but on heterologous erythrocytes that restrict the human AP and in FH transgenic mice. We find that FH contains mostly α2-6-linked sialic acid, making an intramolecular interaction with its α2-3-sialic acid specific binding site and an associated self-lock mechanism unlikely, substantiate that there is only a single sialic acid binding site in FH and none in FHL-1, and demonstrate direct binding of FH to the nonhuman sialic acid Neu5Gc, supporting the use of FH transgenic mouse models for studies of complement-related diseases.

Polysialic acid is a cellular receptor for human adenovirus 52.

Human adenovirus 52 (HAdV-52) is one of only three known HAdVs equipped with both a long and a short fiber protein. While the long fiber binds to the coxsackie and adenovirus receptor, the function of the short fiber in the virus life cycle is poorly understood. Here, we show, by glycan microarray analysis and cellular studies, that the short fiber knob (SFK) of HAdV-52 recognizes long chains of α-2,8-linked polysialic acid (polySia), a large posttranslational modification of selected carrier proteins, and that HAdV-52 can use polySia as a receptor on target cells. X-ray crystallography, NMR, molecular dynamics simulation, and structure-guided mutagenesis of the SFK reveal that the nonreducing, terminal sialic acid of polySia engages the protein with direct contacts, and that specificity for polySia is achieved through subtle, transient electrostatic interactions with additional sialic acid residues. In this study, we present a previously unrecognized role for polySia as a cellular receptor for a human viral pathogen. Our detailed analysis of the determinants of specificity for this interaction has general implications for protein-carbohydrate interactions, particularly concerning highly charged glycan structures, and provides interesting dimensions on the biology and evolution of members of Human mastadenovirus G.

The lectin self of complement factor H.

Complement, a part of the humoral innate immune system, is divided into three pathways. The classical and mannose-binding lectin pathways are triggered by specific recognition of foreign targets. Conversely, the alternative pathway (AP) is actively down-regulated on host tissue. Glycosaminoglycans (GAGs) and sialylated glycans mediate host recognition of the AP as self-associated molecular patterns (SAMPs) to the regulatory protein factor H (FH). This review summarizes the more recent years of research on SAMP recognition by FH from a structural biology point of view and discusses implications for two complement-associated conditions, age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (aHUS). Taking into account crystal structures that elucidated FH binding to a bacterial evasion protein and to the thioester domain of C3b, the target of FH-mediated AP restriction, a novel atomistic model for the mechanism by which FH prevents AP activation on self surfaces is proposed.

Complement Factor H and Simian Virus 40 bind the GM1 ganglioside in distinct conformations.

Mammalian cell surfaces are decorated with a variety of glycan chains that orchestrate development and defense and are exploited by pathogens for cellular attachment and entry. While glycosidic linkages are, in principle, flexible, the conformational space that a given glycan can sample is subject to spatial and electrostatic restrictions imposed by its overall chemical structure. Here, we show how the glycan moiety of the GM1 ganglioside, a branched, monosialylated pentasaccharide that serves as a ligand for various proteins, undergoes differential conformational selection in its interactions with different lectins. Using STD NMR and X-ray crystallography, we found that the innate immune regulator complement Factor H (FH) binds a previously not reported GM1 conformation that is not compatible with the GM1-binding sites of other structurally characterized GM1-binding lectins such as the Simian Virus 40 (SV40) capsid. Molecular dynamics simulations of the free glycan in explicit solvent on the 10 µs timescale reveal that the FH-bound conformation nevertheless corresponds to a minimum in the Gibbs free energy plot. In contrast to the GM1 conformation recognized by SV40, the FH-bound GM1 conformation is associated with poor NOE restraints, explaining how it escaped(1)H-(1)H NOE-restrained modeling in the past and highlighting the necessity for ensemble representations of glycan structures.

Trichodysplasia spinulosa-Associated Polyomavirus Uses a Displaced Binding Site on VP1 to Engage Sialylated Glycolipids.

Trichodysplasia spinulosa-associated Polyomavirus (TSPyV) was isolated from a patient suffering from trichodysplasia spinulosa, a skin disease that can appear in severely immunocompromised patients. While TSPyV is one of the five members of the polyomavirus family that are directly linked to a human disease, details about molecular recognition events, the viral entry pathway, and intracellular trafficking events during TSPyV infection remain unknown. Here we have used a structure-function approach to shed light on the first steps of TSPyV infection. We established by cell binding and pseudovirus infection studies that TSPyV interacts with sialic acids during attachment and/or entry. Subsequently, we solved high-resolution X-ray structures of the major capsid protein VP1 of TSPyV in complex with three different glycans, the branched GM1 glycan, and the linear trisaccharides α2,3- and α2,6-sialyllactose. The terminal sialic acid of all three glycans is engaged in a unique binding site on TSPyV VP1, which is positioned about 18 Å from established sialic acid binding sites of other polyomaviruses. Structure-based mutagenesis of sialic acid-binding residues leads to reduction in cell attachment and pseudovirus infection, demonstrating the physiological relevance of the TSPyV VP1-glycan interaction. Furthermore, treatments of cells with inhibitors of N-, O-linked glycosylation, and glycosphingolipid synthesis suggest that glycolipids play an important role during TSPyV infection. Our findings elucidate the first molecular recognition events of cellular infection with TSPyV and demonstrate that receptor recognition by polyomaviruses is highly variable not only in interactions with sialic acid itself, but also in the location of the binding site.

The Greater Affinity of JC Polyomavirus Capsid for α2,6-Linked Lactoseries Tetrasaccharide c than for Other Sialylated Glycans Is a Major Determinant of Infectivity.

UNLABELLED: The human JC polyomavirus (JCPyV) establishes an asymptomatic, persistent infection in the kidneys of the majority of the population and is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in immunosuppressed individuals. The Mad-1 strain of JCPyV, a brain isolate, was shown earlier to require α2,6-linked sialic acid on the lactoseries tetrasaccharide c (LSTc) glycan for attachment to host cells. In contrast, a JCPyV kidney isolate type 3 strain, WT3, has been reported to interact with sialic acid-containing gangliosides, but the role of these glycans in JCPyV infection has remained unclear. To help rationalize these findings and probe the effects of strain-specific differences on receptor binding, we performed a comprehensive analysis of the glycan receptor specificities of these two representative JCPyV strains using high-resolution X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, and correlated these data with the results of infectivity assays. We show here that capsid proteins of Mad-1 and WT3 JCPyV can both engage LSTc as well as multiple sialylated gangliosides. However, the binding affinities exhibit subtle differences, with the highest affinity observed for LSTc. Engagement of LSTc is a prerequisite for functional receptor engagement, while the more weakly binding gangliosides are not required for productive infection. Our findings highlight the complexity of virus-carbohydrate interactions and demonstrate that subtle differences in binding affinities, rather than the binding event alone, help determine tissue tropism and viral pathogenesis. IMPORTANCE: Viral infection is initiated by attachment to receptors on host cells, and this event plays an important role in viral disease. We investigated the receptor-binding properties of human JC polyomavirus (JCPyV), a virus that resides in the kidneys of the majority of the population and can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in the brains of immunosuppressed individuals. JCPyV has been reported to interact with multiple carbohydrate receptors, and we sought to clarify how the interactions between JCPyV and cellular carbohydrate receptors influenced infection. Here we demonstrate that JCPyV can engage numerous sialylated carbohydrate receptors. However, the virus displays preferential binding to LSTc, and only LSTc mediates a productive infection. Our findings demonstrate that subtle differences in binding affinity, rather than receptor engagement alone, are a key determinant of viral infection.

Glycan engagement dictates hydrocephalus induction by serotype 1 reovirus.

UNLABELLED: Receptors expressed on the host cell surface adhere viruses to target cells and serve as determinants of viral tropism. Several viruses bind cell surface glycans to facilitate entry, but the contribution of specific glycan moieties to viral disease is incompletely understood. Reovirus provides a tractable experimental model for studies of viral neuropathogenesis. In newborn mice, serotype 1 (T1) reovirus causes hydrocephalus, whereas serotype 3 (T3) reovirus causes encephalitis. T1 and T3 reoviruses engage distinct glycans, suggesting that glycan-binding capacity contributes to these differences in pathogenesis. Using structure-guided mutagenesis, we engineered a mutant T1 reovirus incapable of binding the T1 reovirus-specific glycan receptor, GM2. The mutant virus induced substantially less hydrocephalus than wild-type virus, an effect phenocopied by wild-type virus infection of GM2-deficient mice. In comparison to wild-type virus, yields of mutant virus were diminished in cultured ependymal cells, the cell type that lines the brain ventricles. These findings suggest that GM2 engagement targets reovirus to ependymal cells in mice and illuminate the function of glycan engagement in reovirus serotype-dependent disease. IMPORTANCE: Receptor utilization strongly influences viral disease, often dictating host range and target cell selection. Different reovirus serotypes bind to different glycans, but a precise function for these molecules in pathogenesis is unknown. We used type 1 (T1) reovirus deficient in binding the GM2 glycan and mice lacking GM2 to pinpoint a role for glycan engagement in hydrocephalus caused by T1 reovirus. This work indicates that engagement of a specific glycan can lead to infection of specific cells in the host and consequent disease at that site. Since reovirus is being developed as a vaccine vector and oncolytic agent, understanding reovirus-glycan interactions may allow manipulation of reovirus glycan-binding properties for therapeutic applications.

Structural basis for sialic acid-mediated self-recognition by complement factor H.

The serum protein complement factor H (FH) ensures downregulation of the complement alternative pathway, a branch of innate immunity, upon interaction with specific glycans on host cell surfaces. Using ligand-based NMR, we screened a comprehensive set of sialylated glycans for binding to FH and solved the crystal structure of a ternary complex formed by the two C-terminal domains of FH, a sialylated trisaccharide and the complement C3b thioester-containing domain. Key residues in the sialic acid binding site are conserved from mice to men, and residues linked to atypical hemolytic uremic syndrome cluster within this binding site, suggesting a possible role for sialic acid as a host marker also in other mammals and a critical role in human renal complement homeostasis. Unexpectedly, the FH sialic acid binding site is structurally homologous to the binding sites of two evolutionarily unrelated proteins. The crystal structure also advances our understanding of bacterial immune evasion strategies.

Structure analysis of the major capsid proteins of human polyomaviruses 6 and 7 reveals an obstructed sialic acid binding site.

UNLABELLED: Human polyomavirus 6 (HPyV6) and HPyV7 are commonly found on human skin. We have determined the X-ray structures of their major capsid protein, VP1, at resolutions of 1.8 and 1.7 Å, respectively. In polyomaviruses, VP1 commonly determines antigenicity as well as cell-surface receptor specificity, and the protein is therefore linked to attachment, tropism, and ultimately, viral pathogenicity. The structures of HPyV6 and HPyV7 VP1 reveal uniquely elongated loops that cover the bulk of the outer virion surfaces, obstructing a groove that binds sialylated glycan receptors in many other polyomaviruses. In support of this structural observation, interactions of VP1 with α2,3- and α2,6-linked sialic acids could not be detected in solution by nuclear magnetic resonance spectroscopy. Single-cell binding studies indicate that sialylated glycans are likely not required for initial attachment to cultured human cells. Our findings establish distinct antigenic properties of HPyV6 and HPyV7 capsids and indicate that these two viruses engage nonsialylated receptors. IMPORTANCE: Eleven new human polyomaviruses, including the skin viruses HPyV6 and HPyV7, have been identified during the last decade. In contrast to better-studied polyomaviruses, the routes of infection, cell tropism, and entry pathways of many of these new viruses remain largely mysterious. Our high-resolution X-ray structures of major capsid proteins VP1 from HPyV6 and from HPyV7 reveal critical differences in surface morphology from those of all other known polyomavirus structures. A groove that engages specific sialic acid-containing glycan receptors in related polyomaviruses is obstructed, and VP1 of HPyV6 and HPyV7 does not interact with sialylated compounds in solution or on cultured human cells. A comprehensive comparison with other structurally characterized polyomavirus VP1 proteins enhances our understanding of molecular determinants that underlie receptor specificity, antigenicity, and, ultimately, pathogenicity within the polyomavirus family and highlight the need for structure-based analysis to better define phylogenetic relationships within the growing polyomavirus family and perhaps also for other viruses.

BIR2 affects complex formation of BAK1 with ligand binding receptors in plant defense.

BAK1 is a multifunctional leucine-rich repeat receptor kinase (LRR-RLK) that exerts its function by interacting with multiple ligand binding receptors and thereby influences diverse processes varying from brassinosteroid perception via PAMP and DAMP perception to cell death control. We recently identified a new BAK1 interacting protein, BIR2, that is also a LRR-RLK but, in contrast to BAK1, negatively regulates BAK1-dependent PAMP responses. While brassinosteroid responses are not affected by BIR2, cell death is negatively regulated as described for BAK1. BIR2 is released from BAK1 after ligand perception, increasing the pool of free BAK1 that is available to form complexes with activated ligand binding receptors. Individual ligands can only partially release BAK1 from BIR2. After exposition to a cocktail of ligands, almost the complete amount of BAK1 can be released indicating that BAK1 exists, together with BIR2, in subpools that can be individually addressed by specific ligands. These data support the idea that BAK1 exists in preformed complexes with its ligand binding receptor partners. Overexpression of BIR2 results in reduced complex formation of BAK1 with FLS2, showing that BIR2 negatively regulates BAK1 complex formation with ligand binding receptors.

Structure of the pseudokinase domain of BIR2, a regulator of BAK1-mediated immune signaling in Arabidopsis.

The BAK1-interacting receptor-like kinase 2 (BIR2) belongs to the large family of leucine-rich repeat receptor-like kinases (LRR-RLKs) that mediate development and innate immunity in plants and form a monophyletic gene family with the Drosophila Pelle and human interleukin-1 receptor-associated kinases (IRAK). BIR2 is a negative regulator of BAK1-mediated defense mechanisms and cell death responses, yet key residues that are typically required for kinase activity are not present in the BIR2 kinase domain. We have determined the crystal structure of the BIR2 cytosolic domain and show that its nucleotide binding site is occluded. NMR spectroscopy confirmed that neither wild type nor phosphorylation-mimicking mutants of BIR2 bind ATP-analogues in solution, suggesting that BIR2 is a genuine enzymatically inactive pseudokinase. BIR2 is, however, phosphorylated by its target of regulation, BAK1. Using nano LC-MS/MS analysis for site-specific analysis of phosphorylation, we found a high density of BAK1-transphosphorylation sites in the BIR2 juxta membrane domain, a region previously implicated in regulation of RLKs. Our findings provide a structural basis to better understand signaling through kinase-dead domains that are predicted to account for 20% of all Arabidopsis RLKs and 10% of all human kinases.