Different inositol phospholipids (PIPs) distribute to distinct subcellular organelles, creating an addressing system that dictates the sites of action of PIP-binding proteins, including components of the Endosomal Sorting Complexes Required for Transport (ESCRT). The ESCRT machinery is recruited to remodel many different cellular membranes through combinatorial binding interactions made by the early-acting ESCRT-I and ESCRT-II complexes with PIPs, ubiquitin modifications, and membrane-specific adaptors. Membrane remodeling, constriction, and fission are then mediated by membrane-associated filaments formed by subunits of the late-acting ESCRT-III complexes, together with their associated VPS4 AAA ATPases. Here, we describe two different classes of helical ESCRT-III filaments that can surround and tubulate membranes containing PIP 2 lipids. Cryo-EM reconstructions revealed that protofilaments comprising closed IST1 subunits formed 8-stranded nanotubes that encase membrane monolayers. The nanotube coordinates exposed PI(4,5)P 2 or PI(3,5)P 2 headgroups within a basic pocket formed at the junction of three IST1 subunits, and our structures reveal how the pocket can accommodate either PIP 2 isomer with minimal adjustment. In contrast, protofilaments comprising open CHMP1A subunits formed one start helices that encase membrane bilayers and bind exposed PI(4,5)P 2 headgroups across a basic surface that spans adjacent subunits of the CHMP1A protofilament. These two different structures extend the known plasticity of ESCRT-III polymers, reveal how PIP 2 lipids can promote ESCRT-III filament assembly and membrane remodeling, and define the molecular contacts that underlie specific ESCRT-III/PIP 2 interactions.

In recent years, cryo-electron microscopy (cryo-EM) has become a practical and effective method of determining structures at previously unattainable resolutions due to advances in detection, automation, and data processing. However, sample preparation remains a major bottleneck in the cryo-EM workflow. Even after the arduous process of biochemical sample optimization, it often takes several iterations of grid vitrification and screening to determine the optimal grid freezing parameters that yield suitable ice thickness and particle distribution for data collection. Since a high-quality sample is imperative for high-resolution structure determination, grid optimization is a vital step. For researchers who rely on cryo-EM facilities for grid screening, each iteration of this optimization process may delay research progress by a matter of months. Therefore, a more strategic and efficient approach should be taken to ensure that the grid optimization process can be completed in as few iterations as possible. Here, we present an implementation of Design of Experiments (DOE) to expedite and strategize the grid optimization process. A Fractional Factorial Design (FFD) guides the determination of a limited set of experimental conditions which can model the full parameter space of interest. Grids are frozen with these conditions and screened for particle distribution and ice thickness. Quantitative scores are assigned to each of these grid characteristics based on a qualitative rubric. Input conditions and response scores are used to generate a least-squares regression model of the parameter space in JMP, which is used to determine the conditions which should, in theory, yield optimal grids. Upon testing this approach on apoferritin and L-glutamate dehydrogenase on both the Vitrobot Mark IV and the Leica GP2 plunge freezers, the resulting grid conditions reliably yielded grids with high-quality ice and particle distribution that were suitable for collecting large overnight datasets on a Krios. We conclude that a DOE-based approach is a cost-effective and time-saving tool for cryo-EM grid preparation.

Matriglycan is a linear glycan (xylose-β1,3-glucuronate)n, which binds proteins in the extracellular matrix that contain laminin-globular domains and Lassa Fever Virus. It is indispensable for neuromuscular function. Matriglycan of insufficient length can cause muscular dystrophy with abnormal brain and eye development. LARGE1 (Like-acetylglucosaminyltransferase-1) uniquely synthesizes matriglycan on dystroglycan. The mechanism of matriglycan synthesis is not obvious from cryo-EM reconstructions of LARGE1. However, by reconstituting activity in vitro on recombinant prodystroglycan we show that the presence of the dystroglycan N-terminal domain (DGN), phosphorylated core M3, and a xylose-glucuronate primer are necessary for matriglycan polymerization by LARGE1. By introducing active site mutations, we demonstrate that LARGE1 processively polymerizes matriglycan on prodystroglycan, with its length regulated by the dystroglycan prodomain, DGN. Our enzymatic analysis of LARGE1 uncovers the mechanism of matriglycan synthesis on dystroglycan, which can form the basis for therapeutic strategies to treat matriglycan-deficient neuromuscular disorders and arenaviral infections.

SufR is an iron-sulfur ([4Fe-4S]) cluster-containing transcription factor belonging to an uncharacterized domain family (COG2345). It has been shown to negatively regulate the sulfur utilization factor (SUF) Fe-S biogenesis system in several Gram-negative and Gram-positive bacteria including Cyanobacteria, Mycobacteria and Streptomyces. The structural basis for its DNA recognition and transcriptional regulation by the SufR-like proteins remains enigmatic. In this study, we present the cryo-EM structure of Mycobacterium tuberculosis SufR bound to its promoter, revealing a new domain architecture. Our structural, biochemical and molecular analyses show that SufR possesses an unusual [4Fe-4S] cluster coordination environment in the sensory domain and recognizes its promoter via a dual-module mechanism using both the AT-hook and the helix-turn-helix (HTH) DNA-binding motif in the DNA-binding domain. This DNA recognition strategy differs from a canonical winged HTH transcription factor. Moreover, our bioinformatic analysis and structural modeling suggest that SufR and SufR-like proteins in the COG2345 family represent a large, previously uncharacterized family of transcription factors widely distributed across bacteria and archaea. Together, these findings establish SufR-like proteins as a new model for transcriptional regulation by Fe-S transcription factors in prokaryotes and uncover the underappreciated evolutionary versatility of AT-hooks.

BACKGROUND:Hemophilia A arises from dysfunctional or deficient coagulation factor (F)VIII and leads to inefficient fibrin clot formation and uncontrolled bleeding events. The development of antibody inhibitors is a clinical complication in hemophilia A patients receiving FVIII replacement therapy. LE2E9 is an anti-C1 domain inhibitor previously isolated from a mild/moderate hemophilia A patient and disrupts FVIII interactions with von Willebrand factor and FIXa, though the intermolecular contacts that underpin LE2E9-mediated FVIII neutralization are undefined. OBJECTIVES:To determine the structure of the complex between FVIII and LE2E9 and characterize its mechanism of inhibition. METHODS:FVIII was bound to the antigen binding fragment (Fab) of NB2E9, a recombinant construct of LE2E9, and its structure was determined by cryogenic electron microscopy. RESULTS:This report communicates the 3.46 Å structure of FVIII bound to NB2E9, with its epitope comprising FVIII residues S2040 to Y2043, K2065 to W2070, and R2150 to H2155. Structural analysis reveals that the LE2E9 epitope overlaps with portions of the epitope for 2A9, a murine-derived inhibitor, suggesting that these residues represent a shared antigenic region on the C1 domain between FVIII-/- mice and hemophilia A patients. Furthermore, the FVIII:NB2E9 structure elucidates the orientation of the LE2E9 glycan, illustrating how the glycan sterically blocks interactions between the FVIII C1 domain and the von Willebrand factor D' domain. A putative model of the FVIIIa:FIXa complex suggests potential clashing between the NB2E9 glycan and FIXa light chain. CONCLUSION:These results describe an antigenic "hotspot" on the FVIII C1 domain and provide a structural basis for engineering FVIII replacement therapeutics with reduced antigenicity.

Blood coagulation factor VIII (FVIII) is a key regulator of fibrin clot formation. Once activated by thrombin, activated FVIII (FVIIIa) binds to activated platelet surfaces and forms a complex with activated factor IX (FIXa) termed the intrinsic tenase (Xase) complex which sustains fibrin clot development. Excessive Xase complex activity is clinically linked with venous thromboembolism. Conversely, dysfunctional or deficient levels of FVIII or FIX is associated with hemophilia A or B, respectively. Despite decades of biochemical and structural research, the structural determinants for Xase complex assembly remain elusive. Here, we describe the structures of FVIIIa and the Xase complex, providing the first structural glimpse into thrombin-catalyzed activation of FVIII and assembly of the Xase complex. We utilized single-particle cryogenic electron microscopy (cryoEM) to determine the structures of FVIIIa and the Xase complex using ET3i, a bioengineered FVIII human/porcine chimera with enhanced stability suitable for cryoEM experiments, and wildtype human FIXa treated with a protease inhibitor to prevent degradation of the Xase complex. CryoEM maps of FVIIIa and the Xase complex were determined to nominal resolutions of 3.56 Å and 3.64 Å, respectively. Leveraging recent advancements in heterogeneous cryoEM data analysis, our results reveal continuous conformational changes to isolated FVIIIa that are concomitant with thrombin-catalyzed activation. One of the most pronounced structural rearrangements is a novel ~20° bending centered at the interface between the A domains and C domains. This tilted conformer is only present in the FVIIIa structure with no FIXa bound, suggesting that this conformation of FVIIIa is not conducive for forming the active tenase complex. We are also able to visualize continuous dissociation of the A2 domain, allowing for structural investigation into how mutations at the A2 interface with the A1 and A3 domains may stabilize or destabilize FVIII/FVIIIa. Furthermore, we identified a separate set of particles consisting of intact FVIIIa bound to FIXa. Our structure of the Xase complex reveals for the first time how the FIXa catalytic domain binds to the FVIIIa A2 domain, centered on FVIIIa residues 558-565. This structural arrangement places FIXa residue p.Arg384 (legacy numbering) docked onto several hydrophilic FVIIIa residues, neighboring a hydrophobic patch, on the A2 domain, providing a structure-based rationale for FIXa variants p.Arg384Leu (Padua) and p.Arg384Gln (Shanghai) which have been identified in patients with thrombosis. The Xase structure also suggests that the acidic C-terminal tail of the A2 domain anchors FVIIIa to FIXa through electrostatic interactions with FIXa exosite II. Further inspection of the cryoEM map of the Xase complex reveals how the FIXa light chain wraps around FVIIIa A3-C1 domains for optimal binding to lipid membranes. Correlating these results with the CDC Hemophilia Mutation Projects (CHAMP and CHBMP) databases, we are able to hypothesize how hemophilia A or B missense mutations disrupt Xase complex assembly and/or activity.

The development of pathogenic antibody inhibitors against coagulation factor VIII (FVIII) occurs in approximately 30% of congenital hemophilia A patients receiving FVIII replacement therapy as well as in all cases of acquired hemophilia A. KM33 is an anti-C1 domain antibody inhibitor previously isolated from a severe hemophilia A patient. In addition to potently blocking FVIII binding to von Willebrand factor and phospholipid surfaces, KM33 disrupts FVIII binding to lipoprotein receptor-related protein 1 (LRP1), which drives FVIII hepatic clearance and antigen presentation in dendritic cells. Here, we report on the structure of FVIII bound to NB33, a recombinant derivative of KM33, by single-particle cryo-electron microscopy. Structural analysis reveals the NB33 epitope localizes to FVIII residues R2090-S2094 and I2158-R2159 which constitute membrane-binding loops in the C1 domain. Further analysis reveals multiple FVIII lysine and arginine residues, previously shown to mediate binding to LRP1, dock onto an acidic cleft at the NB33 variable domain interface, thus blocking a putative LRP1 binding site. Together, these results demonstrate a novel mechanism of FVIII inhibition by a patient-derived antibody inhibitor and provide structural evidence toward engineering FVIII with reduced LRP1-mediated clearance.

Adeno-associated virus (AAV) has a single-stranded DNA genome encapsidated in a small icosahedrally symmetric protein shell with 60 subunits. AAV is the leading delivery vector in emerging gene therapy treatments for inherited disorders, so its structure and molecular interactions with human hosts are of intense interest. A wide array of electron microscopic approaches have been used to visualize the virus and its complexes, depending on the scientific question, technology available, and amenability of the sample. Approaches range from subvolume tomographic analyses of complexes with large and flexible host proteins to detailed analysis of atomic interactions within the virus and with small ligands at resolutions as high as 1.6 Å. Analyses have led to the reclassification of glycan receptors as attachment factors, to structures with a new-found receptor protein, to identification of the epitopes of antibodies, and a new understanding of possible neutralization mechanisms. AAV is now well-enough characterized that it has also become a model system for EM methods development. Heralding a new era, cryo-EM is now also being deployed as an analytic tool in the process development and production quality control of high value pharmaceutical biologics, namely AAV vectors.

Matriglycan is a linear polysaccharide of alternating xylose and glucuronate that binds extracellular matrix proteins and acts as a receptor for Lassa fever virus. LARGE1 synthesizes matriglycan on dystroglycan and mutations in LARGE1 cause muscular dystrophy with abnormal brain development. However, the mechanism of matriglycan polymerization by LARGE1 is unknown. Here, we report the cryo-EM structure of LARGE1. We show that LARGE1 functions as a dimer to polymerize matriglycan by alternating activities between the xylose transferase domain on one protomer and the glucuronate transferase domain on the other protomer. Biochemical analyses using a recombinant Golgi form of dystroglycan reveal that LARGE1 polymerizes matriglycan processively. Our results provide mechanistic insights into LARGE1 function and may facilitate novel therapeutic strategies for treating neuromuscular disorders or arenaviral infections. One-Sentence Summary Dimeric LARGE1 processively polymerizes matriglycan on dystroglycan using orthogonal active sites on alternate protomers.

Matriglycan is a linear polysaccharide of alternating xylose and glucuronate that is required for muscle function and brain development. It binds several extracellular matrix proteins that contain laminin-globular domains with high affinity and is a receptor for Old-World arenaviruses such as Lassa fever virus. In skeletal muscle, LARGE1 polymerizes an extended form of matriglycan, which acts as an extracellular matrix scaffold to prevent muscular dystrophy. However, how matriglycan is polymerized is unknown. Here, we report a cryo-EM structure of LARGE1 and show that it forms a dimer with xylose and glucuronate transferase sites from different protomers facing the same direction. Moreover, using in vivo and in vitro assays we show that matriglycan is processively polymerized on α-dystroglycan. Collectively, our results correlate the orientation of LARGE1 active sites in its quaternary structure with the mechanism of matriglycan polymerization, which may facilitate novel therapeutic strategies to treat neuromuscular disorders or arenaviral infections.Funding Information: This project was supported by grants P41 GM103622 and P30 GM138395 from the National Institute of General Medical Sciences of the National Institutes of Health.Declaration of Interests: Authors declare that they have no competing interests.

Adeno‐associated virus (AAV) is a small human single‐stranded parvovirus. Interest centers upon the use of recombinant forms (rAAV) as delivery vectors in gene therapy, the first of which have been gaining FDA approval. An understanding of the molecular interactions on cell entry is a foundation for the design of vectors with greater tissue specificity. In 2016, we revisited the identity of cell receptors, with a genome‐wide deep‐sequencing of haploid human cells selected from gene‐trap mutant library. We enriched the population of AAV‐resistant cells, thorough fluorescence activated cell‐sorting, using an rAAV encoding mCherry, allowing us to identify host‐factors needed for AAV transduction. Foremost was an uncharacterized membrane protein, AAVR, that is needed for trafficking from the cell surface to the perinuclear trans Golgi network. After identifying the extracellular domains of AAVR that interact with AAV, we have expressed soluble domain fragments and have recently obtained structures of complexes at about 2.5 Å by cryo‐electron microscopy (EM). The highest resolution structures have come from complexes with just the two N‐terminal PKD domains, allowing us to minimize conflicts between receptors bound at adjacent sites on the virus surface related by symmetry. However, the high resolution structures are fully consistent with cryo‐electron tomography, where we see (at about 3nm resolution) not only the domains bound directly, but those linked by flexible hinges as they adopt several configurations. Thus, this is an illustration of a holistic understanding of a flexible complex coming from different constructs amenable to studies at different resolutions. AAV serotype 2 (AAV‐2) interacts primarily with AAVR’s polycystic kidney disease (PKD) domain 2 at a relatively conserved part of the viral surface which is also targeted by some of AAV’s most neutralizing monoclonal antibodies. Intriguingly, binding studies, mutation, and now EM structure, show that AAV‐5 interacts more exclusively with PKD domain 1 at a non‐overlapping site on the virus. In addition to providing a lead in understanding the differential cell specificities of different AAVs, these unexpected results pose interesting questions regarding the evolution of virus‐host interactions, specifically in the emergence of distinct sets of molecular interactions among related viruses that still target the same receptor.Support or Funding InformationFunded by R35 GM122564 (MSC).

Adeno-associated virus (AAV) is a promising gene therapy vector and the biophysical characterization of its interactions with host proteins is a critical foundation for engineering tissue targeting and immune escape. Presented here are protocols for the production of: (a) the outer protein shells (virus-like particles or VLPs) for serotype 2 (AAV-2) and (b) two fragments from the binding ectodomain of AAV's cellular receptor, AAVR. His6PKD1-2 comprises the first two polycystic kidney disease (PKD) domains, the minimal required for efficient binding of AAV, expressed with an N-terminal histidine tag. MBP-PKD1-5 is a fusion of the maltose binding protein with all five of the PKD domains of the AAVR receptor. Presented are the expression and purification of milligram quantities, ample for in vitro analyses. For AAV-2, the protocol offers an alternative to the use of (infectious) wild-type virus or transducing vectors. One of the methods for producing transducing vector is in Sf9 cells, and the production of VLPs is based on this. For AAVR, the protocols enable biochemical and biophysical characterization of virus-binding. The minimal two-domain construct allows more saturated binding to symmetry-equivalent sites on the virus, while the larger construct might be better expected to reflect the native receptor.

Adeno-associated virus (AAV) vectors are preeminent in emerging clinical gene therapies. Generalizing beyond the most tractable genetic diseases will require modulation of cell specificity and immune neutralization. Interactions of AAV with its cellular receptor, AAVR, are key to understanding cell-entry and trafficking with the rigor needed to engineer tissue-specific vectors. Cryo-electron tomography shows ordered binding of part of the flexible receptor to the viral surface, with distal domains in multiple conformations. Regions of the virus and receptor in close physical proximity can be identified by cross-linking/mass spectrometry. Cryo-electron microscopy with a two-domain receptor fragment reveals the interactions at 2.4 Å resolution. AAVR binds between AAV's spikes on a plateau that is conserved, except in one clade whose structure is AAVR-incompatible. AAVR's footprint overlaps the epitopes of several neutralizing antibodies, prompting a re-evaluation of neutralization mechanisms. The structure provides a roadmap for experimental probing and manipulation of viral-receptor interactions.

Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.