Phoebe L. Stewart

The Mannose Receptor Delivers Lipoglycan Antigens to Endosomes for Presentation to T Cells by CD1b Molecules

We have characterized the CD1b-mediated presentation pathway for the mycobacterial lipoglycan lipoarabinomannan (LAM) in monocyte-derived antigen-presenting cells. The macrophage mannose receptor (MR) was responsible for uptake of LAM. Antagonism of MR function inhibited both the internalization of LAM and the presentation of this antigen to LAM-reactive T cells. Intracellular MRs were most abundant in early endosomes, but they also were located in the compartment for MHC class II antigen loading (MIIC). Internalized LAM was transported to late endosomes, lysosomes, and MIICs. MRs colocalized with CD1b molecules, suggesting that the MR could deliver LAM to late endosomes for loading onto CD1b. LAM and CD1b colocalized in organelles that may be sites of lipoglycan antigen loading. This pathway links recognition of microbial antigens by a receptor of the innate immune system to the induction of adaptive T cell responses.

Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo

Human species C adenovirus serotype 5 (Ad5) is the most common viral vector used in clinical studies worldwide. Ad5 vectors infect liver cells in vivo with high efficiency via a poorly defined mechanism, which involves virus binding to vitamin K-dependent blood coagulation factors. Here, we report that the major Ad5 capsid protein, hexon, binds human coagulation factor X (FX) with an affinity of 229 pM. This affinity is 40-fold stronger than the reported affinity of Ad5 fiber for the cellular receptor coxsackievirus and adenovirus receptor, CAR. Cryoelectron microscopy and single-particle image reconstruction revealed that the FX attachment site is localized to the central depression at the top of the hexon trimer. Hexon-mutated virus bearing a large insertion in hexon showed markedly reduced FX binding in vitro and failed to deliver a transgene to hepatocytes in vivo. This study describes the mechanism of FX binding to Ad5 and demonstrates the critical role of hexon for virus infection of hepatocytes in vivo.

Structure and Mechanism of Protein Stability Sensors: Chaperone Activity of Small Heat Shock Proteins

Small heat shock proteins (sHSP) make up a remarkably diverse group of molecular chaperones possessing a degree of structural plasticity unparalleled in other protein superfamilies. In the absence of chemical energy input, these stability sensors can sensitively recognize and bind destabilized proteins, even in the absence of gross misfolding. Cellular conditions regulate affinity toward client proteins, allowing tightly controlled switching and tuning of sHSP chaperone capacity. Perturbations of this regulation, through chemical modification or mutation, directly lead to a variety of disease states. This review explores the structural basis of sHSP oligomeric flexibility and the corresponding functional consequences in the context of a model describing sHSP activity with a set of three coupled thermodynamic equilibria. As current research illuminates many novel physiological roles for sHSP outside of their traditional duties as molecular chaperones, such a conceptual framework provides a sound foundation for describing these emerging functions in physiological and pathological processes.

Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization

Interaction of the adenovirus penton base protein with αV integrins promotes virus entry into host cells. The location of the integrin binding sequence Arg‐Gly‐Asp (RGD) on human type 2 adenovirus (Ad2) was visualized by cryo‐electron microscopy (cryo‐EM) and image reconstruction using a mAb (DAV‐1) which recognizes a linear epitope, IRGDTFATR. The sites for DAV‐1 binding corresponded to the weak density above each of the five 22 Å protrusions on the adenovirus penton base protein. Modeling of a Fab fragment crystal structure into the adenovirus‐Fab cryo‐EM density indicated a large amplitude of motion for the Fab and the RGD epitope. An unexpected finding was that Fab fragments, but not IgG antibody molecules, inhibited adenovirus infection. Steric hindrance from the adenovirus fiber and a few bound IgG molecules, as well as epitope mobility, most likely prevent binding of IgG antibodies to all five RGD sites on the penton base protein within the intact virus. These studies indicate that the structure of the adenovirus particle facilitates interaction with cell integrins, whilst restricting binding of potentially neutralizing antibodies.

Crystal Structure of Human Adenovirus at 3.5 Å Resolution

Human Adenovirus Structures Human adenoviruses may be a common cause of acute infections in humans, but they can also be used as vectors for vaccine and therapeutic gene transfer. Rational engineering of safe adenovirus vectors has been hampered by a lack of high-resolution structural information. Two papers now describe the structure of human adenovirus using complementary techniques. Reddy et al. (p. 1071; see the Perspective by Harrison) have determined the crystal structure at 3.5 angstrom resolution, while Liu et al. (p. 1038; see the Perspective by Harrison) solved the structure to 3.6 angstrom resolution by electron microscopy. Together the structures provide insights into viral assembly, stabilization, and cell entry mechanisms. High-resolution structures provide a basis for optimizing adenovirus as a vaccine and gene-therapy vector. Rational development of adenovirus vectors for therapeutic gene transfer is hampered by the lack of accurate structural information. Here, we report the x-ray structure at 3.5 angstrom resolution of the 150-megadalton adenovirus capsid containing nearly 1 million amino acids. We describe interactions between the major capsid protein (hexon) and several accessory molecules that stabilize the capsid. The virus structure also reveals an altered association between the penton base and the trimeric fiber protein, perhaps reflecting an early event in cell entry. The high-resolution structure provides a substantial advance toward understanding the assembly and cell entry mechanisms of a large double-stranded DNA virus and provides new opportunities for improving adenovirus-mediated gene transfer.

Flexibility of the adenovirus fiber is required for efficient receptor interaction

ABSTRACT The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.

Visualization of α-Helices in a 6-Ångstrom Resolution Cryoelectron Microscopy Structure of Adenovirus Allows Refinement of Capsid Protein Assignments

ABSTRACT The structure of adenovirus was determined to a resolution of 6 Å by cryoelectron microscopy (cryoEM) single-particle image reconstruction. Docking of the hexon and penton base crystal structures into the cryoEM density established that α-helices of 10 or more residues are resolved as rods. A difference map was calculated by subtracting a pseudoatomic capsid from the cryoEM reconstruction. The resulting density was analyzed in terms of observed α-helices and secondary structure predictions for the additional capsid proteins that currently lack atomic resolution structures (proteins IIIa, VI, VIII, and IX). Protein IIIa, which is predicted to be highly α-helical, is assigned to a cluster of helices observed below the penton base on the inner capsid surface. Protein VI is present in ∼1.5 copies per hexon trimer and is predicted to have two long α-helices, one of which appears to lie inside the hexon cavity. Protein VIII is cleaved by the adenovirus protease into two fragments of 7.6 and 12.1 kDa, and the larger fragment is predicted to have one long α-helix, in agreement with the observed density for protein VIII on the inner capsid surface. Protein IX is predicted to have one long α-helix, which also has a strongly indicated propensity for coiled-coil formation. A region of density near the facet edge is now resolved as a four-helix bundle and is assigned to four copies of the C-terminal α-helix from protein IX.

Structure of the vault, a ubiquitous celular component

BACKGROUND The vault is a ubiquitous and highly conserved ribonucleoprotein particle of approximately 13 MDa. This particle has been shown to be upregulated in certain multidrug-resistant cancer cell lines and to share a protein component with the telomerase complex. Determination of the structure of the vault was undertaken to provide a first step towards understanding the role of this cellular component in normal metabolism and perhaps to shed some light on its role in mediating drug resistance. RESULTS Over 1300 particle images were combined to calculate an approximately 31 A resolution structure of the vault. Rotational power spectra did not yield a clear symmetry peak, either because of the thin, smooth walls or inherent flexibility of the vault. Although cyclic eightfold (C8) symmetry was imposed, the resulting reconstruction may be partially cylindrically averaged about the eightfold axis. Our results reveal the vault to be a hollow, barrel-like structure with two protruding caps and an invaginated waist. CONCLUSIONS Although the normal cellular function of the vault is as yet undetermined, the structure of the vault is consistent with either a role in subcellular transport, as previously suggested, or in sequestering macromolecular assemblies.

Lens α-crystallin: Function and structure

α-Crystallin is a major lens protein, comprising up to 40% of total lens proteins, where its structural function is to assist in maintaining the proper refractive index in the lens. In addition to its structural role, it has been shown to function in a chaperone-like manner. The chaperone-like function of α-crystallin will help prevent the formation of large light-scattering aggregates and possibly cataract. In the lens, α-crystallin is a polydisperse molecule consisting of a 3:1 ratio of αA to αB subunits. In this study, we expressed recombinant αA- and αB-crystallin in E. coli and compared the polydispersity, structure and aggregation state between each other and native bovine lens α-crystallin. Using gel permeation chromatography to assay for polydispersity, we found native α-crystallin to be significantly more polydisperse than either recombinant αA- or αB-crystallin, with αB-crystallin having the most homogeneous structure of the three. Reconstructed images of αB-crystallin obtained with cryo-electron microscopy support the concept that αB-crystallin is an extremely dynamic molecule and demonstrated that it has a hollow interior. Interestingly, we present evidence that native α-crystallin is significantly more thermally stable than either αA- or αB-crystallin alone. In fact, our experiments suggest that a 3:1 ratio of αA to αB subunit composition in an α-crystallin molecule is optimal in terms of thermal stability. This fascinating result explains the stoichiometric ratios of αA- and αB-crystallin subunits in the mammalian lens.

Tilt-pair analysis of images from a range of different specimens in single-particle electron cryomicroscopy.

The comparison of a pair of electron microscope images recorded at different specimen tilt angles provides a powerful approach for evaluating the quality of images, image-processing procedures, or three-dimensional structures. Here, we analyze tilt-pair images recorded from a range of specimens with different symmetries and molecular masses and show how the analysis can produce valuable information not easily obtained otherwise. We show that the accuracy of orientation determination of individual single particles depends on molecular mass, as expected theoretically since the information in each particle image increases with molecular mass. The angular uncertainty is less than 1° for particles of high molecular mass (∼ 50 MDa), several degrees for particles in the range 1–5 MDa, and tens of degrees for particles below 1 MDa. Orientational uncertainty may be the major contributor to the effective temperature factor (B-factor) describing contrast loss and therefore the maximum resolution of a structure determination. We also made two unexpected observations. Single particles that are known to be flexible showed a wider spread in orientation accuracy, and the orientations of the largest particles examined changed by several degrees during typical low-dose exposures. Smaller particles presumably also reorient during the exposure; hence, specimen movement is a second major factor that limits resolution. Tilt pairs thus enable assessment of orientation accuracy, map quality, specimen motion, and conformational heterogeneity. A convincing tilt-pair parameter plot, where 60% of the particles show a single cluster around the expected tilt axis and tilt angle, provides confidence in a structure determined using electron cryomicroscopy.