Robert J. C. Gilbert

The Interaction Properties of Costimulatory Molecules Revisited

B7-1 and B7-2 are generally thought to have comparable structures and affinities for their receptors, CD28 and CTLA-4, each of which is assumed to be bivalent. We show instead (1) that B7-2 binds the two receptors more weakly than B7-1, (2) that, relative to its CTLA-4 binding affinity, B7-2 binds CD28 2- to 3-fold more effectively than B7-1, (3) that, unlike B7-1, B7-2 does not self-associate, and (4) that, in contrast to CTLA-4 homodimers, which are bivalent, CD28 homodimers are monovalent. Our results indicate that B7-1 markedly favors CTLA-4 over CD28 engagement, whereas B7-2 exhibits much less bias. We propose that the distinct structures and binding properties of B7-1 and B7-2 account for their overlapping but distinct effects on T cell responses.

The Long and Short of MicroRNA

MicroRNAs (miRNAs) are versatile regulators of gene expression in higher eukaryotes. In order to silence many different mRNAs in a precise manner, miRNA stability and efficacy is controlled by highly developed regulatory pathways and fine-tuning mechanisms both affecting miRNA processing and altering mature miRNA target specificity.

Structural Basis of Pore Formation by the Bacterial Toxin Pneumolysin

The bacterial toxin pneumolysin is released as a soluble monomer that kills target cells by assembling into large oligomeric rings and forming pores in cholesterol-containing membranes. Using cryo-EM and image processing, we have determined the structures of membrane-surface bound (prepore) and inserted-pore oligomer forms, providing a direct observation of the conformational transition into the pore form of a cholesterol-dependent cytolysin. In the pore structure, the domains of the monomer separate and double over into an arch, forming a wall sealing the bilayer around the pore. This transformation is accomplished by substantial refolding of two of the four protein domains along with deformation of the membrane. Extension of protein density into the bilayer supports earlier predictions that the protein inserts beta hairpins into the membrane. With an oligomer size of up to 44 subunits in the pore, this assembly creates a transmembrane channel 260 A in diameter lined by 176 beta strands.

A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting.

The triplet-based genetic code requires that translating ribosomes maintain the reading frame of a messenger RNA faithfully to ensure correct protein synthesis. However, in programmed -1 ribosomal frameshifting, a specific subversion of frame maintenance takes place, wherein the ribosome is forced to shift one nucleotide backwards into an overlapping reading frame and to translate an entirely new sequence of amino acids. This process is indispensable in the replication of numerous viral pathogens, including HIV and the coronavirus associated with severe acute respiratory syndrome, and is also exploited in the expression of several cellular genes. Frameshifting is promoted by an mRNA signal composed of two essential elements: a heptanucleotide ‘slippery’ sequence and an adjacent mRNA secondary structure, most often an mRNA pseudoknot. How these components operate together to manipulate the ribosome is unknown. Here we describe the observation of a ribosome–mRNA pseudoknot complex that is stalled in the process of -1 frameshifting. Cryoelectron microscopic imaging of purified mammalian 80S ribosomes from rabbit reticulocytes paused at a coronavirus pseudoknot reveals an intermediate of the frameshifting process. From this it can be seen how the pseudoknot interacts with the ribosome to block the mRNA entrance channel, compromising the translocation process and leading to a spring-like deformation of the P-site transfer RNA. In addition, we identify movements of the likely eukaryotic ribosomal helicase and confirm a direct interaction between the translocase eEF2 and the P-site tRNA. Together, the structural changes provide a mechanical explanation of how the pseudoknot manipulates the ribosome into a different reading frame.

Structure and dimerization of a soluble form of B7-1.

B7-1 (CD80) and B7-2 (CD86) are glycoproteins expressed on antigen-presenting cells. The binding of these molecules to the T cell homodimers CD28 and CTLA-4 (CD152) generates costimulatory and inhibitory signals in T cells, respectively. The crystal structure of the extracellular region of B7-1 (sB7-1), solved to 3 A resolution, consists of a novel combination of two Ig-like domains, one characteristic of adhesion molecules and the other previously seen only in antigen receptors. In the crystal lattice, sB7-1 unexpectedly forms parallel, 2-fold rotationally symmetric homodimers. Analytical ultracentrifugation reveals that sB7-1 also dimerizes in solution. The structural data suggest a mechanism whereby the avidity-enhanced binding of B7-1 and CTLA-4 homodimers, along with the relatively high affinity of these interactions, favors the formation of very stable inhibitory signaling complexes.

Two Structural Transitions in Membrane Pore Formation by Pneumolysin, the Pore-Forming Toxin of Streptococcus pneumoniae

The human pathogen Streptococcus pneumoniae produces soluble pneumolysin monomers that bind host cell membranes to form ring-shaped, oligomeric pores. We have determined three-dimensional structures of a helical oligomer of pneumolysin and of a membrane-bound ring form by cryo-electron microscopy. Fitting the four domains from the crystal structure of the closely related perfringolysin reveals major domain rotations during pore assembly. Oligomerization results in the expulsion of domain 3 from its original position in the monomer. However, domain 3 reassociates with the other domains in the membrane pore form. The base of domain 4 contacts the bilayer, possibly along with an extension of domain 3. These results reveal a two-stage mechanism for pore formation by the cholesterol-binding toxins.

Pore-forming toxins

Abstract. Pore-forming toxins are widely distributed proteins which form lesions in biological membranes. In this review, bacterial pore-forming toxins are treated as a paradigm and discussed in terms of the structural principles on which they work. Then, a large family of bacterial toxins, the cholesterol-binding toxins, are analyzed in depth to provide an overview of the processes involved in pore formation. The ways in which the cholesterol-binding toxins (cholesterol-dependent cytolysins) interact with membranes and form pores, the structure of the monomeric soluble and oligomeric pore-forming states, and the effects of the toxin on membrane structure are discussed. By surveying the range of work which has been done on cholesterol-binding toxins, a working model is elaborated which reconciles two current, apparently diametrically opposed, models for their mechanism.

Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1

Type 5 adenovirus (Ad5) is a human pathogen that has been widely developed for therapeutic uses, with only limited success to date. We report here the novel finding that human erythrocytes present Coxsackie virus-adenovirus receptor (CAR) providing an Ad5 sequestration mechanism that protects against systemic infection. Interestingly, erythrocytes from neither mice nor rhesus macaques present CAR. Excess Ad5 fiber protein or anti-CAR antibody inhibits the binding of Ad5 to human erythrocytes and cryo-electron microscopy shows attachment via the fiber protein of Ad5, leading to close juxtaposition with the erythrocyte membrane. Human, but not murine, erythrocytes also present complement receptor (CR1), which binds Ad5 in the presence of antibodies and complement. Transplantation of human erythrocytes into nonobese diabetic/severe combined immunodeficiency mice extends blood circulation of intravenous Ad5 but decreases its extravasation into human xenograft tumors. Ad5 also shows extended circulation in transgenic mice presenting CAR on their erythrocytes, although it clears rapidly in transgenic mice presenting erythrocyte CR1. Hepatic infection is inhibited in both transgenic models. Erythrocytes may therefore restrict Ad5 infection (natural and therapeutic) in humans, independent of antibody status, presenting a formidable challenge to Ad5 therapeutics. “Stealthing” of Ad5 using hydrophilic polymers may enable circumvention of these natural virus traps.

A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results

An initial tranche of results from day-to-day use of a robotic system for setting up 100 nl-scale vapour-diffusion sitting-drop protein crystallizations has been surveyed. The database of over 50 unrelated samples represents a snapshot of projects currently at the stage of crystallization trials in Oxford research groups and as such encompasses a broad range of proteins. The results indicate that the nanolitre-scale methodology consistently identifies more crystallization conditions than traditional hand-pipetting-style methods; however, in a number of cases successful scale-up is then problematic. Crystals grown in the initial 100 nl-scale drops have in the majority of cases allowed useful characterization of x-ray diffraction, either in-house or at synchrotron beamlines. For a significant number of projects, full x-ray diffraction data sets have been collected to 3 A resolution or better (either in-house or at the synchrotron) from crystals grown at the 100 nl scale. To date, five structures have been determined by molecular replacement directly from such data and a further three from scale-up of conditions established at the nanolitre scale.

Structural basis of Nipah and Hendra virus attachment to their cell-surface receptor ephrin-B2

Nipah and Hendra viruses are emergent paramyxoviruses, causing disease characterized by rapid onset and high mortality rates, resulting in their classification as Biosafety Level 4 pathogens. Their attachment glycoproteins are essential for the recognition of the cell-surface receptors ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3). Here we report crystal structures of both Nipah and Hendra attachment glycoproteins in complex with human EFNB2. In contrast to previously solved paramyxovirus attachment complexes, which are mediated by sialic acid interactions, the Nipah and Hendra complexes are maintained by an extensive protein-protein interface, including a crucial phenylalanine side chain on EFNB2 that fits snugly into a hydrophobic pocket on the viral protein. By analogy with the development of antivirals against sialic acid binding viruses, these results provide a structural template to target antiviral inhibition of protein-protein interactions.