Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.

Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery.

Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ ∼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.

Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase.

Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.

Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle.

The multidrug transporter P-glycoprotein (P-gp, ABCB1) is an ATP-dependent pump that mediates the efflux of structurally diverse drugs and xenobiotics across cell membranes, affecting drug pharmacokinetics and contributing to the development of multidrug resistance. Structural information about the conformational changes in human P-gp during the ATP hydrolysis cycle has not been directly demonstrated, although mechanistic information has been inferred from biochemical and biophysical studies conducted with P-gp and its orthologs, or from structures of other ATP-binding cassette transporters. Using single-particle cryo-electron microscopy, we report the surprising discovery that, in the absence of the transport substrate and nucleotides, human P-gp can exist in both open [nucleotide binding domains (NBDs) apart; inward-facing] and closed (NBDs close; outward-facing) conformations. We also probe conformational states of human P-gp during the catalytic cycle, and demonstrate that, following ATP hydrolysis, P-gp transitions through a complete closed conformation to a complete open conformation in the presence of ADP.

Three-dimensional imaging of HIV-1 virological synapses reveals membrane architectures involved in virus transmission.

UNLABELLED: HIV transmission efficiency is greatly increased when viruses are transmitted at virological synapses formed between infected and uninfected cells. We have previously shown that virological synapses formed between HIV-pulsed mature dendritic cells (DCs) and uninfected T cells contain interdigitated membrane surfaces, with T cell filopodia extending toward virions sequestered deep inside invaginations formed on the DC membrane. To explore membrane structural changes relevant to HIV transmission across other types of intercellular conjugates, we used a combination of light and focused ion beam scanning electron microscopy (FIB-SEM) to determine the three-dimensional (3D) architectures of contact regions between HIV-1-infected CD4(+) T cells and either uninfected human CD4(+) T cells or human fetal astrocytes. We present evidence that in each case, membrane extensions that originate from the uninfected cells, either as membrane sheets or filopodial bridges, are present and may be involved in HIV transmission from infected to uninfected cells. We show that individual virions are distributed along the length of astrocyte filopodia, suggesting that virus transfer to the astrocytes is mediated, at least in part, by processes originating from the astrocyte itself. Mechanisms that selectively disrupt the polarization and formation of such membrane extensions could thus represent a possible target for reducing viral spread. IMPORTANCE: Our findings lead to new insights into unique aspects of HIV transmission in the brain and at T cell-T cell synapses, which are thought to be a predominant mode of rapid HIV transmission early in the infection process.

Multiple members of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family are essential for viability in Drosophila.

Mucin-type O-glycosylation represents a major form of post-translational modification that is conserved across most eukaryotic species. This type of glycosylation is initiated by a family of enzymes (GalNAc-Ts in mammals and PGANTs in Drosophila) whose members are expressed in distinct spatial and temporal patterns during development. Previous work from our group demonstrated that one member of this family is essential for viability and another member modulates extracellular matrix composition and integrin-mediated cell adhesion during development. To investigate whether other members of this family are essential, we employed RNA interference (RNAi) to each gene in vivo. Using this approach, we identified 4 additional pgant genes that are required for viability. Ubiquitous RNAi to pgant4, pgant5, pgant7, or the putative glycosyltransferase CG30463 resulted in lethality. Tissue-specific RNAi was also used to define the specific organ systems and tissues in which each essential family member is required. Interestingly, each essential pgant had a unique complement of tissues in which it was required. Additionally, certain tissues (mesoderm, digestive system, and tracheal system) required more than one pgant, suggesting unique functions for specific enzymes in these tissues. Expanding upon our RNAi results, we found that conventional mutations in pgant5 resulted in lethality and specific defects in specialized cells of the digestive tract, resulting in loss of proper digestive system acidification. In summary, our results highlight essential roles for O-glycosylation and specific members of the pgant family in many aspects of development and organogenesis.

N- and O-glycans modulate galectin-1 binding, CD45 signaling, and T cell death.

Galectin-1, a beta-galactoside-binding protein highly expressed in the thymus, induces apoptosis of specific thymocyte subsets and activated T cells. Galectin-1 binds to N- and O-glycans on several glycoprotein receptors, including CD7, CD43, and CD45. Here we show that galectin-1 signaling through CD45, which carries both N- and O-glycans, is regulated by CD45 isoform expression, core 2 O-glycan formation and the balance of N-glycan sialylation. Regulation of galectin-1 T cell death by O-glycans is mediated through CD45 phosphatase activity. While galectin-1 signaling in cells expressing low molecular weight isoforms of CD45 requires expression of core 2 O-glycans (high affinity ligands for galectin-1), galectin-1 signaling in cells expressing a high molecular weight isoform of CD45 does not require core 2 O-glycans, suggesting that a larger amount of core 1 O-glycans (low affinity ligands for galectin-1) is sufficient to overcome lack of core 2 O-glycans. Furthermore, regulation of galectin-1 signaling by alpha2,6-sialylation of N-glycans is not solely dependent on CD45 phosphatase activity and can be modulated by the relative expression of enzymes that attach sialic acid in an alpha2,6- or alpha2,3-linkage. Thus, N- and O-glycans modulate galectin-1 T cell death by distinct mechanisms, and different glycosylation events can render thymocytes susceptible or resistant to galectin-1.

Galectin multimerization and lattice formation are regulated by linker region structure.

Galectins regulate cellular functions by binding to glycan ligands on cell surface glycoprotein receptors. Prototype galectins, such as galectin-1, are one carbohydrate recognition domain (CRD) monomers that noncovalently dimerize, whereas tandem-repeat galectins, such as galectin-9, have two non-identical CRDs connected by a linker domain. Dimerization of prototype galectins, or both CRDs in tandem-repeat galectins, is typically required for the crosslinking of glycoprotein receptors and subsequent cellular signaling. Several studies have found that tandem-repeat galectins are more potent than prototype galectins in triggering many cell responses, including cell death. These differences could be due to CRD specificity, the presence or absence of a linker domain between CRDs, or both. To interrogate the basis for the increased potency of tandem-repeat galectins compared with prototype galectins in triggering cell death, we created three tandem-repeat galectin constructs with different linker regions joining identical galectin-1 CRDs, so that any differences we observed would be due to the contribution of the linker region rather than due to CRD specificity. We found that random-coil or rigid α-helical linkers that permit separation of the two galectin-1 CRDs facilitated the formation of higher-order galectin multimers and that these galectins were more potent in binding to glycan ligands and cell surface glycoprotein receptors, as well as triggering T cell death, compared with native galectin-1 or a construct with a short rigid linker. Thus, the increased potency of tandem-repeat galectins compared with prototype galectins is likely due to the ability of the linker domain to permit intermolecular CRD interactions, resulting in the formation of higher-order multimers with increased valency, rather than differences in CRD specificity.

Microbiology catches the cryo-EM bug.

Over the past few years, the advances in technology and methods that have revolutionized cryo-EM are allowing for key insights in a variety of areas in biology, and microbiology is no exception. A wide range of important macromolecular assemblies in prokaryotic and eukaryotic cells, as well as intact viruses, have now become accessible to investigation by new methods in 3D electron microscopy. We focus here on selected examples that illustrate this breadth, and review the application of methods in single particle cryo-EM and cryo-electron tomography to progress in the structural biology of CRISPR systems, visualization of small molecule drugs in membrane proteins, in situ visualization of bacterial nanomachines, and the analysis of antigen-antibody interactions to drive vaccine design.

Atomic Resolution Cryo-EM Structure of ß-Galactosidase.

The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for ß-galactosidase bound to the inhibitor phenylethyl ß-D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at ∼ 1.5 Å resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the non-covalently bound inhibitor and offers further opportunities for structure-guided inhibitor design.

Cryo-EM: beyond the microscope.

The pace at which cryo-EM is being adopted as a mainstream tool in structural biology has continued unabated over the past year. Initial successes in obtaining near-atomic resolution structures with cryo-EM were enabled to a large extent by advances in microscope and detector technology. Here, we review some of the complementary technical improvements that are helping sustain the cryo-EM revolution. We highlight advances in image processing that permit high resolution structure determination even in the presence of structural and conformational heterogeneity. We also review selected examples where biochemical strategies for membrane protein stabilization facilitate cryo-EM structure determination, and discuss emerging approaches for further improving the preparation of reliable plunge-frozen specimens.

Resolution advances in cryo-EM enable application to drug discovery.

The prospect that the structures of protein assemblies, small and large, can be determined using cryo-electron microscopy (cryo-EM) is beginning to transform the landscape of structural biology and cell biology. Great progress is being made in determining 3D structures of biological assemblies ranging from icosahedral viruses and helical arrays to small membrane proteins and protein complexes. Here, we review recent advances in this field, focusing especially on the emerging use of cryo-EM in mapping the binding of drugs and inhibitors to protein targets, an application that requires structure determination at the highest possible resolutions. We discuss methods used to evaluate the information contained in cryo-EM density maps and consider strengths and weaknesses of approaches currently used to measure map resolution.

Maturation of the HIV-1 core by a non-diffusional phase transition.

The formation of the HIV-1 core is the final step in the viral maturation pathway, resulting in the formation of infectious virus. Most current models for HIV-1 core formation suggest that, upon proteolytic cleavage from the immature Gag, capsid (CA) dissociates into the viral interior before reforming into the core. Here we present evidence for an alternate view of core formation by taking advantage of our serendipitous observation of large membrane-enclosed structures in HIV-1 supernatants from infected cells. Cryo-electron tomographic studies show that these structures, which contain ordered arrays of what is likely the membrane-associated matrix protein, contain multiple cores that can be captured at different stages of maturation. Our studies suggest that HIV maturation involves a non-diffusional phase transition in which the detaching layer of the cleaved CA lattice is gradually converted into a roll that ultimately forms the surface of the mature conical core.

Catching HIV 'in the act' with 3D electron microscopy.

The development of a safe, effective vaccine to prevent HIV infection is a key step for controlling the disease on a global scale. However, many aspects of HIV biology make vaccine design problematic, including the sequence diversity and structural variability of the surface envelope glycoproteins and the poor accessibility of neutralization-sensitive epitopes on the virus. In this review, we discuss recent progress in understanding HIV in a structural context using emerging tools in 3D electron microscopy, and outline how some of these advances could be important for a better understanding of mechanisms of viral entry and for vaccine design.

Cryo-electron microscopy--a primer for the non-microscopist.

Cryo-electron microscopy (cryo-EM) is increasingly becoming a mainstream technology for studying the architecture of cells, viruses and protein assemblies at molecular resolution. Recent developments in microscope design and imaging hardware, paired with enhanced image processing and automation capabilities, are poised to further advance the effectiveness of cryo-EM methods. These developments promise to increase the speed and extent of automation, and to improve the resolutions that may be achieved, making this technology useful to determine a wide variety of biological structures. Additionally, established modalities for structure determination, such as X-ray crystallography and nuclear magnetic resonance spectroscopy, are being routinely integrated with cryo-EM density maps to achieve atomic-resolution models of complex, dynamic molecular assemblies. In this review, which is directed towards readers who are not experts in cryo-EM methodology, we provide an overview of emerging themes in the application of this technology to investigate diverse questions in biology and medicine. We discuss the ways in which these methods are being used to study structures of macromolecular assemblies that range in size from whole cells to small proteins. Finally, we include a description of how the structural information obtained by cryo-EM is deposited and archived in a publicly accessible database.

Structural features of galectin-9 and galectin-1 that determine distinct T cell death pathways.

The galectin family of lectins regulates multiple biologic functions, such as development, inflammation, immunity, and cancer. One common function of several galectins is the ability to trigger T cell death. However, differences among the death pathways triggered by various galectins with regard to glycoprotein receptors, intracellular death pathways, and target cell specificity are not well understood. Specifically, galectin-9 and galectin-1 both kill thymocytes, peripheral T cells, and T cell lines; however, we have found that galectin-9 and galectin-1 require different glycan ligands and glycoprotein receptors to trigger T cell death. The two galectins also utilize different intracellular death pathways, as galectin-9, but not galectin-1, T cell death was blocked by intracellular Bcl-2, whereas galectin-1, but not galectin-9, T cell death was blocked by intracellular galectin-3. Target cell susceptibility also differed between the two galectins, as galectin-9 and galectin-1 killed different subsets of murine thymocytes. To define structural features responsible for distinct activities of the tandem repeat galectin-9 and dimeric galectin-1, we created a series of bivalent constructs with galectin-9 and galectin-1 carbohydrate recognition domains connected by different peptide linkers. We found that the N-terminal carbohydrate recognition domain and linker peptide contributed to the potency of these constructs. However, we found that the C-terminal carbohydrate recognition domain was the primary determinant of receptor recognition, death pathway signaling, and target cell susceptibility. Thus, carbohydrate recognition domain specificity, presentation, and valency make distinct contributions to the specific effects of different galectins in initiating T cell death.