Eva Nogales

Structure of the alpha beta tubulin dimer by electron crystallography.

The αβ tubulin heterodimer is the structural subunit of microtubules, which are cytoskeletal elements that are essential for intracellular transport and cell division in all eukaryotes. Each tubulin monomer binds a guanine nucleotide, which is non-exchangeable when it is bound in the α subunit, or N site, and exchangeable when bound in the β subunit, or E site. The α- and β-tubulins share 40% amino-acid sequence identity, both exist in several isotype forms, and both undergo a variety of post-translational modifications. Limited sequence homology has been found with the proteins FtsZ and Misato, which are involved in cell division in bacteria and Drosophila, respectively. Here we present an atomic model of the αβ tubulin dimer fitted to a 3.7-Å density map obtained by electron crystallography of zinc-induced tubulin sheets. The structures of α- and β-tubulin are basically identical: each monomer is formed by a core of two β-sheets surrounded by α-helices. The monomer structure is very compact, but can be divided into three functional domains: the amino-terminal domain containing the nucleotide-binding region, an intermediate domain containing the Taxol-binding site, and the carboxy-terminal domain, which probably constitutes the binding surface for motor proteins.

High-Resolution Model of the Microtubule

A high-resolution model of the microtubule has been obtained by docking the crystal structure of tubulin into a 20 A map of the microtubule. The excellent fit indicates the similarity of the tubulin conformation in both polymers and defines the orientation of the tubulin structure within the microtubule. Long C-terminal helices form the crest on the outside of the protofilament, while long loops define the microtubule lumen. The exchangeable nucleotide in beta-tubulin is exposed at the plus end of the microtubule, while the proposed catalytic residue in alpha-tubulin is exposed at the minus end. Extensive longitudinal interfaces between monomers have polar and hydrophobic components. At the lateral contacts, a nucleotide-sensitive helix interacts with a loop that contributes to the binding site of taxol in beta-tubulin.

Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation.

Introduction Bacteria and archaea defend themselves against invasive DNA using adaptive immune systems comprising CRISPR (clustered regularly interspaced short palindromic repeats) loci and CRISPR-associated (Cas) genes. In association with Cas proteins, small CRISPR RNAs (crRNAs) guide the detection and cleavage of complementary DNA sequences. Type II CRISPR systems employ the RNA-guided endonuclease Cas9 to recognize and cleave double-stranded DNA (dsDNA) targets using conserved RuvC and HNH nuclease domains. Cas9-mediated cleavage is strictly dependent on the presence of a protospacer adjacent motif (PAM) in the target DNA. Recently, the biochemical properties of Cas9–guide RNA complexes have been harnessed for various genetic engineering applications and RNA-guided transcriptional control. Despite these ongoing successes, the structural basis for guide RNA recognition and DNA targeting by Cas9 is still unknown. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. (A) Crystal structures of S. pyogenes (SpyCas9) and A. naeslundii (AnaCas9) Cas9 proteins. (B) Left: Negative-stain EM reconstructions of apo-SpyCas9 (top) and SpyCas9-RNA-target DNA complex (bottom) show that nucleic acid binding causes a reorientation of the nuclease (blue) and α-helical (gray) lobes in SpyCas9. Right: Cartoon representations of the structures. tracrRNA, trans-activating crRNA. Rationale To compare the architectures and domain organization of diverse Cas9 proteins, the atomic structures of Cas9 from Streptococcus pyogenes (SpyCas) and Actinomyces naeslundii (AnaCas9) were determined by x-ray crystallography. Crosslinking of target DNA containing 5-bromodeoxyuridines was conducted to identify PAM-interacting regions in SpyCas9. To test functional interactions with nucleic acid ligands, structure-based mutant SpyCas9 proteins were assayed for endonuclease activity with radiolabeled oligonucleotide dsDNA targets, and target DNA binding was monitored by electrophoretic mobility shift assays. To compare conformations of Cas9 in different states of nucleic acid binding, three-dimensional reconstructions of apo-SpyCas9, SpyCas9:RNA, and SpyCas9:RNA:DNA were obtained by negative-stain single-particle electron microscopy. Guide RNA and target DNA positions were determined with streptavidin labeling. Exonuclease protection assays were carried out to determine the extent of Cas9–target DNA interactions. Results The 2.6 Å–resolution structure of apo-SpyCas9 reveals a bilobed architecture comprising a nuclease domain lobe and an α-helical lobe. Both lobes contain conserved clefts that may function in nucleic acid binding. Photocrosslinking experiments show that the PAM in target DNA is engaged by two tryptophan-containing flexible loops, and mutations of both loops impair target DNA binding and cleavage. The 2.2 Å–resolution crystal structure of AnaCas9 reveals the conserved structural core shared by all Cas9 enzyme subtypes, and both SpyCas9 and AnaCas9 adopt autoinhibited conformations in their apo forms. The electron microscopic (EM) reconstructions of SpyCas9:RNA and SpyCas9:RNA:DNA complexes reveal that guide RNA binding results in a conformational rearrangement and formation of a central channel for target DNA binding. Site-specific labeling of guide RNA and target DNA define the orientations of nucleic acids in the target-bound complex. Conclusion The SpyCas9 and AnaCas9 structures define the molecular architecture of the Cas9 enzyme family in which a conserved structural core encompasses the two nuclease domains responsible for DNA cleavage, while structurally divergent regions, including the PAM recognition loops, are likely responsible for distinct guide RNA and PAM specificities. Cas9 enzymes adopt a catalytically inactive conformation in the apo state, necessitating structural activation for DNA recognition and cleavage. Our EM analysis shows that by triggering a conformational rearrangement in Cas9, the guide RNA acts as a critical determinant of target DNA binding. Cas9 Solved Clustered regularly interspaced short palindromic repeats (CRISPR)–associated (Cas) loci allow prokaryotes to identify and destroy invading DNA. Not only important to bacteria, the universal value of Cas endonuclease specificity has also resulted in Cas9 being exploited as a tool for genome editing. Jinek et al. (10.1126/science.1247997, published online 6 February) determined the 2.6 and 2.2 angstrom resolution crystal structures of two Cas9 enzymes to reveal a common structural core with distinct peripheral elaborations. The enzymes are autoinhibited, undergo large conformational changes on binding RNA, and have channels lined with basic residues that are candidates for an RNA-DNA binding groove. Based on these and other insights from the structures, this work provides important revelations both for the CRISPR mechanism and for genome editing. Binding of a guide RNA triggers structural changes in a set of DNA-cleaving enzymes. Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA–induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

Tubulin and FtsZ form a distinct family of GTPases

Tubulin and FtsZ share a common fold of two domains connected by a central helix. Structure-based sequence alignment shows that common residues localize in the nucleotide-binding site and a region that interacts with the nucleotide of the next tubulin subunit in the protofilament, suggesting that tubulin and FtsZ use similar contacts to form filaments. Surfaces that would make lateral interactions between protofilaments or interact with motor proteins are, however, different. The highly conserved nucleotide-binding sites of tubulin and FtsZ clearly differ from those of EF-Tu and other GTPases, while resembling the nucleotide site of glyceraldehyde-3-phosphate dehydrogenase. Thus, tubulin and FtsZ form a distinct family of GTP-hydrolyzing proteins.

Complete subunit architecture of the proteasome regulatory particle.

The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes polyubiquitinated substrates. Here we used electron microscopy and a new heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle from yeast. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes and the protein unfolding machinery at subnanometre resolution, outlining the substrate’s path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.

Microtubule structure at 8 A resolution.

We have obtained a 3D reconstruction of intact microtubules, using cryoelectron microscopy and image processing, at a resolution of about 8 A, sufficient to resolve much of the secondary structure. The level of detail in the map allows docking of the tubulin structure previously determined by electron crystallography, with very strong constraints, providing several important insights not previously available through docking tubulin into lower-resolution maps. This work provides an improved picture of the interactions between adjacent protofilaments, which are responsible for microtubule stability, and also suggests that some structural features are different in microtubules from those in the zinc sheets with which the tubulin structure was determined.

The binding conformation of Taxol in β-tubulin: A model based on electron crystallographic density

The chemotherapeutic drug Taxol is known to interact within a specific site on β-tubulin. Although the general location of the site has been defined by photoaffinity labeling and electron crystallography, the original data were insufficient to make an absolute determination of the bound conformation. We have now correlated the crystallographic density with analysis of Taxol conformations and have found the unique solution to be a T-shaped Taxol structure. This T-shaped or butterfly structure is optimized within the β-tubulin site and exhibits functional similarity to a portion of the B9-B10 loop in the α-tubulin subunit. The model provides structural rationalization for a sizeable body of Taxol structure–activity relationship data, including binding affinity, photoaffinity labeling, and acquired mutation in human cancer cells.

Structures of the RNA-guided surveillance complex from a bacterial immune system

Bacteria and archaea acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA into clustered regularly interspaced short palindromic repeats (CRISPRs). These repetitive loci maintain a genetic record of all prior encounters with foreign transgressors. CRISPRs are transcribed and the long primary transcript is processed into a library of short CRISPR-derived RNAs (crRNAs) that contain a unique sequence complementary to a foreign nucleic-acid challenger. In Escherichia coli, crRNAs are incorporated into a multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defence), which is required for protection against bacteriophages. Here we use cryo-electron microscopy to determine the subnanometre structures of Cascade before and after binding to a target sequence. These structures reveal a sea-horse-shaped architecture in which the crRNA is displayed along a helical arrangement of protein subunits that protect the crRNA from degradation while maintaining its availability for base pairing. Cascade engages invading nucleic acids through high-affinity base-pairing interactions near the 5′ end of the crRNA. Base pairing extends along the crRNA, resulting in a series of short helical segments that trigger a concerted conformational change. This conformational rearrangement may serve as a signal that recruits a trans-acting nuclease (Cas3) for destruction of invading nucleic-acid sequences.

Structural insight into microtubule function.

Microtubules are polymers that are essential for, among other functions, cell transport and cell division in all eukaryotes. The regulation of the microtubule system includes transcription of different tubulin isotypes, folding of alpha/beta-tubulin heterodimers, post-translation modification of tubulin, and nucleotide-based microtubule dynamics, as well as interaction with numerous microtubule-associated proteins that are themselves regulated. The result is the precise temporal and spatial pattern of microtubules that is observed throughout the cell cycle. The recent high-resolution analysis of the structure of tubulin and the microtubule has brought new insight to the study of microtubule function and regulation, as well as the mode of action of antimitotic drugs that disrupt normal microtubule behavior. The combination of structural, genetic, biochemical, and biophysical data should soon give us a fuller understanding of the exquisite details in the regulation of the microtubule cytoskeleton.

Structural roles for human translation factor eIF3 in initiation of protein synthesis.

Protein synthesis in mammalian cells requires initiation factor eIF3, a ∼750-kilodalton complex that controls assembly of 40S ribosomal subunits on messenger RNAs (mRNAs) bearing either a 5′-cap or an internal ribosome entry site (IRES). Cryo–electron microscopy reconstructions show that eIF3, a five-lobed particle, interacts with the hepatitis C virus (HCV) IRES RNA and the 5′-cap binding complex eIF4F via the same domain. Detailed modeling of eIF3 and eIF4F onto the 40S ribosomal subunit reveals that eIF3 uses eIF4F or the HCV IRES in structurally similar ways to position the mRNA strand near the exit site of 40S, promoting initiation complex assembly.