Jelle B. Bultema

Structural basis for CRISPR RNA-guided DNA recognition by Cascade

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions.

Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated (cas) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1–4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a “seed sequence” at the 5′ end of the CRISPR RNA spacer. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli, termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems.

Light-responsive capture and release of DNA in a ternary supramolecular complex.

The wavelength determines whether DNA is captured in a light-responsive ternary supramolecular complex or released (see scheme). The reversible binding of DNA is triggered by a photoisomerization, which switches the complex from a multivalent to a monovalent binding mode.

High-light vs. low-light: Effect of light acclimation on photosystem II composition and organization in Arabidopsis thaliana

The structural response of photosystem II (PSII) and its light-harvesting proteins (LHCII) in Arabidopis thaliana after long-term acclimation to either high or low light intensity was characterized. Biochemical and structural analysis of isolated thylakoid membranes by electron microscopy indicates a distinctly different response at the level of PSII and LHCII upon plant acclimation. In high light acclimated plants, the C(2)S(2)M(2) supercomplex, which is the dominating form of PSII in Arabidopsis, is a major target of structural re-arrangement due to the down-regulation of Lhcb3 and Lhcb6 antenna proteins. The PSII ability to form semi-crystalline arrays in the grana membrane is strongly reduced compared to plants grown under optimal light conditions. This is due to the structural heterogeneity of PSII supercomplexes rather than to the action of PsbS protein as its level was unexpectedly reduced in high light acclimated plants. In low light acclimated plants, the architecture of the C(2)S(2)M(2) supercomplex and its ability to form semi-crystalline arrays remained unaffected but the density of PSII in grana membranes is reduced due to the synthesis of additional LHCII proteins. However, the C(2)S(2)M(2) supercomplexes in semi-crystalline arrays are more densely packed, which can be important for efficient energy transfer between PSII under light limiting conditions.

The Vesicle-inducing Protein 1 from Synechocystis sp. PCC 6803 Organizes into Diverse Higher-Ordered Ring Structures

The vesicle-inducing protein in plastids 1 (Vipp1) was found to be involved in thylakoid membrane formation in chloroplasts and cyanobacteria. In contrast to chloroplasts, it has been suggested that in cyanobacteria the protein is only tightly associated with the cytoplasmic membrane. In the present study we analyze and describe the subcellular localization and the oligomeric organization of Vipp1 from the cyanobacterium Synechocystis PCC 6803. Vipp1 forms stable dimers and higher-ordered oligomers in the cytoplasm as well as at both the cytoplasmic and thylakoid membrane. Vipp1 oligomers are organized in ring structures with a variable diameter of 25-33 nm and corresponding calculated molecular masses of approximately 1.6-2.2 MDa. Six different types of rings were found with an unusual 12-17-fold symmetrical conformation. The simultaneous existence of multiple types of rings is very unusual and suggests a special function of Vipp1. Involvement of diverse ring structures in vesicle formation is suggested.

Three-way stabilization of the covalent intermediate in amylomaltase, an alpha-amylase-like transglycosylase.

Amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77 that are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides. Using protein crystallography, we have generated a flip book movie of the amylomaltase catalytic cycle in atomic detail. The structures include a covalent glycosyl enzyme intermediate and a covalent intermediate in complex with an analogue of a co-substrate and show how the structures of both enzyme and substrate respond to the changes required by the catalytic cycle as it proceeds. Notably, the catalytic nucleophile changes conformation dramatically during the reaction. Also, Gln-256 on the 250s loop is involved in orienting the substrate in the +1 site. The absence of a suitable base in the covalent intermediate structure explains the low hydrolysis activity.

Crystal Structure and Biochemical Properties of a Novel Thermostable Esterase Containing an Immunoglobulin-Like Domain

Comparative analysis of the genome of the hyperthermophilic bacterium Thermotoga maritima revealed a hypothetical protein (EstA) with typical esterase features. The EstA protein was functionally produced in Escherichia coli and purified to homogeneity. It indeed displayed esterase activity with optima at or above 95 degrees C and at pH 8.5, with a preference for esters with short acyl chains (C2-C10). Its 2.6-A-resolution crystal structure revealed a classical alpha/beta hydrolase domain with a catalytic triad consisting of a serine, an aspartate, and a histidine. EstA is irreversibly inhibited by the organophosphate paraoxon. A 3.0-A-resolution structure confirmed that this inhibitor binds covalently to the catalytic serine residue of EstA. Remarkably, the structure also revealed the presence of an N-terminal immunoglobulin (Ig)-like domain, which is unprecedented among esterases. EstA forms a hexamer both in the crystal and in solution. Electron microscopy showed that the hexamer in solution is identical with the hexamer in the crystal, which is formed by two trimers, with the N-terminal domains facing each other. Mutational studies confirmed that residues Phe89, Phe112, Phe116, Phe246, and Trp377 affect enzyme activity. A truncated mutant of EstA, in which the Ig-like domain was removed, showed only 5% of wild-type activity, had lower thermostability, and failed to form hexamers. These data suggest that the Ig-like domain plays an important role in the enzyme multimerization and activity of EstA.

Imaging of organelles by electron microscopy reveals protein-protein interactions in mitochondria and chloroplasts

Ongoing progress in electron microscopy (EM) offers now an opening to visualize cells at the nanoscale by cryo‐electron tomography (ET). Large protein complexes can be resolved at near‐atomic resolution by single particle averaging. Some examples from mitochondria and chloroplasts illustrate the possibilities with an emphasis on the membrane organization. Cryo‐ET performed on non‐chemically fixed, unstained, ice‐embedded material can visualize specific large membrane protein complexes. In combination with averaging methods, 3D structures were calculated of mitochondrial ATP synthase at 6 nm resolution and of chloroplast photosystem II at 3.5 nm.

Efficient Light Harvesting in a Dark, Hot, Acidic Environment: The Structure and Function of PSI-LHCI from Galdieria sulphuraria

Photosystem I-light harvesting complex I (PSI-LHCI) was isolated from the thermoacidophilic red alga Galdieria sulphuraria, and its structure, composition, and light-harvesting function were characterized by electron microscopy, mass spectrometry, and ultrafast optical spectroscopy. The results show that Galdieria PSI is a monomer with core features similar to those of PSI from green algae, but with significant differences in shape and size. A comparison with the crystal structure of higher plant (pea) PSI-LHCI indicates that Galdieria PSI binds seven to nine light-harvesting proteins. Results from ultrafast optical spectroscopy show that the functional coupling of the LHCI proteins to the PSI core is tighter than in other eukaryotic PSI-LHCI systems reported thus far. This tight coupling helps Galdieria perform efficient light harvesting under the low-light conditions present in its natural endolithic habitat.

Vipp1 and PspA: Related but not twins

The Vesicle Inducing Protein in Plastids 1 (Vipp1) was suggested to be involved in thylakoid membrane formation in both chloroplasts and cyanobacteria. The protein shows sequence homology to the Phage Shock Protein A (PspA) from bacteria, and both proteins have similar secondary structures. 2D-structures of PspA and of Vipp1 have been determined by electron microscopy in the recent years. Both PspA and Vipp1 form large homooligomeric rings with high molecular masses but their ring dimensions differ significantly. Furthermore, Vipp1 forms rings with different rotational symmetries whereas PspA appears to form rings with singular rotational symmetry. In this article addendum we compare the structures of PspA and Vipp1. Furthermore, we suggest a spatial structural model of the observed Vipp1 rings.