Robert D. Fagerlund

The extrinsic proteins of Photosystem II

In this review we examine the structure and function of the extrinsic proteins of Photosystem II. These proteins include PsbO, present in all oxygenic organisms, the PsbP and PsbQ proteins, which are found in higher plants and eukaryotic algae, and the PsbU, PsbV, CyanoQ, and CyanoP proteins, which are found in the cyanobacteria. These proteins serve to optimize oxygen evolution at physiological calcium and chloride concentrations. They also shield the Mn(4)CaO(5) cluster from exogenous reductants. Numerous biochemical, genetic and structural studies have been used to probe the structure and function of these proteins within the photosystem. We will discuss the most recent proposed functional roles for these components, their structures (as deduced from biochemical and X-ray crystallographic studies) and the locations of their proposed binding domains within the Photosystem II complex. This article is part of a Special Issue entitled: Photosystem II.

A mutation of human cytochrome c enhances the intrinsic apoptotic pathway but causes only thrombocytopenia.

We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis.

CRISPR-Cas: Adapting to change

Variation in prokaryote adaptive immunity To repel infection by phage and mobile genetic elements, prokaryotes have a form of adaptive immune response and memory invested in clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas). This molecular machinery can recognize and remember foreign nucleic acids by capturing and retaining small nucleotide sequences. On subsequent encounters, the cognate CRISPR-Cas marshals enzymatic defenses to destroy infecting elements that contain the same sequences. Jackson et al. review the molecular mechanisms by which diverse CRISPR-Cas systems adapt and anticipate novel threats and evasive countermeasures from mobile genetic elements. Science, this issue p. eaal5056 BACKGROUND The arms race between prokaryotes and their perpetually evolving predators has fueled the evolution of a defense arsenal. The so-called CRISPR-Cas systems—clustered regularly interspaced short palindromic repeats and associated proteins—are adaptive immune defense systems found in bacteria and archaea. The recent exponential growth of research in the CRISPR field has led to the discovery of a diverse range of CRISPR-Cas systems and insight into their defense functions. These systems are divided into two major classes and six types. Each system consists of two components: a locus for memory storage (the CRISPR array) and cas genes that encode the machinery driving immunity. Information stored within CRISPR arrays is used to direct the sequence-specific destruction of invading genetic elements, including viruses and plasmids. As such, all CRISPR-Cas immune systems are reliant on the formation of CRISPR memories, known as spacers, to facilitate future defense. To form these memories, small fragments of invader nucleic acids are added as spacers to the CRISPR memory banks in a process termed CRISPR adaptation. The genetic basis of immunity means that CRISPR adaptation provides heritable benefits, an attribute that is unparalleled in eukaryotic immune systems. There is widespread evidence of highly active CRISPR adaptation in nature, and it is clear that these systems play important roles in shaping microbial evolution and global ecological networks. ADVANCES CRISPR adaptation requires several processes, including selection and processing of spacer precursors and their subsequent localization to, and integration into, the CRISPR loci. Although our understanding of all facets of the CRISPR adaptation pathway is not yet complete, considerable progress has been made in the past few years. At the heart of CRISPR adaptation is a protein complex, the Cas1-Cas2 “workhorse,” which catalyzes the addition of new spacers to CRISPR memory banks. A combination of functional assays and high-resolution structures of Cas1-Cas2 complexes has recently led to major advances. There is now a sound understanding of how foreign DNA is converted to prespacer substrates and captured by the Cas1-Cas2 complex. After this, Cas1-Cas2 locates the genomic CRISPR locus and docks in the appropriate position for insertion of the new spacer into the CRISPR array, while duplicating a CRISPR repeat. The cues directing the docking of substrate-laden Cas1-Cas2 differ between systems, with some relying on intrinsic sequence specificity and others assisted by host proteins. Before integration, accurate processing of the spacer precursors is required to ensure that the new spacers are compatible with the protein machinery in order to elicit CRISPR-Cas defense. For a given CRISPR-Cas system, spacers must typically be of a certain length and be inserted into the CRISPR in a specific orientation. It is becoming increasingly apparent that Cas1-Cas2 complexes from diverse systems are capable of ensuring that these system-specific factors are met with high fidelity. New findings also account for the ordering of stored memories: Typically, the insertion of new spacers is directed to one end of CRISPR arrays, and it has been shown that this enhances immunity against recently encountered invaders. The chronological ordering of new spacers has enabled insights into the temporal dynamics of interactions between hosts and invaders that are constantly changing. Some CRISPR-Cas systems use existing spacers to recognize previously encountered elements and promote the formation of new CRISPR memories, a process known as primed CRISPR adaptation. Viruses and plasmids that have escaped previous CRISPR-Cas defenses through genetic mutations trigger primed CRISPR adaptation. Several recent studies have revealed that primed CRISPR adaptation is also strongly promoted by recurrent invaders, even in the absence of escape mutations. This has led to previously separate paradigms of invader destruction and primed CRISPR adaptation beginning to converge into a unified model. OUTLOOK CRISPR adaptation is crucial for ensuring both population-level protection through spacer diversity and protection of the host through invader clearance. Although many studies have explored CRISPR adaptation in a broad range of host-specific and metagenomic contexts, much of the mechanistic detail has been gleaned from studying a relatively small subset of systems. Thus, despite the relative wealth of mechanistic information about CRISPR adaptation in a few specific types, work in other systems continues to reveal distinct modes of operation for spacer acquisition. Therefore, studies of CRISPR adaptation in alternative systems are necessary to determine which processes are conserved and which are system-specific. An important remaining question is why the enhanced primed CRISPR adaptation commonly found in type I systems has not yet been observed in other types. Do other systems possess analogous mechanisms that have yet to be discovered, or does the absence of priming in these systems explain the prevalence of type I systems in nature? Future expansion of our understanding of how CRISPR adaptation is carried out in the diverse repertoire of CRISPR-Cas systems is vital for maximizing the potential for repurposing the spacer acquisition machinery in biotechnological applications. Commandeering CRISPR adaptation for on-demand memory formation will usher in a new era of biological information storage, with many applications that await discovery. Many hues of the CRISPR-Cas adaptation machinery. The complex of the Cas1 and Cas2 proteins, which is the workhorse of CRISPR adaptation in diverse CRISPR-Cas prokaryotic immune systems, is depicted with a DNA substrate. Despite the near-ubiquitous Cas1-Cas2 molecular machinery, type-specific differences in the insertion of new information into CRISPR memory banks are beginning to come to light. Bacteria and archaea are engaged in a constant arms race to defend against the ever-present threats of viruses and invasion by mobile genetic elements. The most flexible weapons in the prokaryotic defense arsenal are the CRISPR-Cas adaptive immune systems. These systems are capable of selective identification and neutralization of foreign DNA and/or RNA. CRISPR-Cas systems rely on stored genetic memories to facilitate target recognition. Thus, to keep pace with a changing pool of hostile invaders, the CRISPR memory banks must be regularly updated with new information through a process termed CRISPR adaptation. In this Review, we outline the recent advances in our understanding of the molecular mechanisms governing CRISPR adaptation. Specifically, the conserved protein machinery Cas1-Cas2 is the cornerstone of adaptive immunity in a range of diverse CRISPR-Cas systems.

The Cpf1 CRISPR-Cas protein expands genome-editing tools

CRISPR-Cas systems have immense biotechnological utility. A recent study reveals the potential of the Cpf1 nuclease to complement and extend the existing CRISPR-Cas9 genome-editing tools.

The Proapoptotic G41S Mutation to Human Cytochrome c Alters the Heme Electronic Structure and Increases the Electron Self-Exchange Rate.

The naturally occurring G41S mutation to human (Hs) cytochrome (cyt) c enhances apoptotic activity based upon previous in vitro and in vivo studies, but the molecular mechanism underlying this enhancement remains unknown. Here, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and density functional theory (DFT) calculations have been used to identify the structural and electronic differences between wild-type (WT) and G41S Hs cyt c. S41 is part of the hydrogen bonding network for propionate 7 of heme pyrrole ring A in the X-ray structure of G41S Hs cyt c and, compared to WT, G41S Hs cyt c has increased spin density on pyrrole ring C and a faster electron self-exchange rate. DFT calculations illustrate an electronic mechanism where structural changes near ring A can result in electronic changes at ring C. Since ring C is part of the solvent-exposed protein surface, we propose that this heme electronic structure change may ultimately be responsible for the enhanced proapoptotic activity of G41S Hs cyt c.

Crystal Structure of PsbQ from Synechocystis sp. PCC 6803 at 1.8 Å: Implications for Binding and Function in Cyanobacterial Photosystem II

In Synechocystis sp. PCC 6803, PsbQ is associated with photosystem II (PSII) complexes with the highest activity and stability. However, this subunit is not found in PSII X-ray crystallographic structures from Thermosynechococcus elongatus or Thermosynechococcus vulcanus. We present the crystal structure of cyanobacterial PsbQ determined in the presence and absence of Zn(2+). The protein has a well-defined helical core, containing four helices arranged in an up-down-up-down fold. A conserved potential interaction site composed of a divalent metal binding site and adjacent hydrophobic pocket has been identified.

Spacer capture and integration by a type I-F Cas1?Cas2-3 CRISPR adaptation complex

Significance CRISPR-Cas systems provide prokaryotic adaptive immunity against invading genetic elements. For immunity, fragments of invader DNA are integrated into CRISPR arrays by Cas1 and Cas2 proteins. Type I-F systems contain a unique fusion of Cas2 to Cas3, the enzyme responsible for destruction of invading DNA. Structural, biophysical, and biochemical analyses of Cas1 and Cas2-3 from Pectobacterium atrosepticum demonstrated that they form a 400-kDa complex with a Cas14:Cas2-32 stoichiometry. Cas1–Cas2-3 binds, processes, and catalyzes the integration of DNA into CRISPR arrays independent of Cas3 activity. The arrangement of Cas3 in the complex, together with its redundant role in processing and integration, supports a scenario where Cas3 couples invader destruction with immunization—a process recently demonstrated in vivo. CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1–Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas14–Cas2-32 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1–Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1–Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3′-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1–Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1–Cas2-3 adaptation complexes and their integration mechanism.

Soluble expression and purification of tumor suppressor WT1 and its zinc finger domain

Full length murine WT1 and its zinc finger domain were separately inserted into Escherichia coli expression vectors with various fusion tags on either terminus by Gateway technology (Invitrogen) and expression of soluble protein was assessed. Fusion proteins including the four zinc finger domains of WT1 were used to optimize expression and purification conditions and to characterize WT1:DNA interactions in the absence of WT1:WT1 interactions. Zinc finger protein for in vitro characterization was prepared by IMAC purification of WT1 residues 321-443 with a thioredoxin-hexahistidine N-terminal fusion, followed by 3C protease cleavage to liberate the zinc fingers and cation exchange chromatography to isolate the zinc fingers and reduce the level of the truncated forms. Titration of zinc finger domain with a binding site from the PDGFA promoter gave a K(d) of 100±30nM for the -KTS isoform and 130±40nM for the +KTS isoform. The zinc finger domain was also co-crystallized with a double-stranded DNA oligonucleotide, yielding crystals that diffract to 5.5Å. Using protocols established for the zinc finger domain, we expressed soluble full-length WT1 with an N-terminal thioredoxin domain and purified the fusion protein by IMAC. In electro-mobility shift assays, purified full-length WT1 bound double-stranded oligonucleotides containing known WT1 binding sites, but not control oligonucleotides. Two molecules of WT1 bind an oligonucleotide presenting the full PDGFA promoter, demonstrating that active full-length WT1 can be produced in E. coli and used to investigate WT1 dimerization in complex with DNA in vitro.

The importance of the hydrophilic region of PsbL for the plastoquinone electron acceptor complex of Photosystem II

The PsbL protein is a 4.5kDa subunit at the monomer-monomer interface of Photosystem II (PS II) consisting of a single membrane-spanning domain and a hydrophilic stretch of ~15 residues facing the cytosolic (or stromal) side of the photosystem. Deletion of conserved residues in the N-terminal region has been used to investigate the importance of this hydrophilic extension. Using Synechocystis sp. PCC 6803, three deletion strains: ∆(N6-N8), ∆(P11-V12) and ∆(E13-N15), have been created. The ∆(N6-N8) and ∆(P11-V12) strains remained photoautotrophic but were more susceptible to photodamage than the wild type; however, the ∆(E13-N15) cells had the most severe phenotype. The Δ(E13-N15) mutant showed decreased photoautotrophic growth, a reduced number of PS II centers, impaired oxygen evolution in the presence of PS II-specific electron acceptors, and was highly susceptible to photodamage. The decay kinetics of chlorophyll a variable fluorescence after a single turnover saturating flash and the sensitivity to low concentrations of PS II-directed herbicides in the Δ(E13-N15) strain indicate that the binding of plastoquinone to the QB-binding site had been altered such that the affinity of QB is reduced. In addition, the PS II-specific electron acceptor 2,5-dimethyl-p-benzoquinone was found to inhibit electron transfer through the quinone-acceptor complex of the ∆(E13-N15) strain. The PsbL Y20A mutant was also investigated and it exhibited increased susceptibility to photodamage and increased herbicide sensitivity. Our data suggest that the N-terminal hydrophilic region of PsbL influences forward electron transfer from QA through indirect interactions with the D-E loop of the D1 reaction center protein. Our results further indicate that disruption of interactions between the N-terminal region of PsbL and other PS II subunits or lipids destabilizes PS II dimer formation. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Structure-Function Studies of the Photosystem II Extrinsic Subunits PsbQ and PsbP from the Cyanobacterium Synechocystis sp. PCC 6803

The oxygen-evolving centre of Photosystem II (PS II) is located on the lumenal side of the PS II complex and is surrounded by a group of polypeptides known as the extrinsic proteins. In PS II of the cyanobacterium Synechocystis sp. PCC 6803 six extrinsic proteins have been identified: PsbO, PsbP, PsbQ, PsbU, PsbV and Psb27. We have obtained two X-ray crystallographic structures of PsbQ, from crystals grown in either the presence or absence of Zn2+ ions. The structures were solved by multiple wavelength anomalous dispersion phasing using data obtained from a selenomethionine derivative and have essentially identical structures. The protein was found to consist of a four-helix bundle with an up-down-up-down fold. His76 (present in a unique HisGlyPro motif which forms a kink in helix 2 of cyanobacterial PsbQ) together with Asp116 (helix 3), coordinates Zn adjacent to a hydrophobic cavity on the H2/H3 face. We hypothesize this metal binding site and cavity may play a role in a protein-protein interaction with another PS II subunit. Similar structure-function studies are underway for the PsbP subunit; to facilitate solving the structure of PsbP in solution we have determined the NMR backbone chemical shift values of isotopically labelled recombinant PsbP.