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1.  Crystal structure of the antimicrobial peptidase lysostaphin from Staphylococcus simulans 
The Febs Journal  2014;281(18):4112-4122.
Staphylococcus simulans biovar staphylolyticus lysostaphin efficiently cleaves Staphylococcus aureus cell walls. The protein is in late clinical trials as a topical anti-staphylococcal agent, and can be used to prevent staphylococcal growth on artificial surfaces. Moreover, the gene has been both stably engineered into and virally delivered to mice or livestock to obtain resistance against staphylococci. Here, we report the first crystal structure of mature lysostaphin and two structures of its isolated catalytic domain at 3.5, 1.78 and 1.26 Å resolution, respectively. The structure of the mature active enzyme confirms its expected organization into catalytic and cell-wall-targeting domains. It also indicates that the domains are mobile with respect to each other because of the presence of a highly flexible peptide linker. The high-resolution structures of the catalytic domain provide details of Zn2+ coordination and may serve as a starting point for the engineering of lysostaphin variants with improved biotechnological characteristics.
Structured digital abstract
lysostaphin by x-ray crystallography (1,2).
PMCID: PMC4286107  PMID: 25039253
crystal structure; lysis; lysostaphin; Staphylococcus aureus; Staphylococcus simulans
2.  Carboxy terminus of GATA4 transcription factor is required for its cardiogenic activity and interaction with CDK4 
Mechanisms of Development  2014;134:31-41.
•Carboxy terminal region of GATA4 is required for cardiogenesis in Xenopus pluripotent explants and in embryos.•Carboxy terminus of GATA4 interacts with CDK4.•CDK4 enhances transcriptional and cardiogenic activity of GATA4.•GATA4-Tbx5 and GATA4-FOG2 interactions are not required for cardiogenesis.
GATA4-6 transcription factors regulate numerous aspects of development and homeostasis in multiple tissues of mesodermal and endodermal origin. In the heart, the best studied of these factors, GATA4, has multiple distinct roles in cardiac specification, differentiation, morphogenesis, hypertrophy and survival. To improve understanding of how GATA4 achieves its numerous roles in the heart, here we have focused on the carboxy-terminal domain and the residues required for interaction with cofactors FOG2 and Tbx5. We present evidence that the carboxy terminal region composed of amino acids 362–400 is essential for mediating cardiogenesis in Xenopus pluripotent explants and embryos. In contrast, the same region is not required for endoderm-inducing activity of GATA4. Further evidence is presented that the carboxy terminal cardiogenic region of GATA4 does not operate as a generic transcriptional activator. Potential mechanism of action of the carboxy terminal end of GATA4 is provided by the results showing physical and functional interaction with CDK4, including the enhancement of cardiogenic activity of GATA4 by CDK4. These results establish CDK4 as a GATA4 partner in cardiogenesis. The interactions of GATA4 with its other well described cofactors Tbx5 and FOG2 are known to be involved in heart morphogenesis, but their requirement for cardiac differentiation is unknown. We report that the mutations that disrupt interactions of GATA4 with Tbx5 and FOG2, G295S and V217G, respectively, do not impair cardiogenic activity of GATA4. These findings add support to the view that distinct roles of GATA4 in the heart are mediated by different determinants of the protein. Finally, we show that the rat GATA4 likely induces cardiogenesis cell autonomously or directly as it does not require activity of endodermal transcription factor Sox17, a GATA4 target gene that induces cardiogenesis non-cell autonomously.
PMCID: PMC4259525  PMID: 25241353
GATA4; Cardiogenesis; CDK4
3.  Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase 
Open Biology  2014;4(8):140110.
In mammals, a family of TET enzymes producing oxidized forms of 5-methylcytosine (5mC) plays an important role in modulating DNA demethylation dynamics. In contrast, nothing is known about the function of a single TET orthologue present in invertebrates. Here, we show that the honeybee TET (AmTET) catalytic domain has dioxygenase activity and converts 5mC to 5-hydroxymethylcytosine (5hmC) in a HEK293T cell assay. In vivo, the levels of 5hmC are condition-dependent and relatively low, but in testes and ovaries 5hmC is present at approximately 7–10% of the total level of 5mC, which is comparable to that reported for certain mammalian cells types. AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains. Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species. In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.
PMCID: PMC4150289  PMID: 25100549
epigenetic code; epigenomics; brain plasticity; phenotypic polymorphism; demethylation; social insect
4.  Structural basis of the methylation specificity of R.DpnI 
Nucleic Acids Research  2014;42(13):8745-8754.
R.DpnI consists of N-terminal catalytic and C-terminal winged helix domains that are separately specific for the Gm6ATC sequences in Dam-methylated DNA. Here we present a crystal structure of R.DpnI with oligoduplexes bound to the catalytic and winged helix domains and identify the catalytic domain residues that are involved in interactions with the substrate methyl groups. We show that these methyl groups in the Gm6ATC target sequence are positioned very close to each other. We further show that the presence of the two methyl groups requires a deviation from B-DNA conformation to avoid steric conflict. The methylation compatible DNA conformation is complementary with binding sites of both R.DpnI domains. This indirect readout of methylation adds to the specificity mediated by direct favorable interactions with the methyl groups and solvation/desolvation effects. We also present hydrogen/deuterium exchange data that support ‘crosstalk’ between the two domains in the identification of methylated DNA, which should further enhance R.DpnI methylation specificity.
PMCID: PMC4117772  PMID: 24966351
5.  Crystal structure of the 5hmC specific endonuclease PvuRts1I 
Nucleic Acids Research  2014;42(9):5929-5936.
PvuRts1I is a prototype for a larger family of restriction endonucleases that cleave DNA containing 5-hydroxymethylcytosine (5hmC) or 5-glucosylhydroxymethylcytosine (5ghmC), but not 5-methylcytosine (5mC) or cytosine. Here, we report a crystal structure of the enzyme at 2.35 Å resolution. Although the protein has been crystallized in the absence of DNA, the structure is very informative. It shows that PvuRts1I consists of an N-terminal, atypical PD-(D/E)XK catalytic domain and a C-terminal SRA domain that might accommodate a flipped 5hmC or 5ghmC base. Changes to predicted catalytic residues of the PD-(D/E)XK domain or to the putative pocket for a flipped base abolish catalytic activity. Surprisingly, fluorescence changes indicative of base flipping are not observed when PvuRts1I is added to DNA substrates containing pyrrolocytosine in place of 5hmC (5ghmC). Despite this caveat, the structure suggests a model for PvuRts1I activity and presents opportunities for protein engineering to alter the enzyme properties for biotechnological applications.
PMCID: PMC4027163  PMID: 24634440
6.  Isolation of monomeric photosystem II that retains the subunit PsbS 
Photosynthesis Research  2013;118(3):199-207.
Photosystem II has been purified from a transplastomic strain of Nicotiana tabacum according to two different protocols. Using the procedure described in Piano et al. (Photosynth Res 106:221–226, 2010) it was possible to isolate highly active PSII composed of monomers and dimers but depleted in their PsbS protein content. A “milder” procedure than the protocol reported by Fey et al. (Biochim Biophys Acta 1777:1501–1509, 2008) led to almost exclusively monomeric PSII complexes which in part still bind the PsbS protein. This finding might support a role for PSII monomers in higher plants.
PMCID: PMC3825537  PMID: 23975205
Photosystem II; Photosynthesis; PsbS; Thylakoid membranes; Nicotiana tabacum; Oligomeric state
7.  Anti-staphylococcal activities of lysostaphin and LytM catalytic domain 
BMC Microbiology  2012;12:97.
Lysostaphin and the catalytic domain of LytM cleave pentaglycine crossbridges of Staphylococcus aureus peptidoglycan. The bacteriocin lysostaphin is secreted by Staphylococcus simulans biovar staphylolyticus and directed against the cell walls of competing S. aureus. LytM is produced by S. aureus as a latent autolysin and can be activated in vitro by the removal of an N-terminal domain and occluding region.
We compared the efficacies of the lysostaphin and LytM catalytic domains using a newly developed model of chronic S. aureus infected eczema. Lysostaphin was effective, like in other models. In contrast, LytM was not significantly better than control. The different treatment outcomes could be correlated with in vitro properties of the proteins, including proteolytic stability, affinity to cell wall components other than peptidoglycan, and sensitivity to the ionic milieu.
Although lysostaphin and LytM cleave the same peptide bond in the peptidoglycan, the two enzymes have very different environmental requirements what is reflected in their contrasting performance in mouse eczema model.
PMCID: PMC3413552  PMID: 22672475
8.  Crystal structure and mechanism of action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI 
Nucleic Acids Research  2012;40(15):7563-7572.
DNA methylation-dependent restriction enzymes have many applications in genetic engineering and in the analysis of the epigenetic state of eukaryotic genomes. Nevertheless, high-resolution structures have not yet been reported, and therefore mechanisms of DNA methylation-dependent cleavage are not understood. Here, we present a biochemical analysis and high-resolution DNA co-crystal structure of the N6-methyladenine (m6A)-dependent restriction enzyme R.DpnI. Our data show that R.DpnI consists of an N-terminal catalytic PD-(D/E)XK domain and a C-terminal winged helix (wH) domain. Surprisingly, both domains bind DNA in a sequence- and methylation-sensitive manner. The crystal contains R.DpnI with fully methylated target DNA bound to the wH domain, but distant from the catalytic domain. Independent readout of DNA sequence and methylation by the two domains might contribute to R.DpnI specificity or could help the monomeric enzyme to cut the second strand after introducing a nick.
PMCID: PMC3424567  PMID: 22610857
9.  RIBER/DIBER: a software suite for crystal content analysis in the studies of protein–nucleic acid complexes 
Bioinformatics  2012;28(6):880-881.
Summary: Co-crystallization experiments of proteins with nucleic acids do not guarantee that both components are present in the crystal. We have previously developed DIBER to predict crystal content when protein and DNA are present in the crystallization mix. Here, we present RIBER, which should be used when protein and RNA are in the crystallization drop. The combined RIBER/DIBER suite builds on machine learning techniques to make reliable, quantitative predictions of crystal content for non-expert users and high-throughput crystallography.
Availability: The program source code, Linux binaries and a web server are available at RIBER/DIBER requires diffraction data to at least 3.0 Å resolution in MTZ or CIF (web server only) format. The RIBER/DIBER code is subject to the GNU Public License.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3307108  PMID: 22238259
10.  Amyloid-Associated Nucleic Acid Hybridisation 
PLoS ONE  2011;6(5):e19125.
Nucleic acids promote amyloid formation in diseases including Alzheimer's and Creutzfeldt-Jakob disease. However, it remains unclear whether the close interactions between amyloid and nucleic acid allow nucleic acid secondary structure to play a role in modulating amyloid structure and function. Here we have used a simplified system of short basic peptides with alternating hydrophobic and hydrophilic amino acid residues to study nucleic acid - amyloid interactions. Employing biophysical techniques including X-ray fibre diffraction, circular dichroism spectroscopy and electron microscopy we show that the polymerized charges of nucleic acids concentrate and enhance the formation of amyloid from short basic peptides, many of which would not otherwise form fibres. In turn, the amyloid component binds nucleic acids and promotes their hybridisation at concentrations below their solution Kd, as shown by time-resolved FRET studies. The self-reinforcing interactions between peptides and nucleic acids lead to the formation of amyloid nucleic acid (ANA) fibres whose properties are distinct from their component polymers. In addition to their importance in disease and potential in engineering, ANA fibres formed from prebiotically-produced peptides and nucleic acids may have played a role in early evolution, constituting the first entities subject to Darwinian evolution.
PMCID: PMC3098241  PMID: 21625537
11.  Crystallization of the Photosystem II core complex and its chlorophyll binding subunit CP43 from transplastomic plants of Nicotianatabacum 
Photosynthesis Research  2010;106(3):221-226.
Photosystem II from transplastomic plants of Nicotiana tabacum with a hexahistidine tag at the N-terminal end of the PsbE subunit (α-chain of the cytochrome b559) was purified according to the protocol of Fey et al. (BBA 12:1501–1509, 2008). The protein sample was then subjected to two additional gel filtration runs in order to increase its homogeneity and to standardize the amount of detergent. Large three dimensional crystals of the core complex were obtained. Crystals of one of its chlorophyll binding subunits (CP43) in isolation grew in very similar conditions that differed only in the concentration of the detergent. Diffraction of Photosystem II and CP43 crystals at various synchrotron beamlines was limited to a resolution of 7 and 14 Å, respectively. In both cases the diffraction quality was insufficient for an unambiguous assignment of the crystallographic lattice or space group.
PMCID: PMC3005121  PMID: 21063907
Photosystem II core complex; Higher plants; Nicotiana tabacum; CP43; 3D crystallization
12.  Hpy188I–DNA pre- and post-cleavage complexes—snapshots of the GIY-YIG nuclease mediated catalysis 
Nucleic Acids Research  2010;39(4):1554-1564.
The GIY-YIG nuclease domain is present in all kingdoms of life and has diverse functions. It is found in the eukaryotic flap endonuclease and Holliday junction resolvase Slx1–Slx4, the prokaryotic nucleotide excision repair proteins UvrC and Cho, and in proteins of ‘selfish’ genetic elements. Here we present the structures of the ternary pre- and post-cleavage complexes of the type II GIY-YIG restriction endonuclease Hpy188I with DNA and a surrogate or catalytic metal ion, respectively. Our structures suggest that GIY-YIG nucleases catalyze DNA hydrolysis by a single substitution reaction. They are consistent with a previous proposal that a tyrosine residue (which we expect to occur in its phenolate form) acts as a general base for the attacking water molecule. In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine. A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site. This metal ion contacts the phosphate proS oxygen atom and the leaving group 3′-oxygen atom, presumably to facilitate its departure. Taken together, our data reveal striking analogy in the absence of homology between GIY-YIG and ββα-Me nucleases.
PMCID: PMC3045582  PMID: 20935048
13.  DNA intercalation without flipping in the specific ThaI–DNA complex 
Nucleic Acids Research  2010;39(2):744-754.
The PD-(D/E)XK type II restriction endonuclease ThaI cuts the target sequence CG/CG with blunt ends. Here, we report the 1.3 Å resolution structure of the enzyme in complex with substrate DNA and a sodium or calcium ion taking the place of a catalytic magnesium ion. The structure identifies Glu54, Asp82 and Lys93 as the active site residues. This agrees with earlier bioinformatic predictions and implies that the PD and (D/E)XK motifs in the sequence are incidental. DNA recognition is very unusual: the two Met47 residues of the ThaI dimer intercalate symmetrically into the CG steps of the target sequence. They approach the DNA from the minor groove side and penetrate the base stack entirely. The DNA accommodates the intercalating residues without nucleotide flipping by a doubling of the CG step rise to twice its usual value, which is accompanied by drastic unwinding. Displacement of the Met47 side chains from the base pair midlines toward the downstream CG steps leads to large and compensating tilts of the first and second CG steps. DNA intercalation by ThaI is unlike intercalation by HincII, HinP1I or proteins that bend or repair DNA.
PMCID: PMC3025569  PMID: 20861000
14.  Crystal structure of the ββα-Me type II restriction endonuclease Hpy99I with target DNA 
Nucleic Acids Research  2009;37(11):3799-3810.
The ββα-Me restriction endonuclease (REase) Hpy99I recognizes the CGWCG target sequence and cleaves it with unusual stagger (five nucleotide 5′-recessed ends). Here we present the crystal structure of the specific complex of the dimeric enzyme with DNA. The Hpy99I protomer consists of an antiparallel β-barrel and two β4α2 repeats. Each repeat coordinates a structural zinc ion with four cysteine thiolates in two CXXC motifs. The ββα-Me region of the second β4α2 repeat holds the catalytic metal ion (or its sodium surrogate) via Asp148 and Asn165 and activates a water molecule with the general base His149. In the specific complex, Hpy99I forms a ring-like structure around the DNA that contacts DNA bases on the major and minor groove sides via the first and second β4α2 repeats, respectively. Hpy99I interacts with the central base pair of the recognition sequence only on the minor groove side, where A:T resembles T:A and G:C is similar to C:G. The Hpy99I–DNA co-crystal structure provides the first detailed illustration of the ββα-Me site in REases and complements structural information on the use of this active site motif in other groups of endonucleases such as homing endonucleases (e.g. I-PpoI) and Holliday junction resolvases (e.g. T4 endonuclease VII).
PMCID: PMC2699513  PMID: 19380375
15.  Central base pair flipping and discrimination by PspGI 
Nucleic Acids Research  2008;36(19):6109-6117.
PspGI is a representative of a group of restriction endonucleases that recognize a pentameric sequence related to CCNGG. Unlike the previously investigated Ecl18kI, which does not have any specificity for the central base pair, PspGI prefers A/T over G/C in its target site. Here, we present a structure of PspGI with target DNA at 1.7 Å resolution. In this structure, the bases at the center of the recognition sequence are extruded from the DNA and flipped into pockets of PspGI. The flipped thymine is in the usual anti conformation, but the flipped adenine takes the normally unfavorable syn conformation. The results of this and the accompanying manuscript attribute the preference for A/T pairs over G/C pairs in the flipping position to the intrinsically lower penalty for flipping A/T pairs and to selection of the PspGI pockets against guanine and cytosine. Our data show that flipping can contribute to the discrimination between normal bases. This adds a new role to base flipping in addition to its well-known function in base modification and DNA damage repair.
PMCID: PMC2577326  PMID: 18829716
16.  How PspGI, catalytic domain of EcoRII and Ecl18kI acquire specificities for different DNA targets 
Nucleic Acids Research  2008;36(19):6101-6108.
Restriction endonucleases Ecl18kI and PspGI/catalytic domain of EcoRII recognize CCNGG and CCWGG sequences (W stands for A or T), respectively. The enzymes are structurally similar, interact identically with the palindromic CC:GG parts of their recognition sequences and flip the nucleotides at their centers. Specificity for the central nucleotides could be influenced by the strength/stability of the base pair to be disrupted and/or by direct interactions of the enzymes with the flipped bases. Here, we address the importance of these contributions. We demonstrate that wt Ecl18kI cleaves oligoduplexes containing canonical, mismatched and abasic sites in the central position of its target sequence CCNGG with equal efficiencies. In contrast, substitutions in the binding pocket for the extrahelical base alter the Ecl18kI preference for the target site: the W61Y mutant prefers only certain mismatched substrates, and the W61A variant cuts exclusively at abasic sites, suggesting that pocket interactions play a major role in base discrimination. PspGI and catalytic domain of EcoRII probe the stability of the central base pair and the identity of the flipped bases in the pockets. This ‘double check’ mechanism explains their extraordinary specificity for an A/T pair in the flipping position.
PMCID: PMC2577355  PMID: 18820295
17.  Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence 
Nucleic Acids Research  2007;35(14):4792-4799.
Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI–DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution.
PMCID: PMC1950555  PMID: 17617640
18.  Restriction endonuclease MvaI is a monomer that recognizes its target sequence asymmetrically 
Nucleic Acids Research  2007;35(6):2035-2046.
Restriction endonuclease MvaI recognizes the sequence CC/WGG (W stands for A or T, ‘/’ designates the cleavage site) and generates products with single nucleotide 5′-overhangs. The enzyme has been noted for its tolerance towards DNA modifications. Here, we report a biochemical characterization and crystal structures of MvaI in an apo-form and in a complex with target DNA at 1.5 Å resolution. Our results show that MvaI is a monomer and recognizes its pseudosymmetric target sequence asymmetrically. The enzyme consists of two lobes. The catalytic lobe anchors the active site residues Glu36, Asp50, Glu55 and Lys57 and contacts the bases from the minor grove side. The recognition lobe mediates all major grove interactions with the bases. The enzyme in the crystal is bound to the strand with T at the center of the recognition sequence. The crystal structure with calcium ions and DNA mimics the prereactive state. MvaI shows structural similarities to BcnI, which cleaves the related sequence CC/SGG and to MutH enzyme, which is a component of the DNA repair machinery, and nicks one DNA strand instead of making a double-strand break.
PMCID: PMC1874612  PMID: 17344322

Results 1-18 (18)