The essential function of HP1 beta: a case of the tail wagging the dog?

A large body of work in various organisms has shown that the presence of HP1 structural proteins and methylated lysine 9 of histone H3 (H3K9me) represent the characteristic hallmarks of heterochromatin. We propose that a more critical assessment of the physiological importance of the H3K9me-HP1 interaction is warranted in light of recent studies on the mammalian HP1 beta protein. Based on this new research, we conclude that the essential function of HP1 beta (and perhaps that of its orthologues in other species) lies outside the canonical heterochromatic H3K9me-HP1 interaction. We suggest instead that binding of a small fraction of HP1 beta to the H3 histone fold performs a critical role in heterochromatin function and organismal survival.

Crystallographic snapshots of the complete reaction cycle of nicotine degradation by an amine oxidase of the monoamine oxidase (MAO) family.

FAD-linked oxidases constitute a class of enzymes which catalyze dehydrogenation as a fundamental biochemical reaction, followed by reoxidation of reduced flavin. Here, we present high-resolution crystal structures showing the flavoenzyme 6-hydroxy-l-nicotine oxidase in action. This enzyme was trapped during catalytic degradation of the native substrate in a sequence of discrete reaction states corresponding to the substrate-reduced enzyme, a complex of the enzyme with the intermediate enamine product and formation of the final aminoketone product. The inactive d-stereoisomer binds in mirror symmetry with respect to the catalytic axis, revealing absolute stereospecificity of hydrogen transfer to the flavin. The structural data suggest deprotonation of the substrate when bound at the active site, an overall binary complex mechanism and oxidation by direct hydride transfer. The amine nitrogen has a critical role in the dehydrogenation step and may activate carbocation formation at the α-carbon via delocalization from the lone pair to σ* C(α)-H. Enzymatically assisted hydrolysis of the intermediate product occurs at a remote (P site) cavity. Substrate entry and product exit follow different paths. Structural and kinetic data suggest that substrate can also bind to the reduced enzyme, associated with slower reoxidation as compared to the rate of reoxidation of free enzyme. The results are of general relevance for the mechanisms of flavin amine oxidases.

Crystal structure of D-serine dehydratase from Escherichia coli.

D-Serine dehydratase from Escherichia coli is a member of the ß-family (fold-type II) of the pyridoxal 5'-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97Å-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55Å resolution. The structure of DSD reveals a larger pyridoxal 5'-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the ß-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.

Mechanism of phosphoryl transfer catalyzed by shikimate kinase from Mycobacterium tuberculosis.

The structural mechanism of the catalytic functioning of shikimate kinase from Mycobacterium tuberculosis was investigated on the basis of a series of high-resolution crystal structures corresponding to individual steps in the enzymatic reaction. The catalytic turnover of shikimate and ATP into the products shikimate-3-phosphate and ADP, followed by release of ADP, was studied in the crystalline environment. Based on a comparison of the structural states before initiation of the reaction and immediately after the catalytic step, we derived a structural model of the transition state that suggests that phosphoryl transfer proceeds with inversion by an in-line associative mechanism. The random sequential binding of shikimate and nucleotides is associated with domain movements. We identified a synergic mechanism by which binding of the first substrate may enhance the affinity for the second substrate.

Crystallization and preliminary X-ray diffraction analysis of the small subunit of the heterodimeric restriction endonuclease R.BspD6I.

The heterodimeric restriction endonuclease R.BspD6I is composed of a small subunit with a cleavage site and a large subunit, containing a recognition domain and a cleavage domain, that may function separately as a monomeric nicking endonuclease. Here, the crystallization of the small subunit and diffraction data collection to 1.5 A resolution are reported.

Structure of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum.

Cyclase-associated proteins (CAPs) are widely distributed and highly conserved proteins that regulate actin remodeling in response to cellular signals. The N termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C termini bind to G-actin and thereby alter the dynamic rearrangements of the microfilament system. We report here the X-ray structure of the core of the N-terminal domain of the CAP from Dictyostelium discoideum, which comprises residues 51-226, determined by a combination of single isomorphous replacement with anomalous scattering (SIRAS). The overall structure of this fragment is an alpha helix bundle composed of six antiparallel helices. Results from gel filtration and crosslinking experiments for CAP(1-226), CAP(255-464), and the full-length protein, together with the CAP N-terminal domain structure and the recently determined CAP C-terminal domain structure, provide evidence that the functional structure of CAP is multimeric.

Coenzyme F420-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) from Methanopyrus kandleri: a methanogenic enzyme with an unusual quarternary structure.

The fourth reaction step of CO(2)-reduction to methane in methanogenic archaea is catalyzed by coenzyme F(420)-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd). We have structurally characterized this enzyme in the selenomethionine-labelled form from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri at 1.54A resolution using the single wavelength anomalous dispersion method for phase determination. Mtd was found to be a homohexameric protein complex that is organized as a trimer of dimers. The fold of the individual subunits is composed of two domains: a larger alpha,beta domain and a smaller helix bundle domain with a short C-terminal beta-sheet segment. In the homohexamer the alpha,beta domains are positioned at the outside of the enzyme, whereas, the helix bundle domains assemble towards the inside to form an unusual quarternary structure with a 12-helix bundle around a 3-fold axis. No structural similarities are detectable to other enzymes with F(420) and/or substituted tetrahydropterins as substrates. The substrate binding sites of F(420) and methylenetetrahydromethanopterin are most likely embedded into a crevice between the domains of one subunit, their isoalloxazine and tetrahydropterin rings being placed inside a pocket formed by this crevice and a loop segment of the adjacent monomer of the dimer. Mtd revealed the highest stability at low salt concentrations of all structurally characterized enzymes from M.kandleri. This finding might be due to the compact quaternary structure that buries 36% of the monomer surface and to the large number of ion pairs.

Crystallization and preliminary crystallographic analysis of the site-specific DNA nickase Nb.BspD6I.

Crystals of site-specific DNA nickase Nb.BspD6I (of molecular weight 70.8 kDa) have been grown at 291 K using PEG 8000 as precipitant. The diffraction pattern of the crystal extends to 3.3 A resolution at 100 K. The crystal belongs to space group P2(1), with unit-cell parameters a = 57.76, b = 90.67, c = 71.71, beta = 110.1 degrees. There is one molecule in the asymmetric unit and the solvent content is estimated to be 53% by volume.

Crystallization and preliminary crystallographic analysis of the (cytosine-5)-DNA methyltransferase NlaX from Neisseria lactamica.

Crystals of the (cytosine-5)-DNA methyltransferase NlaX from Neisseria lactamica (molecular weight 36.5 kDa) have been grown at 291 K using 2.5 M NaCl as precipitant. The crystals diffract to 3.0 A resolution at 100 K. The crystals belong to space group P321, with unit-cell parameters a = 121.98, b = 121.98, c = 56.71 A. There is one molecule in the asymmetric unit and the solvent content is estimated to be 62.1% by volume.

Flexibility of the linker between the domains of DNA methyltransferase SsoII revealed by small-angle X-ray scattering: implications for transcription regulation in SsoII restriction-modification system.

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) consists of a methyltransferase domain (residues 72-379) and an N-terminal region (residues 1-71) which regulates transcription in SsoII restriction-modification system. Small-angle X-ray scattering (SAXS) is employed here to study the low resolution structure of M.SsoII and its complex with DNA containing the methylation site. The shapes reconstructed ab initio from the SAXS data reveal two distinct protein domains of unequal size. The larger domain matches the crystallographic structure of a homologous DNA methyltransferase HhaI (M.HhaI), and the cleft in this domain is occupied by DNA in the model of the complex reconstructed from the SAXS data. This larger domain can thus be identified as the methyltransferase domain whereas the other domain represents the N-terminal region. Homology modeling of the M.SsoII structure is performed by using the model of M.HhaI for the methyltransferase domain and representing the N-terminal region either as a flexible chain of dummy residues or as a rigid structure of a homologous protein (phage 434 repressor) connected to the methyltransferase domain by a short flexible linker. Both models are compatible with the SAXS data and demonstrate high mobility of the N-terminal region. The linker flexibility might play an important role in the function of M.SsoII as a transcription factor.

Structural comparison of the poplar plastocyanin isoforms PCa and PCb sheds new light on the role of the copper site geometry in interactions with redox partners in oxygenic photosynthesis.

Plastocyanin (PC) from poplar leaves is present in two isoforms, PCa and PCb, which differ in sequence by amino acid replacements at locations remote from the copper center and simultaneously act in the photosynthetic electron-transport chain. We describe ultra-high resolution structures of PCa and high-resolution structures of PCb, both under oxidizing and reducing conditions at pH 4, 6 and 8. The docking on cytochrome f and photosystem I, respectively, has been modeled for both isoforms. PCa and PCb exhibit closely similar overall and active-site structures, except for a difference in the relative orientation of the acidic patches. The isoforms exhibit substantial differences in the dependence of the reduced (Cu(I)) geometry on pH. In PCa, the decrease in pH causes a gradual dissociation of His87 from Cu(I) at low pH, probably adopting a neutral tautomeric state. In PCb, the histidine remains covalently bound to Cu(I) and may adopt a doubly protonated state at low pH. The fact that both isoforms have similar although not identical functions in photosynthetic electron flows suggests that the His87 imidazole does not play a crucial role for the pathway of electron transport from cytochrome f to oxidized PC.

Crystal structure analysis of free and substrate-bound 6-hydroxy-L-nicotine oxidase from Arthrobacter nicotinovorans.

The pathway for oxidative degradation of nicotine in Arthrobacter nicotinovorans includes two genetically and structurally unrelated flavoenzymes, 6-hydroxy-L-nicotine oxidase (6HLNO) and 6-hydroxy-D-nicotine oxidase, which act with absolute stereospecificity on the L- and D-forms, respectively, of 6-hydroxy-nicotine. We solved the crystal structure of 6HLNO at 1.95 A resolution by combined isomorphous/multiple-wavelength anomalous dispersion phasing. The overall structure of each subunit of the 6HLNO homodimer and the folds of the individual domains are closely similar as in eukaryotic monoamine oxidases. Unexpectedly, a diacylglycerophospholipid molecule was found to be non-covalently bound to each protomer of 6HLNO. The fatty acid chains occupy hydrophobic channels that penetrate deep into the interior of the substrate-binding domain of each subunit. The solvent-exposed glycerophosphate moiety is located at the subunit-subunit interface. We further solved the crystal structure of a complex of dithionite-reduced 6HLNO with the natural substrate 6-hydroxy-L-nicotine at 2.05 A resolution. The location of the substrate in a tight cavity suggests that the binding geometry of this unproductive complex may be closely similar as under oxidizing conditions. The observed orientation of the bound substrate relative to the isoalloxazine ring of the flavin adenine dinucleotide cofactor is suitable for hydride-transfer dehydrogenation at the carbon atom that forms the chiral center of the substrate molecule. A comparison of the substrate-binding modes of 6HLNO and 6-hydroxy-D-nicotine oxidase, based on models of complexes with the D-substrate, suggests an explanation for the stereospecificity of both enzymes. The two enzymes are proposed to orient the enantiomeric substrates in mirror symmetry with respect to the plane of the flavin.

Structural analysis of the heterodimeric type IIS restriction endonuclease R.BspD6I acting as a complex between a monomeric site-specific nickase and a catalytic subunit.

The heterodimeric restriction endonuclease R.BspD6I from Bacillus species D6 recognizes a pseudosymmetric sequence and cuts both DNA strands outside the recognition sequence. The large subunit, Nt.BspD6I, acts as a type IIS site-specific monomeric nicking endonuclease. The isolated small subunit, ss.BspD6I, does not bind DNA and is not catalytically active. We solved the crystal structures of Nt.BspD6I and ss.BspD6I at high resolution. Nt.BspD6I consists of three domains, two of which exhibit structural similarity to the recognition and cleavage domains of FokI. ss.BspD6I has a fold similar to that of the cleavage domain of Nt.BspD6I, each containing a PD-(D/E)XK motif and a histidine as an additional putative catalytic residue. In contrast to the DNA-bound FokI structure, in which the cleavage domain is rotated away from the DNA, the crystal structure of Nt.BspD6I shows the recognition and cleavage domains in favorable orientations for interactions with DNA. Docking models of complexes of Nt.BspD6I and R.BspD6I with cognate DNA were constructed on the basis of structural similarity to individual domains of FokI, R.BpuJI and HindIII. A three-helix bundle forming an interdomain linker in Nt.BspD6I acts as a rigid spacer adjusting the orientations of the spatially separated domains to match the distance between the recognition and cleavage sites accurately.

The endoproteinase furin contains two essential Ca2+ ions stabilizing its N-terminus and the unique S1 specificity pocket.

The mammalian prohormone/proprotein convertase (PC) furin is responsible for the maturation of a great variety of homeostatic but also many pathogenic proteins within the secretory pathway and the endosomal pathway and at the cell surface. Similar to other members of the PC family, furin requires calcium for catalytic activity. In a previous paper, the structural association of the catalytic and the P-domain of furin was shown and data were presented indicating two or three calcium-binding sites. The exact number and the three-dimensional localization of the essential calcium sites within furin have now been determined by collecting X-ray diffraction data on either side of the Ca K absorption edge and by calculating a novel type of double difference map from these anomalous scattering data. Two calcium ions were unambiguously identified: the purely structural Ca-1 also conserved in the bacterial digestive subtilisins and the Ca-2 site specific to PCs and essential for the formation of the P1 specificity-determining S1-binding pocket. In addition, these anomalous diffraction data show that no tightly bound K(+) sites exist in furin.

The 1.9-A crystal structure of the noncollagenous (NC1) domain of human placenta collagen IV shows stabilization via a novel type of covalent Met-Lys cross-link.

Triple-helical collagen IV protomers associate through their N- and C-termini forming a three-dimensional network, which provides basement membranes with an anchoring scaffold and mechanical strength. The noncollagenous (NC1) domain of the C-terminal junction between two adjacent collagen IV protomers from human placenta was crystallized and its 1.9-A structure was solved by multiple anomalous diffraction (MAD) phasing. This hexameric NC1 particle is composed of two trimeric caps, which interact through a large planar interface. Each cap is formed by two alpha 1 fragments and one alpha 2 fragment with a similar previously uncharacterized fold, segmentally arranged around an axial tunnel. Each monomer chain folds into two structurally very similar subdomains, which each contain a finger-like hairpin loop that inserts into a six-stranded beta-sheet of the neighboring subdomain of the same or the adjacent chain. Thus each trimer forms a quite regular, but nonclassical, sixfold propeller. The trimer-trimer interaction is further stabilized by a previously uncharacterized type of covalent cross-link between the side chains of a Met and a Lys residue of the alpha 1 and alpha 2 chains from opposite trimers, explaining previous findings of nonreducible cross-links in NC1. This structure provides insights into NC1-related diseases such as Goodpasture and Alport syndromes.

Structure of adenylylsulfate reductase from the hyperthermophilic Archaeoglobus fulgidus at 1.6-A resolution.

The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5'-phosphosulfate, APS) reductase catalyzes reversibly the reduction of APS to sulfite and AMP. The structures of APS reductase from the hyperthermophilic Archaeoglobus fulgidus in the two-electron reduced state and with sulfite bound to FAD are reported at 1.6- and 2.5- resolution, respectively. The FAD-sulfite adduct was detected after soaking the crystals with APS. This finding and the architecture of the active site strongly suggest that catalysis involves a nucleophilic attack of the N5 atom of reduced FAD on the sulfur atom of APS. In view of the high degree of similarity between APS reductase and fumarate reductase especially with regard to the FAD-binding alpha-subunit, it is proposed that both subunits originate from a common ancestor resembling archaeal APS reductase. The two electrons required for APS reduction are transferred via two [4Fe-4S] clusters from the surface of the protein to FAD. The exceptionally large difference in reduction potential of these clusters (-60 and -500 mV) can be explained by interactions of the clusters with the protein matrix.

Crystal structure of the p14/MP1 scaffolding complex: how a twin couple attaches mitogen-activated protein kinase signaling to late endosomes.

Signaling pathways in eukaryotic cells are often controlled by the formation of specific signaling complexes, which are coordinated by scaffold and adaptor proteins. Elucidating their molecular architecture is essential to understand the spatial and temporal regulation of cellular signaling. p14 and MP1 form a tight (K(d) = 12.8 nM) endosomal adaptor/scaffold complex, which regulates mitogen-activated protein kinase (MAPK) signaling. Here, we present the 1.9-A crystal structure of a biologically functional p14/MP1 complex. The overall topology of the individual MP1 and p14 proteins is almost identical, having a central five-stranded beta-sheet sandwiched between a two-helix and a one-helix layer. Formation of the p14/MP1 heterodimer proceeds by beta-sheet augmentation and yields a unique, almost symmetrical, complex with several potential protein-binding sites on its surface. Mutational analysis allowed identification of the p14 endosomal adaptor motif, which seems to orient the complex relative to the endosomal membrane. Two highly conserved and hydrophobic protein-binding sites are located on the opposite "cytoplasmic" face of the p14/MP1 heterodimer and might therefore function as docking sites for the target proteins extracellular regulated kinase (ERK) 1 and MAPK/ERK kinase 1. Furthermore, detailed sequence analyses revealed that MP1/p14, together with profilins, define a protein superfamily of small subcellular adaptor proteins, named ProflAP. Taken together, the presented work provides insight into the spatial regulation of MAPK signaling, illustrating how p14 and MP1 collaborate as an endosomal adaptor/scaffold complex.