Romanian adjectives at the syntax-semantics interface

In this paper, we argue for the existence of two local domains (phases, cf. Chomsky 2001; 2009; Legate 2003, among others) inside the DP: the n*-phase, parallel to the vP (as in Svenonius 2004), and the d*-phase, parallel to the CP. Two acknowledged phasal properties are discussed. (i) The n*/d*-phases define their own peripheries: peripheries are essentially modal-quantificational spaces, as shown by the decomposition of Topic—Focus features recently proposed (Butler 2004; McNay 2005; 2006). (ii) Phases are assumed to be domains of linearization: after (internal or external) merge, syntactic objects are hierarchical, but not linear, so phases must be linearized before they are sent to PF. The distribution and interpretation of DP-internal adjectives is taken to be indicative of these two domains

Structural basis for the photoconversion of a phytochrome to the activated far-red light-absorbing form

Phytochromes are a collection of bilin-containing photoreceptors that regulate numerous photoresponses in plants and microorganisms through their ability to photointerconvert between a red light-absorbing, ground state Pr and a far-red light-absorbing, photoactivated state Pfr1,2. While the structures of several phytochromes as Pr have been determined3-7, little is known about the structure of Pfr and how it initiates signaling. Here, we describe the three-dimensional solution structure of the bilin-binding domain as Pfr using the cyanobacterial phytochrome from Synechococcus OSB’. Contrary to predictions, light-induced rotation of the A but not the D pyrrole ring is the primary motion of the chromophore during photoconversion. Subsequent rearrangements within the protein then affect intra- and interdomain contact sites within the phytochrome dimer. From our models, we propose that phytochromes act by propagating reversible light-driven conformational changes in the bilin to altered contacts between the adjacent output domains, which in most phytochromes direct differential phosphotransfer

Dss1 is a 26S proteasome ubiquitin receptor

The ubiquitin-proteasome system is the major pathway for protein degradation in eukaryotic cells. Proteins to be degraded are conjugated to ubiquitin chains that act as recognition signals for the 26S proteasome. The proteasome subunits Rpn10 and Rpn13 are known to bind ubiquitin, but genetic and biochemical data suggest the existence of at least one other substrate receptor. Here, we show that the phylogenetically conserved proteasome subunit Dss1 (Sem1) binds ubiquitin chains linked by K63 and K48. Atomic resolution data show that Dss1 is disordered and binds ubiquitin by binding sites characterized by acidic and hydrophobic residues. The complementary binding region in ubiquitin is composed of a hydrophobic patch formed by I13, I44, and L69 flanked by two basic regions. Mutations in the ubiquitin-binding site of Dss1 cause growth defects and accumulation of ubiquitylated proteins

The D-ring, Not the A-ring, Rotates in Synechococcus OS-B' Phytochrome

Phytochrome photoreceptors in plants and microorganisms switch photochromically between two states, controlling numerous important biological processes. Although this phototransformation is generally considered to involve rotation of ring D of the tetrapyrrole chromophore, Ulijasz et al. (Ulijasz, A. T., Cornilescu, G., Cornilescu, C. C., Zhang, J., Rivera, M., Markley, J. L., and Vierstra, R. D. (2010) Nature 463, 250–254) proposed that the A-ring rotates instead. Here, we apply magic angle spinning NMR to the two parent states following studies of the 23-kDa GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domain fragment of phytochrome from Synechococcus OS-B′. Major changes occur at the A-ring covalent linkage to the protein as well as at the protein residue contact of ring D. Conserved contacts associated with the A-ring nitrogen rule out an A-ring photoflip, whereas loss of contact of the D-ring nitrogen to the protein implies movement of ring D. Although none of the methine bridges showed a chemical shift change comparable with those characteristic of the D-ring photoflip in canonical phytochromes, denaturation experiments showed conclusively that the same occurs in Synechococcus OS-B′ phytochrome upon photoconversion. The results are consistent with the D-ring being strongly tilted in both states and the C15=C16 double bond undergoing a Z/E isomerization upon light absorption. More subtle changes are associated with the A-ring linkage to the protein. Our findings thus disprove A-ring rotation and are discussed in relation to the position of the D-ring, photoisomerization, and photochromicity in the phytochrome family

Expression, purification, and crystallization of a plant polyketide cyclase from Cannabis sativa

Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffolds is a key step for diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2/C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide CoA to generate olivetolic acid in the biosynthesis of cannabinoid. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of lack of the functional analysis of the other plant DABB proteins and low sequence identity with the functionally and structually characterized bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized in a vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolutions and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3 Å, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure–function relationship and catalytic mechanism of OAC

Conformational Flexibility in the Enterovirus RNA Replication Platform

A presumed RNA cloverleaf (5′CL), located at the 5′-most end of the noncoding region of the enterovirus genome, is the primary established site for initiation of genomic replication. Stem–loop B (SLB) and stem–loop D (SLD), the two largest stem–loops within the 5′CL, serve as recognition sites for protein interactions that are essential for replication. Here we present the solution structure of rhinovirus serotype 14 5′CL using a combination of nuclear magnetic resonance spectroscopy and small-angle X-ray scattering. In the absence of magnesium, the structure adopts an open, somewhat extended conformation. In the presence of magnesium, the structure compacts, bringing SLB and SLD into close contact, a geometry that creates an extensive accessible major groove surface, and permits interaction between the proteins that target each stem–loop

Solution structure of the inhibitory phosphorylation domain of myosin phosphatase targeting subunit 1.

Cell motility, such as smooth muscle contraction and cell migration, is controlled by the reversible phosphorylation of the regulatory light chain of myosin II and other cytoskeletal proteins. Mounting evidence suggests that in smooth muscle cells and other types of cells in vertebrates, myosin phosphatase (MP) plays an important role in controlling the phosphorylation of myosin II as well as other cytoskeletal proteins, including ezrin, moesin, and radixin.1 MP is a holoenzyme consisting of a catalytic subunit of a type-1 Ser/Thr phosphatase (PP1C) delta isoform, a myosin phosphatase targeting subunit 1 (MYPT1), and an accessory subunit M21. In this ternary complex, MYPT1 is responsible for regulating the phosphatase activity.1 A recent X-ray crystallographic study revealed an allosteric interaction between PP1C and the N-terminal ankyrin repeat domain of MYPT1 that confers the substrate specificity of the enzyme.2 MP activity is suppressed when Thr696 or Thr853 of MYPT1 is phosphorylated by various kinases, such as ROCK, ZIPK, ILK, and PAK.1,3 However, it is still unclear how the phosphorylation of MYPT1 inhibits MP activity. The amino acid sequence around Thr696 of MYPT1 is highly conserved among MYPT1 family members including MYPT2 and MBS85. Therefore, structural insights into the inhibitory domain of MYPT1 are expected to provide new clues to fully elucidate the mechanism that controls phosphatase activity via the phosphorylation of MYPT1 or other family members involved in kinase-phosphatase crosstalk in cytoskeletal regulation. Here, we prepared a bacterial recombinant fragment of MYPT1 corresponding to residues 658 to 714, including the phosphorylation site Thr696, and determined its three-dimensional structure through the use of computer-assisted distance geometry and a simulated annealing protocol combined with stable-isotope-aided multi-dimensional NMR techniques

The structure of a resuscitation-promoting factor domain from Mycobacterium tuberculosis shows homology to lysozymes

Resuscitation-promoting factor (RPF) proteins reactivate stationary-phase cultures of (G+C)-rich Gram-positive bacteria including the causative agent of tuberculosis, Mycobacterium tuberculosis. We report the solution structure of the RPF domain from M. tuberculosis Rv1009 (RpfB) solved by heteronuclear multidimensional NMR. Structural homology with various glycoside hydrolases suggested that RpfB cleaved oligosaccharides. Biochemical studies indicate that a conserved active site glutamate is important for resuscitation activity. These data, as well as the presence of a clear binding pocket for a large molecule, indicate that oligosaccharide cleavage is probably the signal for revival from dormancy

High-resolution NMR studies of structure and dynamics of human ERp27 indicate extensive interdomain flexibility

ERp27 (endoplasmic reticulum protein 27.7 kDa) is a homologue of PDI (protein disulfide-isomerase) localized to the endoplasmic reticulum. ERp27 is predicted to consist of two thioredoxinfold domains homologous with the non-catalytic b and b domains of PDI. The structure in solution of the N-terminal blike domain of ERp27 was solved using high-resolution NMR data. The structure confirms that it has the thioredoxin fold and that ERp27 is a member of the PDI family. 15N-NMR relaxation data were obtained and ModelFree analysis highlighted limited exchange contributions and slow internal motions, and indicated that the domain has an average order parameter S 2 of 0.79. Comparison of the single-domain structure determined in the present study with the equivalent domain within fulllength ERp27, determined independently by X-ray diffraction, indicated very close agreement. The domain interface inferred from NMR data in solution was much more extensive than that observed in the X-ray structure, suggesting that the domains flex independently and that crystallization selects one specific interdomain orientation. This led us to apply a new rapid method to simulate the flexibility of the full-length protein, establishing that the domains show considerable freedom to flex (tilt and twist) about the interdomain linker, consistent with the NMR data

Solution structure of the inner DysF domain of myoferlin and implications for limb girdle muscular dystrophy type 2b

Mutations in the protein dysferlin, a member of the ferlin family, lead to limb girdle muscular dystrophy type 2B and Myoshi myopathy. The ferlins are large proteins characterised by multiple C2 domains and a single C-terminal membrane-spanning helix. However, there is sequence conservation in some of the ferlin family in regions outside the C2 domains. In one annotation of the domain structure of these proteins, an unusual internal duplication event has been noted where a putative domain is inserted in between the N- and C-terminal parts of a homologous domain. This domain is known as the DysF domain. Here, we present the solution structure of the inner DysF domain of the dysferlin paralogue myoferlin, which has a unique fold held together by stacking of arginine and tryptophans, mutations that lead to clinical disease in dysferlin