No abstract

The molecular conformation of silk fibrion is characterized by solid-state 13C NMR before spinning (silk I structure) and after spinning (silk II structure). We compare native silk fibers with the quasi-crystalline Cp-fraction and a synthetic model peptide (Ala-Gly)15, both of which can be converted either into silk I by dialysis from 9 M LiBr or into silk II by treatment with formic acid. Our results demonstrate that silk II fibers are intrinsically heterogeneous, consisting of beta-sheets, distorted beta-turns, and distorted beta-sheets. This higher-order heterogeneity is revealed by the 13C-NMR Cbeta-peak of Ala, indicating that the Ala side chains are stacked partially in parallel and partially face-to-face, at a ratio of 1:2.

No abstract

Post-translational modification by small ubiquitin-like modifier (SUMO) proteins has been implicated in the regulation of a variety of cellular events. The functions of sumoylation are often mediated by downstream effector proteins harboring SUMOinteracting motifs (SIMs) that are composed of a hydrophobic core and a stretch of acidic residues. MBD1-containing chromatin-associated factor 1 (MCAF1), a transcription repressor, interacts with SUMO-2/3 and SUMO-1, with a preference for SUMO-2/3. We used NMR spectroscopy to solve the solution structure of the SIM of MCAF1 bound to SUMO-3. The hydrophobic core of the SIM forms a parallel ␤-sheet pairing with strand ␤2 of SUMO-3, whereas its C-terminal acidic stretch seems to mediate electrostatic interactions with a surface area formed by basic residues of SUMO-3. The significance of these electrostatic interactions was shown by mutations of both SUMO-3 and MCAF1. The present structural and biochemical data suggest that the acidic stretch of the SIM of MCAF1 plays an important role in the binding to SUMO-3. Small ubiquitin-like modifier (SUMO)2 proteins conjugate post-translationally with target proteins through a series of enzymatic reactions that resemble ubiquitination (1-5). In contrast to ubiquitination, which is largely involved in regulating the degradation of target proteins by proteasomes or lysosomes (6), sumoylation appears to regulate a wide variety of cellular events, such as nuclear transport, subnuclear localization, transcriptional regulation, DNA repair, and chromosome segregation (3). In particular, sumoylation of transcription factors represses transcription through a variety of different mechanisms, such as recruitment of histone deacetylases and regulation of nuclear body components (7). It has been suggested that sumoylation generally regulates the functions of target proteins by modulating their proteinprotein or protein-DNA interactions.In mammals, four SUMO paralogues, SUMO-1 through SUMO-4, have been identified, of which SUMO-1 to -3 can serve as protein modifiers (8). Whereas SUMO-2 and SUMO-3 share 97% amino acid identity, they have only 48 and 46% identity with SUMO-1, respectively. This indicates that SUMO-2 and SUMO-3 constitute a subgroup that is distinct from SUMO-1. Although these SUMO paralogues largely share common cellular functions, they also show paralogue-specific properties in regard to cellular localization and substrate specificity (9, 10). For example, SUMO-1 is preferentially found within nucleoli, nuclear envelopes, and cytoplasmic foci (8), whereas SUMO-2/3 accrue on chromosomes early in the nuclear reformation process (11). With respect to substrate specificity, RanGAP1 is preferentially modified by SUMO-1, whereas SUMO-2/3 show preference for topoisomerase 2 during mitosis. However, promyelocytic leukemia protein conjugates to all three SUMO paralogues (12). SUMO-1 is most abundant in the protein-conjugated form, whereas SUMO-2/3 isomers are more abundant in a free pool and are available to conjugate with target proteins...

The multidrug transporter AcrB actively exports a wide variety of noxious compounds using proton-motive force as an energy source in Gram-negative bacteria. AcrB adopts an asymmetric structure comprising three protomers with different conformations that are sequentially converted during drug export; these cyclic conformational changes during drug export are referred to as functional rotation. To investigate functional rotation driven by proton-motive force, all-atom molecular dynamics simulations were performed. Using different protonation states for the titratable residues in the middle of the transmembrane domain, our simulations revealed the correlation between the specific protonation states and the side-chain configurations. Changing the protonation state for Asp408 induced a spontaneous structural transition, which suggests that the proton translocation stoichiometry may be one proton per functional rotation cycle. Furthermore, our simulations demonstrate that alternating the protonation states in the transmembrane domain induces functional rotation in the porter domain, which is primarily responsible for drug transport.

The fiber formation mechanism of Bombyx mori silk fibroin by silkworm is essentially the structural change from silk I (the silk fibroin structure before spinning in the solid state) to silk II (the silk fibroin structure after spinning) under external forces in both silk gland and spinneret of B. mori silkworm. Recently, we proposed structural models for silk I and silk II forms of the model peptide (AlaGly) 15 of B. mori silk fibroin using mainly solid-state NMR methods. In this paper, molecular dynamics (MD) calculation was performed to simulate the structural change of poly(Ala-Gly) from silk I to silk II and to clarify the detailed mechanism of the silk fiber formation. The silk I structure (repeated -turn type II) changes to silk II structure (heterogeneous structure, but mainly antiparallel -sheet) by stretching of the chain with MD simulation, but the change occurs only under very high temperature such as 1000 K and large tensile stress (1.0 GPa). However, the structural change during the MD simulation occurs more easily by taking into account several external forces (the presence of water molecules around the silk chains, and application of both shear and tensile stresses to the silk fibroin) applied to the silk fibroin simultaneously. The heterogeneous structure of the silk fiber determined previously with solid-state NMR could be reproduced well with the MD calculation and then molecular mechanics calculation after removal of water molecules.

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 Å resolution. The TDH tetramerformsacentralporewithdimensionsof23Å indiameterand ϳ50 Å in depth. -Cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside of the pore. Molecular dynamic simulations suggested that water molecules permeate freely through the central and side channel pores. Electron micrographs showed that tetrameric TDH attached to liposomes, and some of the tetramer associated with liposome via one protomer. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.Vibrio parahaemolyticus is a Gram-negative marine bacterium known to be one of the major causes of pandemic seafoodborne gastroenteritis. V. parahaemolyticus possesses two circular replicons of 3.2 and 1.9 megabase pairs, which might confer an advantage by enabling DNA replication in seawater of lower temperature and/or low nutritional value (1, 2). Such an advantage would potentially increase risks of food intoxication by allowing explosive expansion of the population of the microorganism. Hemolysis on Wagatsuma agar (a blood agar), known as the Kanagawa phenomenon, is associated with human pathogenic strains of V. parahaemolyticus. A major virulence factor of this pathogen is the thermostable direct hemolysin (TDH) 7 (3-5), which has a variety of biological activities including hemolytic activity, cardiotoxicity, and enterotoxicity. There are two copies of the tdh gene (or its homologue trh) in pathogenic V. parahaemolyticus, indicating the importance of this exotoxin for survival of the organism (2). The mature form of TDH consists of 165 amino acids, including a single intramolecular disulfide bond, but no close homologue of TDH has been found in other organisms. The significance of Arg 46 , Gly 62 , Trp 65 , and Gly 90 residues on hemolysis was determined by site-directed mutagenesis (3, 6).The common features of the bacterial pore-forming toxin are as follows. 1) It is released as a soluble monomer into the extra-bacterial space. 2) It oligomerizes to form a pore at the host cell membrane (7,8). An earlier study reported that TDH acted as a pore-forming toxin, creating a functional pore ϳ20 Å in diameter (reviewed in Ref. 3). We previously constructed a low resolution C 4 symmetric model of tetrameric TDH in solution based on small angle x-ray scattering (SAXS), transmission electron microscopy (TEM), and analytical ultracentrifugation (9). However, the precise structure and the mechanism for its pore-forming toxicity are still unknown. Several bacterial toxins, including TDH, show paradoxical responses to heat treatment, known as the Arrhenius effect (10, * This study was s...

In an initial attempt to understand the structural organization of Bombyx mori silk fibroin stored in the silk gland using several solid-state NMR techniques, we recently reported the conformation of the crystalline form of silk I (The unprocessed conformation of the silk fibroin before spinning in the solid state) as a repeated type II β-turn structure (Ala, (φ,ψ) = (−60°,130°), and Gly, (φ,ψ ) = (70°,30°)) in a model peptide (Ala-Gly) 15 (Asakura et al. J. Mol. Biol. 2001, 306, 291−305). To examine the favorable secondary structure(s) associated with silk fibroin molecules, we analyzed the results of molecular dynamic (MD) simulations of three model dipeptides of the type Ac-Xxx-NHMe (where Xxx = Gly, Ala and Ser) in explicit water because the concentration of the silk fibroin before spinning in the middle silk gland is about 30% in water. The conformational probability maps constructed for these dipeptides indicate that the torsion angles of Gly, Ala, and Ser residues in the type II β-turn structure are in the stable state even in water, and only our model among silk I models proposed previously can satisfy the stable state for these residues simultaneously. The high possibility of the appearance of β-turn structure is pointed out by the MD simulation of Ac-(Ala-Gly)8-NHMe molecule in water. There is also a high possibility to form intramolecular hydrogen bonding involving the ith Ser OγH functionality as a donor and the (i − 3)th Gly CO group as an acceptor when the side chain conformation of the Ser residue, χ1 = −60°. This is derived from the molecular mechanics simulation of Ac-(Ala-Gly-Ser)-NHMe without water and will further stabilize the type II β-turn conformation.