Kazuki Kawahara

Structural basis for PPARγ transactivation by endocrine-disrupting organotin compounds.

Organotin compounds such as triphenyltin (TPT) and tributyltin (TBT) act as endocrine disruptors through the peroxisome proliferator–activated receptor γ (PPARγ) signaling pathway. We recently found that TPT is a particularly strong agonist of PPARγ. To elucidate the mechanism underlying organotin-dependent PPARγ activation, we here analyzed the interactions of PPARγ ligand-binding domain (LBD) with TPT and TBT by using X-ray crystallography and mass spectroscopy in conjunction with cell-based activity assays. Crystal structures of PPARγ-LBD/TBT and PPARγ-LBD/TPT complexes were determined at 1.95 Å and 1.89 Å, respectively. Specific binding of organotins is achieved through non-covalent ionic interactions between the sulfur atom of Cys285 and the tin atom. Comparisons of the determined structures suggest that the strong activity of TPT arises through interactions with helix 12 of LBD primarily via π-π interactions. Our findings elucidate the structural basis of PPARγ activation by TPT.

Hyperstability and crystal structure of cytochrome c555 from hyperthermophilic Aquifex aeolicus

In order to elucidate the relationship between the stability and the structure of the monohaem cytochrome c(555) (AA c(555)) from the hyperthermophilic bacterium Aquifex aeolicus, chemical denaturation and crystal structure determination were carried out. AA c(555) exhibited higher stability than the thermophilic Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), which is one of the most stable cytochromes c. The three-dimensional crystal structure of AA c(555), which was determined using the multiple anomalous dispersion technique at 1.15 A resolution, included a unique 14-residue extra helix, while the side-chain interactions of several amino-acid residues responsible for the stability of HT c(552) were conserved in AA c(555). The side chain of the Met61 residue in the extra helix was aligned towards the haem, forming a coordination bond between the Met S and haem Fe atoms. In other cytochromes c the corresponding regions always form Omega loops which also include the haem-liganding Met residue and are known to be involved in the initial step in cytochrome c denaturation. The formation of the extra helix in AA c(555) results in the highest helix content, 59.8%, among the monohaem cytochromes c. The extra helix should mainly contribute to the hyperstability of AA c(555) and is presumed to be a novel strategy of cytochromes c for adaptation to a hyperthermophilic environment.

Structure of the CFA/III major pilin subunit CofA from human enterotoxigenic Escherichia coli determined at 0.90 Å resolution by sulfur‐SAD phasing

CofA, a major pilin subunit of colonization factor antigen III (CFA/III), forms pili that mediate small-intestinal colonization by enterotoxigenic Escherichia coli (ETEC). In this study, the crystal structure of an N-terminally truncated version of CofA was determined by single-wavelength anomalous diffraction (SAD) phasing using five sulfurs in the protein. Given the counterbalance between anomalous signal strength and the undesired X-ray absorption of the solvent, diffraction data were collected at 1.5 Å resolution using synchrotron radiation. These data were sufficient to elucidate the sulfur substructure at 1.38 Å resolution. The low solvent content (29%) of the crystal necessitated that density modification be performed with an additional 0.9 Å resolution data set to reduce the phase error caused by the small sulfur anomalous signal. The CofA structure showed the αβ-fold typical of type IVb pilins and showed high structural homology to that of TcpA for toxin-coregulated pili of Vibrio cholerae, including spatial distribution of key residues critical for pilin self-assembly. A pilus-filament model of CofA was built by computational docking and molecular-dynamics simulation using the previously reported filament model of TcpA as a structural template. This model revealed that the CofA filament surface was highly negatively charged and that a 23-residue-long loop between the α1 and α2 helices filled the gap between the pilin subunits. These characteristics could provide a unique binding epitope for the CFA/III pili of ETEC compared with other type IVb pili.

The triple helical structure and stability of collagen model peptide with 4(S)-hydroxyprolyl-pro-gly units

Extensive studies on the structure of collagen have revealed that the hydroxylation of Pro residues in a variety of model peptides with the typical (X‐Y‐Gly)nrepeats (X and Y: Pro and its analogues) represents one of the major factors influencing the stability of triple helices. While(2S,4R)‐hydroxyproline (Hyp) at the position Y stabilizes the triple helix, (2S,4S)‐hydroxyproline (hyp) at the X‐position destabilizes the helix as demonstrated that the triple helix of (hyp‐Pro‐Gly)15 is less stable than that of (Pro‐Pro‐Gly)15 and that a shorter peptide (hyp‐Pro‐Gly)10 does not form the helix. To clarify the role of the hydroxyl group of Pro residues to play in the stabilization mechanism of the collagen triple helix, we synthesized and crystallized a model peptide (Pro‐Hyp‐Gly)4‐(hyp‐Pro‐Gly)2‐(Pro‐Hyp‐Gly)4 and analyzed its structure by X‐ray crystallography and CD spectroscopy. In the crystal, the main‐chain of this peptide forms a typical collagen like triple helix. The majority of hyp residues take down pucker with exceptionally shallow angles probably to relieve steric hindrance, but the remainders protrude the hydroxyl group toward solvent with the less favorable up pucker to fit in a triple helix. There is no indication of the existence of an intra‐molecular hydrogen bond between the hydroxyl moiety and the carbonyl oxygen of hyp supposed to destabilize the triple helix. We also compared the conformational energies of up and down packers of the pyrrolidine ring in Ac‐hyp‐NMe2 by quantum mechanical calculations.

Ordered self-assembly of the collagenous domain of adiponectin with noncovalent interactions via glycosylated lysine residues.

Adiponectin, an anti‐atherogenic and insulin‐sensitizing adipokine, forms multiple isoforms including a trimer, a hexamer and heavier oligomers (mainly octadecamer) that determine their biological activities. We designed 89‐residue peptides containing modifications found in the collagenous domain of native adiponectin. Circular dichroism and analytical ultracentrifugation measurements showed that the peptide bearing glucosyl‐galactosyl‐hydroxylysine residues forms a stable collagen‐like triple helical structure and spontaneously assembled into an octadecamer. An assembly model mediated by noncovalent interactions via glycosylated lysine residues for the octadecamer was constructed. Our findings clarified an essential role of glycosyl modifications to coordinate the ordered self‐assembly of adiponectin.

Polymorphism of Collagen Triple Helix Revealed by 19F NMR of Model Peptide [Pro-4(R)-Hydroxyprolyl-Gly]3-[Pro-4(R)-Fluoroprolyl-Gly]-[Pro-4(R)-Hydroxyprolyl-Gly]3

We have characterized various structures of (Pro-Hyp(R)-Gly)(3)-Pro-fPro(R)-Gly-(Pro-Hyp(R)-Gly)(3) in the process of cis-trans isomerization and helix-coil transition by exploiting the sole (19)F NMR probe in 4(R)-fluoroproline (fPro(R)). Around the transition temperature (T(m)), we detected a species with a triple helical structure distinct from the ordinary one concerning the alignment of three strands. The (19)F-(19)F exchange spectroscopy showed that this misaligned and that the ordinary triple helices were interchangeable only indirectly via an extended monomer strand with all-trans peptide bonds at Pro-fPro(R), Pro-Hyp(R), and Gly-Pro in the central segment. This finding demonstrates that the helix-coil transition of collagen peptides is not described with a simple two-state model. We thus elaborated a scheme for the transition mechanism of (Pro-Hyp(R)-Gly)(n) that the most extended monomer strand can be the sole source both to the misaligned and correctly folded triple-helices. The staggered ends could help misaligned triple helices to self-assemble to higher-order structures. We have also discussed the possible relationship between the misaligned triple helix accumulating maximally at T(m) and the kinetic hysteresis associated with the helix-coil transition of collagen.

Comparative study on stabilization mechanism of monomeric cytochrome c5 from deep-sea piezophilic Shewanella violacea

Monomeric cytochrome c5 from deep-sea piezophilic Shewanella violacea (SVcytc5) was stable against heat and denaturant compared with the homologous protein from shallow-sea piezo-sensitive Shewanella livingstonensis (SLcytc5). Here, the SVcytc5 crystal structure revealed that the Lys-50 side chain on the flexible loop formed a hydrogen bond with heme whereas that of corresponding hydrophobic Leu-50 could not form such a bond in SLcytc5, which appeared to be one of possible factors responsible for the difference in stability between the two proteins. This structural insight was confirmed by a reciprocal mutagenesis study on the thermal stability of these two proteins. As SVcytc5 was isolated from a deep-sea piezophilic bacterium, the present comparative study indicates that adaptation of monomeric SVcytc5 to high pressure environments results in stabilization against heat. Graphical abstract Structure of Shewanella violaceacy to chrome c5 revealed that Lys-50 on a flexible loop was responsible for its stability.

Structural Basis for Dimer Formation of Human Condensin Structural Maintenance of Chromosome Proteins and Its Implications for Single-stranded DNA Recognition

Eukaryotic structural maintenance of chromosome proteins (SMC) are major components of cohesin and condensins that regulate chromosome structure and dynamics during cell cycle. We here determine the crystal structure of human condensin SMC hinge heterodimer with ∼30 residues of coiled coils. The structure, in conjunction with the hydrogen exchange mass spectrometry analyses, revealed the structural basis for the specific heterodimer formation of eukaryotic SMC and that the coiled coils from two different hinges protrude in the same direction, providing a unique binding surface conducive for binding to single-stranded DNA. The characteristic hydrogen exchange profiles of peptides constituted regions especially across the hinge-hinge dimerization interface, further suggesting the structural alterations upon single-stranded DNA binding and the presence of a half-opened state of hinge heterodimer. This structural change potentially relates to the DNA loading mechanism of SMC, in which the hinge domain functions as an entrance gate as previously proposed for cohesin. Our results, however, indicated that this is not the case for condensins based on the fact that the coiled coils are still interacting with each other, even when DNA binding induces structural changes in the hinge region, suggesting the functional differences of SMC hinge domain between condensins and cohesin in DNA recognition.

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli

Colonization factor antigen III (CFA/III) is one of the virulence factors of human enterotoxigenic Escherichia coli (ETEC) that forms the long, thin, proteinaceous fibres of type IV pili through assembly of its major and minor subunits CofA and CofB, respectively. The crystal structure of CofA has recently been reported; however, the lack of structural information for CofB, the largest among the known type IV pilin subunits, hampers a comprehensive understanding of CFA/III pili. In this study, constructs of wild-type CofB with an N-terminal truncation and the corresponding SeMet derivative were cloned, expressed, purified and crystallized. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 103.97, c = 364.57 Å for the wild-type construct and a = b = 103.47, c = 362.08 Å for the SeMet-derivatized form. Although the diffraction quality of these crystals was initially very poor, dehydration of the crystals substantially improved the resolution limit from ∼ 4.0 to ∼ 2.0 Å. The initial phase was solved by the single-wavelength anomalous dispersion (SAD) method using a dehydrated SeMet CofB crystal, which resulted in an interpretable electron-density map.

Conformational preferences of 4-chloroproline residues†

Conformational preferences of the (2S,4R)-4-chloroproline (Clp) and (2S,4S)-4-chloroproline (clp) residues are explored at the M06-2X/cc-pVTZ//M06-2X/6-31+G(d) level of theory in the gas phase and in water, where solvation free energies were calculated using the implicit solvation model, and by an X-ray diffraction study in the solid state. In the gas phase, the down-puckered γ-turn structure with the trans prolyl peptide bond is most preferred for both Ac-Clp-NHMe and Ac-clp-NHMe, in which the C(7) hydrogen bond between two terminal groups seems to play a role, as found for Ac-Pro-NHMe. In water, the Clp residue has a strong preference for the up-puckered PP(II) structure, whereas the up-puckered PP(II) structure prevails a little over the down-puckered PP(II) structure for the clp residue, similar to the Pro residue. Hence, our calculated results on the puckering preference of the Clp and clp residues in water are in accord with the observed results deduced from the relative stabilities of the triple helices of the collagen model peptides. The X-ray structure of Ac-clp-NHMe was found to be the most preferred in water but that of Ac-Clp-NHMe was located as a local minimum with ΔG = 2.0 kcal/mol. In particular, the X-ray structure of Ac-Clp-NHMe was quite different from that of Ac-Clp-OMe but similar to that of Ac-Pro-NHMe. The lowest rotational barriers to the prolyl cis-trans isomerization for Ac-Clp-NHMe become nearly the same as those for Ac-Pro-NHMe in water, whereas the barriers are lower by ∼2 kcal/mol for Ac-clp-NHMe. It was found that the cis-trans isomerization may proceed through the clockwise or anticlockwise rotations for Ac-Clp-NHMe and the anticlockwise rotation for Ac-clp-NHMe and Ac-Pro-NHMe in water.