Aperiodic Crystals at the Mesoscale

Materials with mesoscale structural characteristics have attracted great attention across the fields of chemistry, physics, and materials science. A typical example is mesoporous silica, which are synthesized in water/surfactant/silica systems, and has well-defined mesopores resulting in high surface area. Mesoporous silicas have two defining structural characteristics: (i) disorder at the atomic scale, i.e. only short-range order; and (ii) distinct order at the mesoscale, i.e. long-range order. Atomic-scale structural characterization by common diffraction techniques, such as X-ray single crystal diffraction, is challenging for these partially ordered materials. This is because of the difficulty in obtaining large (> 10 µm) single crystals, and because large-distance periodic features cause diffraction intensities to fall off rapidly with scattering angle, so that only limited small-angle data are available. On the other hand, transmission electron microscopy (TEM) is a powerful tool for structural characterization at the mesoscale level due to the stronger interaction of electrons with matter compared to X-rays, enabling the use of very small crystals. In particular, high-resolution TEM (HRTEM) images give the phase and the amplitude of the crystal structure factors experimentally, leading to a 3D structural model by electron crystallography. Cage-type anionic-surfactant-templated mesoporous silicas display rich structural diversity. Among them, cage-type mesoporous silica with tetrahedrally close-packed (TCP) structures can be described by four types of polyhedra, 5^12, 5^12 6^2, 5^12 6^3, and 5^12 6^4.[1] A variety of structural polymorph have been observed and characterized by TEM. Their structures show a close resemblance to the Frank-Kasper phases, which are well known in intermetallic compounds. We found mesoporous silica with dodecagonal quasicrystalline ordering as one of the TCP structures (Figure).[2] In this presentation, I will discuss structural characterization of aperiodic crystals at the mesoscale, such as mesoporous silicas and binary colloidal crystals, by electron microscopy.

Crystal structure analysis of dipeptidyl amino peptidase from P. mexicana WO24.

Dipeptidyl aminopeptidase (DAP or DPP, EC 3.4.14) catalyses the removal of dipeptides from the amino termini of peptides and proteins. In microorganisms, we have reported the identification, purification, and characterization of DAP BI, DAP BII, DAP BIII, and DAP IV (bacterial DPP4), POP from Pseudoxanthomonas mexicana WO24, and demonstrated that DAP BI, DAP BIII, DAP IV and POP belong to the POP family and they are classified into the clan SC, family S9 in the MEROPS database. On the basis of the enzymological data we obtained, we proposed that bacterial DAPs should be classified in a manner different from that of mammalian DPPs, except for the DAP IV. The DAP IV liberates dipeptides from the free amino terminus and has a specificity for both proline and hydroxyproline residues in the penultimate position of peptides. Here, we report the first structure of the bacterial DPP IV (P. mexicana WO24 DAP IV) complexed with an inhibitor at 2.2 Å resolution. The subunit structure is composed of two domains, the N-terminal eight-bladed β-propeller domain and the C-terminal alpha/beta/alpha sandwich catalytic domain. These structural features are conserved with clan SC S9 family. However, the N-terminal domain contains a unique helix region that extends over the active site acting as a lid, gating substrate or product access. Based upon the structural data, as well as molecular modeling, a model suggesting that the unique helix region is conserved in some kind of bacterial DPP4s except for mammalian DPP4s and some bacterial DPP4s. Some asaccharolytic and anaerobic bacteria can be used protein or peptides as an energy source. Therefore, these bacteria secrete many types of proteases and peptidases. Especially, the elucidation of degradation mechanisms of collagen, including proline and hydroxyproline, are very important from the point of view of host tissue breakdown in pathogens. Our findings suggest that different ligand recognition mechanisms from the bacterial DPP IV to mammalian DPP4 raise the possibility of an antimicrobial development targeting DPP IV from bacteria.

Crystal Structure analyses of dipeptidyl aminopeptidase BII from P. mexicana WO24.

The peptidase family S46 that contains the dipeptidyl aminopeptidase BII (DAP BII) from Pseudoxanthomonas mexicana WO24 is the only exopeptidase family in clan PA peptidases. Our present phylogenetic and experimental studies indicated that the catalytic triad of DAP BII is composed of His 86, Asp 224 and Ser 657 and implied that unknown large helical domains involved in exopeptidase activity[1]. However, three-dimensional structure of a family S46 peptidase has not yet been reported. Thus, the crystal structure of DAP BII is essential not only to understand the catalytic mechanism of family S46 peptidases but also to clarify the structural origin of the exo-type peptidase activities of these enzymes. Recently, we have successfully crystallized the DAP BII and collected X-ray diffraction data to 2.3 Å resolution from the crystal. This crystal belonging to space group P2₁2₁2₁, with unit-cell parameters a = 76.55 Å, b = 130.86 Å, c = 170.87 Å[2]. Structural analysis by the multi-wavelength anomalous diffraction method is underway[3]. Here, we report the first crystallization and structural analysis of the DAP BII from P. mexicana WO24 as family S46 peptidase. Other enzymes that belong to this family are DPP7 and DPP11 from Porphyromonas gingivalis, DPP11 from Porphyromonas endodontalis (periodontal pathogen) and DPP11 from Shewanella putrefaciens (multidrug resistance associated opportunistic pathogen). These gram-negative bacterial pathogens are known to asaccharolytic. Especially, Porphyromonas gingivalis is known to utilize dipeptides, instead of free amino acids, as energy source and cellular material. Since S46 peptidases are not found in mammals, we expect our study will be useful for the discovery of specific inhibitors to S46 peptidases from these pathogens.