Antibody recognition by a novel microgel photonic crystal

In this study, a easy-to-prepare biosensor for the sensitive detection of the antibody (Ab) protein was developed using a novel microgel photonic crystal (MPC). The MPC was fabricated by the spin-coated self-assembly method with the monodisperse Ab-sensitive poly (methyl methacrylate-acrylamide-glutaraldehyde-hapten) (P(MMA-AM-GA-HP)) microgels. Morphology characterization showed that the P(MMA-AM-GA-HP) microgels possessed round shapes and the large specific surface area, and the formed MPC had a highly ordered three dimensional (3D) periodically-ordered structure with the desired structural color. The Ab-response event of the P(MMA-AM-GA-HP) microgels can be directly transferred into a readable optical signal through a change in Bragg reflection of the periodic structure of the MPC. With the sensory system, the sensitive and selective detection of Ab was achieved without labeling techniques and expensive instruments. Therefore, this easy and sensitive detection system has great potential for next generation of the bioassay platform for clinical diagnosis and other applications.     

Great spotted cuckoo eggshell microstructure characteristics can make eggs stronger

Obligate avian brood parasites lay stronger eggs than their hosts or non‐parasitic relatives because they are rounder and have a thicker eggshell. Additionally, some other characteristics of the brood parasitic eggshells related to their microstructure such as size and orientation of calcite crystal units could also contribute to generating even stronger shells. An eggshell microstructure formed by small randomly oriented calcite crystal units can increase the robustness of the eggshells of birds. Here, the eggshell microstructure of avian brood parasites as well as their hosts have been characterized in detail, using X‐ray diffraction analyses to estimate the size and degree of orientation of calcite crystal units making the eggshell. Specifically, the brood parasitic great spotted cuckoo Clamator glandarius and two hosts (jackdaws Corvus monedula and magpie Pica pica) and one non‐host species (the pigeon, Columba livia domestica) were considered. Calcite crystal of the eggshell of the brood parasitic species was smaller and more randomly oriented than those of the eggshells of non‐parasitic species, which suggest that eggshell microstructure would contribute to explain why parasitic eggs are more resistant to breakage than those of their hosts.     

Structures and activities of widely conserved small prokaryotic aminopeptidases-P clarify classification of M24B peptidases

M24B peptidases cleaving Xaa-Pro bond in dipeptides are prolidases whereas those cleaving this bond in longer peptides are aminopeptidases-P. Bacteria have small aminopeptidases-P (36-39 kDa), which are diverged from canonical aminopeptidase-P of Escherichia coli (50 kDa). Structure-function studies of small aminopeptidases-P are lacking. We report crystal structures of small aminopeptidases-P from E. coli and Deinococcus radiodurans, and report substrate-specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are aminopeptidases-P, structurally close to small prolidases except for absence of dipeptide-selectivity loop. We noticed absence of this loop and conserved arginine in canonical archaeal prolidase (Maher et al., Biochemistry. 43, 2004, 2771-2783) and questioned its classification. Our enzymatic assays show that this enzyme is an aminopeptidase-P. Further, our mutagenesis studies illuminate importance of DXRY sequence motif in bacterial small aminopeptidases-P and suggest common evolutionary origin with human XPNPEP1/XPNPEP2. Our analyses reveal sequence/structural features distinguishing small aminopeptidases-P from other M24B peptidases.     

Structural insights into the specific interaction between Geobacillus stearothermophilus tryptophanyl-tRNA synthetase and antimicrobial Chuangxinmycin

The potential antimicrobial compound Chuangxinmycin (CXM) targets the tryptophanyl-tRNA synthetase (TrpRS) of both Gram-negative and Gram-positive bacteria. However, the specific steric recognition mode and interaction mechanism between CXM and TrpRS is unclear. Here, we studied this interaction using recombinant GsTrpRS from Geobacillus stearothermophilus by X-ray crystallography and molecular dynamics (MD) simulations. The crystal structure of the recombinant GsTrpRS in complex with CXM was experimentally determined to a resolution at 2.06 Å. After analysis using a complex-structure probe, MD simulations, and site-directed mutation verification through isothermal titration calorimetry, the interaction between CXM and GsTrpRS was determined to involve the key residues M129, D132, I133, and V141 of GsTrpRS. We further evaluated binding affinities between GsTrpRS WT/mutants and CXM; GsTrpRS was found to bind CXM through hydrogen bonds with D132 and hydrophobic interactions between the lipophilic tricyclic ring of CXM and M129, I133, and V141 in the substrate-binding pockets. This study elucidates the precise interaction mechanism between CXM and its target GsTrpRS at the molecular level and provides a theoretical foundation and guidance for the screening and rational design of more effective CXM analogs against both Gram-negative and Gram-positive bacteria.     

Structural analysis of P450 AmphL from Streptomyces nodosus provides insights into substrate selectivity of polyene macrolide antibiotic biosynthetic P450s

AmphL is a cytochrome P450 enzyme that catalyzes the C8 oxidation of 8-deoxyamphotericin B to the polyene macrolide antibiotic, amphotericin B. To understand this substrate selectivity, we solved the crystal structure of AmphL to a resolution of 2.0 Å in complex with amphotericin B and performed molecular dynamics (MD) simulations. A detailed comparison with the closely related P450, PimD, which catalyzes the epoxidation of 4,5-desepoxypimaricin to the macrolide antibiotic, pimaricin, reveals key catalytic structural features responsible for stereo- and regio-selective oxidation. Both P450s have a similar access channel that runs parallel to the active site I helix over the surface of the heme. Molecular dynamics simulations of substrate binding reveal PimD can “pull” substrates further into the P450 access channel owing to additional electrostatic interactions between the protein and the carboxyl group attached to the hemiketal ring of 4,5-desepoxypimaricin. This substrate interaction is absent in AmphL although the additional substrate -OH groups in 8-deoxyamphotericin B help to correctly position the substrate for C8 oxidation. Simulations of the oxy-complex indicates that these -OH groups may also participate in a proton relay network required for O₂ activation as has been suggested for two other macrolide P450s, PimD and P450eryF. These findings provide experimentally testable models that can potentially contribute to a new generation of novel macrolide antibiotics with enhanced antifungal and/or antiprotozoal efficacy.     

The mechanism of aconitase: 1.8 A resolution crystal structure of the S642a:citrate complex

The crystal structure of the S642A mutant of mitochondrial aconitase (mAc) with citrate bound has been determined at 1.8 A resolution and 100 K to capture this binding mode of substrates to the native enzyme. The 2.0 A resolution, 100 K crystal structure of the S642A mutant with isocitrate binding provides a control, showing that the Ser --> Ala replacement does not alter the binding of substrates in the active site. The aconitase mechanism requires that the intermediate product, cis-aconitate, flip over by 180 degrees about the C alpha-C beta double bond. Only one of these two alternative modes of binding, that of the isocitrate mode, has been previously visualized. Now, however, the structure revealing the citrate mode of binding provides direct support for the proposed enzyme mechanism.     

Bicelle-based liquid crystals for NMR-measurement of dipolar couplings at acidic and basic pH values

It is demonstrated that mixtures of ditetradecyl- phosphatidylcholine or didodecyl-phoshatidylcholine and dihexyl- phosphatidylcholine in water form lyotropic liquid crystalline phases under similar conditions as previously reported for bicelles consisting of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl- phosphatidylcholine (DHPC). The carboxy-ester bonds present in DMPC and DHPC are replaced by ether linkages in their alkyl analogs, which prevents acid- or base-catalyzed hydrolysis of these compounds. 15N-1H dipolar couplings measured for ubiquitin over the 2.3–10.4pH range indicate that this protein retains a backbone conformation which is very similar to its structure at pH 6.5 over this entire range.     

Recent advances in the structure and function of isopenicillin N synthase

Isopenicillin N synthase is a key enzyme in the biosynthesis of penicillin and cephalosporin antibiotics, catalyzing the oxidative ring closure of -(L--aminoadipoyl)-L-cysteinyl-D-valine to form isopenicillin N. Recent advances in our understanding of the unique chemistry of this enzyme have come through the combined application of spectroscopic, molecular genetic and crystallographic approaches and led to important new insights into the structure and function of this enzyme. Here we review new information on the nature of the endogenous ligands that constitute the ferrous iron active site, sequence evidence for a novel structural motif involved in iron binding in this and related non-heme iron dependent dioxygenases, crystal structure studies on the enzyme and its substrate complex and the impact of these and site-directed mutagenesis studies for unraveling the mechanism of the isopenicillin N synthase reaction.     

Crystal structures of adenine phosphoribosyltransferase from Leishmania donovani.

The enzyme adenine phosphoribosyltransferase (APRT) functions to salvage adenine by converting it to adenosine-5-monophosphate (AMP). APRT deficiency in humans is a well characterized inborn error of metabolism, and APRT may contribute to the indispensable nutritional role of purine salvage in protozoan parasites, all of which lack de novo purine biosynthesis. We determined crystal structures for APRT from Leishmania donovani in complex with the substrate adenine, the product AMP, and sulfate and citrate ions that appear to mimic the binding of phosphate moieties. Overall, these structures are very similar to each other, although the adenine and AMP complexes show different patterns of hydrogen-bonding to the base, and the active site pocket opens slightly to accommodate the larger AMP ligand. Whereas AMP adopts a single conformation, adenine binds in two mutually exclusive orientations: one orientation providing adenine-specific hydrogen bonds and the other apparently positioning adenine for the enzymatic reaction. The core of APRT is similar to that of other phosphoribosyltransferases, although the adenine-binding domain is quite different. A C-terminal extension, unique to Leishmania APRTs, extends an extensive dimer interface by wrapping around the partner molecule. The active site involves residues from both subunits of the dimer, indicating that dimerization is essential for catalysis.     

Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: effects of side-chain size on reduction potential

Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles. Here, the high-resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding. Our crystal structures of [V44L] (at 1.8 A resolution), [V44A] (1.6 A), [V44G] (2.0 A) and [V44A, G45P] (1.5 A) Rd (all in their oxidized states) show that there is a gradual decrease in the distance between Fe and the amide nitrogen of residue 44 upon reduction in the size of the side chain of residue 44; the decrease occurs from leucine to valine, alanine or glycine and is accompanied by a gradual increase in their reduction potentials. Mutation of Cp Rd at position 44 also changes the hydrogen-bond distance between the amide nitrogen of residue 44 and the sulfur of cysteine 42 in a size-dependent manner. Our results suggest that residue 44 is an important determinant of Rd reduction potential in a manner dictated by side-chain size. Along with the electric dipole moment of the 43-44 peptide bond and the 44-42 NH--S type hydrogen bond, a modulation mechanism for solvent accessibility through residue 41 might regulate the redox reaction of the Rds.