David H. Dyer

3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili.

Type IV pili (Tfp) are widespread filamentous bacterial organelles that mediate multiple virulence-related phenotypes. They are composed mainly of pilin subunits, which are processed before filament assembly by dedicated prepilin peptidases. Other proteins processed by these peptidases, whose molecular nature and mode of action remain enigmatic, play critical roles in Tfp biology. We have performed a detailed structure/function analysis of one such protein, PilX from Neisseria meningitidis, which is crucial for formation of bacterial aggregates and adhesion to human cells. The x-ray crystal structure of PilX reveals the α/β roll fold shared by all pilins, and we show that this protein colocalizes with Tfp. These observations suggest that PilX is a minor, or low abundance, pilin that assembles within the filaments in a similar way to pilin. Deletion of a PilX distinctive structural element, which is predicted to be exposed on the filament surface, abolishes aggregation and adhesion. Our results support a model in which surface-exposed motifs in PilX subunits stabilize bacterial aggregates against the disruptive force of pilus retraction and illustrate how a minor pilus component can enhance the functional properties of pili of rather simple composition and structure.

The Structural Determination of an Insect Sterol Carrier Protein-2 with a Ligand-bound C16 Fatty Acid at 1.35-Å Resolution

Yellow fever mosquito sterol carrier protein (SCP-2) is known to bind to cholesterol. We report here the three-dimensional structure of the complex of SCP-2 from Aedes aegypti with a C16 fatty acid to 1.35-Å resolution. The protein fold is exceedingly similar to the human and rabbit proteins, which consist of a five-stranded β-sheet that exhibits strand order 3-2-1-4-5 with an accompanying layer of four α-helices that cover the β-sheet. A large cavity exists at the interface of the layer α-helices and the β-sheet, which serves as the fatty acid binding site. The carboxylate moiety of the fatty acid is coordinated by a short loop that connects the first α-helix to the first β-strand, whereas the acyl chain extends deep into the interior of the protein. Interestingly, the orientation of the fatty acid is opposite to the observed orientation for Triton X-100 in the SCP-2-like domain from the peroxisomal multifunctional enzyme (Haapalainen, A. M., van Aalten, D. M., Merilainen, G., Jalonen, J. E., Pirila, P., Wierenga, R. K., Hiltunen, J. K., and Glumoff, T. (2001) J. Mol. Biol. 313, 1127-1138). The present study suggests that the binding pocket in the SCP-2 family of proteins may exhibit conformational flexibility to allow coordination of a variety of lipids.

X-ray structure of putative acyl-ACP desaturase DesA2 from Mycobacterium tuberculosis H37Rv

Genome sequencing showed that two proteins in Mycobacterium tuberculosis H37Rv contain the metal binding motif (D/E)X2HX∼100(D/E)X2H characteristic of the soluble diiron enzyme superfamily. These putative acyl‐ACP desaturase genes desA1 and desA2 were cloned from genomic DNA and expressed in Escherichia coli BL21(DE3). DesA1 was found to be insoluble, but in contrast, DesA2 was a soluble protein amenable to biophysical characterization. Here, we report the 2.0 Å resolution X‐ray structure of DesA2 determined by multiple anomalous dispersion (MAD) phasing from a Se‐met derivative and refinement against diffraction data obtained on the native protein. The X‐ray structure shows that DesA2 is a homodimeric protein with a four‐helix bundle core flanked by five additional helices that overlay with 192 structurally equivalent amino acids in the structure of stearoyl‐ACP Δ9 desaturase from castor plant with an rms difference 1.42 Å. In the DesA2 crystals, one metal (likely Mn from the crystallization buffer) was bound in high occupancy at the B‐site of the conserved metal binding motif, while the A‐site was not occupied by a metal ion. Instead, the amino group of Lys‐76 occupied this position. The relationships between DesA2 and known diiron enzymes are discussed.

Three-dimensional structure/function analysis of SCP-2-like2 reveals differences among SCP-2 family members

Mosquito sterol carrier protein-2 (AeSCP-2) and sterol carrier protein-2-like2 (AeSCP-2L2) are members of the SCP-2 protein family with similar expression profiles in the mosquito life cycle. In an effort to understand how lipids can be transported by different SCP-2 proteins, the three-dimensional crystal structure of AeSCP-2L2 was solved at 1.7 Å resolution. AeSCP-2L2 forms a dimer and binds three fatty acids, one of which resides in a position within the internal cavity at a right angle to the others. This first report of ligand-bound dimerized protein in the SCP-2 protein family indicates that the family has a much more divergent mode of interaction with ligands than previously reported. The potential function of AeSCP-2L2 was investigated via in vivo incorporation of [3H]cholesterol and [3H]palmitic acid. Overexpression of AeSCP-2L2 in mosquito cells leads to an increased uptake of free fatty acid, whereas knockdown of AeSCP-2L2 in adult females decreases the accumulation of free fatty acid in the fat body from a blood meal. In contrast, overexpression or knockdown of AeSCP-2L2 has no effect on cholesterol uptake. Our results suggest that the main function of AeSCP-2L2 is as a general intracellular fatty acid carrier, as opposed to having a dedicated role in cholesterol transport.

A Polyketide Synthase Acyltransferase Domain Structure Suggests a Recognition Mechanism for Its Hydroxymalonyl-Acyl Carrier Protein Substrate

We have previously shown that the acyl transferase domain of ZmaA (ZmaA-AT) is involved in the biosynthesis of the aminopolyol polyketide/nonribosomal peptide hybrid molecule zwittermicin A from cereus UW85, and that it specifically recognizes the precursor hydroxymalonyl-acyl carrier protein (ACP) and transfers the hydroxymalonyl extender unit to a downstream second ACP via a transacylated AT domain intermediate. We now present the X-ray crystal structure of ZmaA-AT at a resolution of 1.7 Å. The structure shows a patch of solvent-exposed hydrophobic residues in the area where the AT is proposed to interact with the precursor ACP. We addressed the significance of the AT/ACP interaction in precursor specificity of the AT by testing whether malonyl- or methylmalonyl-ACP can be recognized by ZmaA-AT. We found that the ACP itself biases extender unit selection. Until now, structural information for ATs has been limited to ATs specific for the CoA-linked precursors malonyl-CoA and (2S)-methylmalonyl-CoA. This work contributes to polyketide synthase engineering efforts by expanding our knowledge of AT/substrate interactions with the structure of an AT domain that recognizes an ACP-linked substrate, the rare hydroxymalonate. Our structure suggests a model in which ACP interaction with a hydrophobic motif promotes secondary structure formation at the binding site, and opening of the adjacent substrate pocket lid to allow extender unit binding in the AT active site.

Aldose reductase from Schistosoma japonicum : crystallization and structure-based inhibitor screening for discovering antischistosomal lead compounds

BackgroundSchistosomiasis is a neglected tropical disease with high morbidity and mortality in the world. Currently, the treatment of this disease depends almost exclusively on praziquantel (PZQ); however, the emergence of drug resistance to PZQ in schistosomes makes the development of novel drugs an urgent task. Aldose reductase (AR), an important component that may be involved in the schistosome antioxidant defense system, is predicted as a potential drug target.MethodsThe tertiary structure of Schistosoma japonicum AR (Sj AR) was obtained through X-ray diffraction method and then its potential inhibitors were identified from the Maybridge HitFinder library by virtual screening based on this structural model. The effects of these identified compounds on cultured adult worms were evaluated by observing mobility, morphological changes and mortality. To verify that Sj AR was indeed the target of these identified compounds, their effects on recombinant Sj AR (rSj AR) enzymatic activity were assessed. The cytotoxicity analysis was performed with three types of human cell lines using a Cell Counting Kit-8.ResultsWe firstly resolved the Sj AR structure and identified 10 potential inhibitors based on this structural model. Further in vitro experiments showed that one of the compounds, renamed as AR9, exhibited significant inhibition in the activity of cultured worms as well as inhibition of enzymatic activity of rSj AR protein. Cytotoxicity analysis revealed that AR9 had relatively low toxicity towards host cells.ConclusionsThe work presented here bridges the gap between virtual screening and experimental validation, providing an effective and economical strategy for the development of new anti-parasitic drugs. Additionally, this study also found that AR9 may become a new potential lead compound for developing novel antischistosomal drugs against parasite AR.

Structure of the minor pseudopilin XcpW from the Pseudomonas aeruginosa type II secretion system

Pseudomonas aeruginosa utilizes the type II secretion machinery to transport virulence factors through the outer membrane into the extracellular space. Five proteins in the type II secretion system share sequence homology with pilin subunits of type IV pili and are called the pseudopilins. The major pseudopilin XcpT(G) assembles into an intraperiplasmic pilus and is thought to act in a piston-like manner to push substrates through an outer membrane secretin. The other four minor pseudopilins, XcpU(H), XcpV(I), XcpW(J) and XcpX(K), play less well defined roles in pseudopilus formation. It was recently discovered that these four minor pseudopilins form a quaternary complex that is presumed to initiate the formation of the pseudopilus and to localize to its tip. Here, the structure of XcpW(J) was refined to 1.85 Å resolution. The structure revealed the type IVa pilin fold with an embellished variable antiparallel β-sheet as also found in the XcpW(J) homologue enterotoxigenic Escherichia coli GspJ(W) and the XcpU(H) homologue Vibrio cholerae EpsU(H). It is proposed that the exposed surface of this sheet may cradle the long N-terminal α1 helix of another pseudopilin. The final 31 amino acids of the XcpW(J) structure are instrinsically disordered. Deletion of this unstructured region of XcpW(J) did not prevent type II secretion in vivo.

Diversity and abundance of ammonia oxidizing archaea in tropical compost systems

Composting is widely used to transform waste materials into valuable agricultural products. In the tropics, large quantities of agricultural wastes could be potentially useful in agriculture after composting. However, while microbiological processes of composts in general are well established, relatively little is known about microbial communities that may be unique to these in tropical systems, particularly nitrifiers. The recent discovery of ammonia oxidizing archaea (AOA) has changed the paradigm of nitrification being initiated solely by ammonia oxidizing bacteria. In the present study, AOA abundance and diversity was examined in composts produced from combinations of plant waste materials common in tropical agriculture (rice straw, sugar cane bagasse, and coffee hulls), which were mixed with either cow- or sheep-manure. The objective was to determine how AOA abundance and diversity varied as a function of compost system and time, the latter being a contrast between the start of the compost process (mesophilic phase) and the finished product (mature phase). The results showed that AOA were relatively abundant in composts of tropical agricultural wastes, and significantly more so than were the ammonia-oxidizing bacteria. Furthermore, while the AOA communities in the composts were predominatly group I.1b, the communities were diverse and exhibited structures that diverged between compost types and phases. These patterns could be taken as indicators of the ecophysiological diversity in the soil AOA (group I.1b), in that significantly different AOA communties developed when exposed to varying physico-chemical environments. Nitrification patterns and levels differed in the composts which, for the mature material, could have significant effects on its performance as a plant growth medium. Thus, it will also be important to determine the association of AOA (and diversity in their communities) with nitrification in these systems.

Crystal structure of gene locus At3g16990 from Arabidopsis thaliana

The Center for Eukaryotic StructuralGenomicsisdedicatedtodeterminingthestructuresofnovelproteins from eukaryotic organisms. Open reading framesare scored using thirteen different categories (i.e. new foldprediction, solubility prediction, small percentage of lowcomplexity sequence, etc.) and then ranked to indicate theirsuitabilityforstudybynuclearmagneticresonance(NMR)orX-raycrystallography.GenelocusAt3g16990from