Scott A. Lesley

Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites

Although it has long been recognized that the enteric community of bacteria that inhabit the human distal intestinal track broadly impacts human health, the biochemical details that underlie these effects remain largely undefined. Here, we report a broad MS-based metabolomics study that demonstrates a surprisingly large effect of the gut “microbiome” on mammalian blood metabolites. Plasma extracts from germ-free mice were compared with samples from conventional (conv) animals by using various MS-based methods. Hundreds of features were detected in only 1 sample set, with the majority of these being unique to the conv animals, whereas ≈10% of all features observed in both sample sets showed significant changes in their relative signal intensity. Amino acid metabolites were particularly affected. For example, the bacterial-mediated production of bioactive indole-containing metabolites derived from tryptophan such as indoxyl sulfate and the antioxidant indole-3-propionic acid (IPA) was impacted. Production of IPA was shown to be completely dependent on the presence of gut microflora and could be established by colonization with the bacterium Clostridium sporogenes. Multiple organic acids containing phenyl groups were also greatly increased in the presence of gut microbes. A broad, drug-like phase II metabolic response of the host to metabolites generated by the microbiome was observed, suggesting that the gut microflora has a direct impact on the drug metabolism capacity of the host. Together, these results suggest a significant interplay between bacterial and mammalian metabolism.

Structural Genomics of the Thermotoga maritima Proteome Implemented in a High-throughput Structure Determination Pipeline

Structural genomics is emerging as a principal approach to define protein structure–function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.

Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts

Successful protein expression, purification, and crystallization for challenging targets typically requires evaluation of a multitude of expression constructs. Often many iterations of truncations and point mutations are required to identify a suitable derivative for recombinant expression. Making and characterizing these variants is a significant barrier to success. We have developed a rapid and efficient cloning process and combined it with a protein microscreening approach to characterize protein suitability for structural studies. The Polymerase Incomplete Primer Extension (PIPE) cloning method was used to rapidly clone 448 protein targets and then to generate 2143 truncations from 96 targets with minimal effort. Proteins were expressed, purified, and characterized via a microscreening protocol, which incorporates protein quantification, liquid chromatography mass spectrometry and analytical size exclusion chromatography (AnSEC) to evaluate suitability of the protein products for X‐ray crystallography. The results suggest that selecting expression constructs for crystal trials based primarily on expression solubility is insufficient. Instead, AnSEC scoring as a measure of protein polydispersity was found to be predictive of ultimate structure determination success and essential for identifying appropriate boundaries for truncation series. Overall structure determination success was increased by at least 38% by applying this combined PIPE cloning and microscreening approach to recalcitrant targets. Proteins 2008.

Three-Dimensional Structural View of the Central Metabolic Network of Thermotoga maritima

Now Shown in 3D With the advent of systems-wide technologies and the development of analytical methods, data produced by analyzing individual or small groups of molecular components can be integrated to reassemble whole biological systems. Zhang et al. (p. 1544) have undertaken a major technological challenge: to integrate biochemical data with experimentally determined or predicted three-dimensional structures of all proteins involved in the central metabolism of a bacterial cell. This integration of large-scale data sets provides evolutionary and functional insights and furthers our understanding of the molecular assembly of complex biological networks. Protein structure and biochemical data generate a three-dimensional view of the metabolic network of a bacterial cell. Metabolic pathways have traditionally been described in terms of biochemical reactions and metabolites. With the use of structural genomics and systems biology, we generated a three-dimensional reconstruction of the central metabolic network of the bacterium Thermotoga maritima. The network encompassed 478 proteins, of which 120 were determined by experiment and 358 were modeled. Structural analysis revealed that proteins forming the network are dominated by a small number (only 182) of basic shapes (folds) performing diverse but mostly related functions. Most of these folds are already present in the essential core (~30%) of the network, and its expansion by nonessential proteins is achieved with relatively few additional folds. Thus, integration of structural data with networks analysis generates insight into the function, mechanism, and evolution of biological networks.

XtalPred: a web server for prediction of protein crystallizability

UNLABELLED XtalPred is a web server for prediction of protein crystallizability. The prediction is made by comparing several features of the protein with distributions of these features in TargetDB and combining the results into an overall probability of crystallization. XtalPred provides: (1) a detailed comparison of the proteins features to the corresponding distribution from TargetDB; (2) a summary of protein features and predictions that indicate problems that are likely to be encountered during protein crystallization; (3) prediction of ligands; and (4) (optional) lists of close homologs from complete microbial genomes that are more likely to crystallize. AVAILABILITY The XtalPred web server is freely available for academic users on http://ffas.burnham.org/XtalPred

Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity

PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.

Crystal structure of the ALK (anaplastic lymphoma kinase) catalytic domain.

ALK (anaplastic lymphoma kinase) is an RTK (receptor tyrosine kinase) of the IRK (insulin receptor kinase) superfamily, which share an YXXXYY autophosphorylation motif within their A-loops (activation loops). A common activation and regulatory mechanism is believed to exist for members of this superfamily typified by IRK and IGF1RK (insulin-like growth factor receptor kinase-1). Chromosomal translocations involving ALK were first identified in anaplastic large-cell lymphoma, a subtype of non-Hodgkins lymphoma, where aberrant fusion of the ALK kinase domain with the NPM (nucleophosmin) dimerization domain results in autophosphosphorylation and ligand-independent activation. Activating mutations within the full-length ALK kinase domain, most commonly R1275Q and F1174L, which play a major role in neuroblastoma, were recently identified. To provide a structural framework for understanding these mutations and to guide structure-assisted drug discovery efforts, the X-ray crystal structure of the unphosphorylated ALK catalytic domain was determined in the apo, ADP- and staurosporine-bound forms. The structures reveal a partially inactive protein kinase conformation distinct from, and lacking, many of the negative regulatory features observed in inactive IGF1RK/IRK structures in their unphosphorylated forms. The A-loop adopts an inhibitory pose where a short proximal A-loop helix (alphaAL) packs against the alphaC helix and a novel N-terminal beta-turn motif, whereas the distal portion obstructs part of the predicted peptide-binding region. The structure helps explain the reported unique peptide substrate specificity and the importance of phosphorylation of the first A-loop Tyr1278 for kinase activity and NPM-ALK transforming potential. A single amino acid difference in the ALK substrate peptide binding P-1 site (where the P-site is the phosphoacceptor site) was identified that, in conjunction with A-loop sequence variation including the RAS (Arg-Ala-Ser)-motif, rationalizes the difference in the A-loop tyrosine autophosphorylation preference between ALK and IGF1RK/IRK. Enzymatic analysis of recombinant R1275Q and F1174L ALK mutant catalytic domains confirms the enhanced activity and transforming potential of these mutants. The transforming ability of the full-length ALK mutants in soft agar colony growth assays corroborates these findings. The availability of a three-dimensional structure for ALK will facilitate future structure-function and rational drug design efforts targeting this receptor tyrosine kinase.

The self-inhibited structure of full-length PCSK9 at 1.9 Å reveals structural homology with resistin within the C-terminal domain

Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) are strongly associated with levels of low-density lipoprotein cholesterol in the blood plasma and, thereby, occurrence or resistance to atherosclerosis and coronary heart disease. Despite this importance, relatively little is known about the biology of PCSK9. Here, the crystal structure of a full-length construct of PCSK9 solved to 1.9-Å resolution is presented. The structure contains a fully folded C-terminal cysteine-rich domain (CRD), showing a distinct structural similarity to the resistin homotrimer, a small cytokine associated with obesity and diabetes. This structural relationship between the CRD of PCSK9 and the resistin family is not observed in primary sequence comparisons and strongly suggests a distant evolutionary link between the two molecules. This three-dimensional homology provides insight into the function of PCSK9 at the molecular level and will help to dissect the link between PCSK9 and CHD.

The challenge of protein structure determination—lessons from structural genomics

The process of experimental determination of protein structure is marred with a high ratio of failures at many stages. With availability of large quantities of data from high‐throughput structure determination in structural genomics centers, we can now learn to recognize protein features correlated with failures; thus, we can recognize proteins more likely to succeed and eventually learn how to modify those that are less likely to succeed. Here, we identify several protein features that correlate strongly with successful protein production and crystallization and combine them into a single score that assesses “crystallization feasibility.” The formula derived here was tested with a jackknife procedure and validated on independent benchmark sets. The “crystallization feasibility” score described here is being applied to target selection in the Joint Center for Structural Genomics, and is now contributing to increasing the success rate, lowering the costs, and shortening the time for protein structure determination. Analyses of PDB depositions suggest that very similar features also play a role in non‐high‐throughput structure determination, suggesting that this crystallization feasibility score would also be of significant interest to structural biology, as well as to molecular and biochemistry laboratories.

The JCSG High-throughput Structural Biology Pipeline

The Joint Center for Structural Genomics high-throughput structural biology pipeline has delivered more than 1000 structures to the community over the past ten years and has made a significant contribution to the overall goal of the NIH Protein Structure Initiative (PSI) of expanding structural coverage of the protein universe.