Publications by authors named "Scott A Lesley"

185 Publications

A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions.

Cell 2020 08;182(4):1027-1043.e17

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA; Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed ∼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.
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http://dx.doi.org/10.1016/j.cell.2020.07.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7440162PMC
August 2020

Structures of single-layer β-sheet proteins evolved from β-hairpin repeats.

Protein Sci 2019 09 2;28(9):1676-1689. Epub 2019 Aug 2.

Joint Center for Structural Genomics, www.jcsg.org.

Free-standing single-layer β-sheets are extremely rare in naturally occurring proteins, even though β-sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single-layer, anti-parallel β-sheet proteins, comprised of three or four twisted β-hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of β-sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent-exposed tyrosine residues, was identified on the concave surface of the β-sheet. These new modular single-layer β-sheet proteins may serve as a new model system for studying folding and design of β-rich proteins.
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http://dx.doi.org/10.1002/pro.3683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699103PMC
September 2019

A High-Throughput System for Transient and Stable Protein Production in Mammalian Cells.

Methods Mol Biol 2019 ;2025:93-142

Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA.

Recombinant protein expression and purification is an essential component of biomedical research and drug discovery. Advances in automation and laboratory robotics have enabled the development of highly parallel and rapid processes for cell culture and protein expression, purification, and analysis. Human embryonic kidney (HEK) cells and Chinese hamster ovary (CHO) cells have emerged as the standard host cell workhorses for producing recombinant secreted mammalian proteins by using both transient and stable production strategies. In this chapter we describe a fully automated custom platform, Protein Expression and Purification Platform (PEPP), used for transient protein production from HEK cells and stable protein production from CHO cells. Central to PEPP operation is a suite of custom robotic and instrumentation platforms designed and built at GNF, custom cell culture ware, and custom scheduling software referred to as Runtime. The PEPP platform enables cost-effective, facile, consistent production of proteins at quantities and quality useful for early stage drug discovery tasks such as screening, bioassays, protein engineering, and analytics.
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http://dx.doi.org/10.1007/978-1-4939-9624-7_5DOI Listing
March 2020

Mammalian cell culture density determination using a laser through-beam sensor.

Biotechniques 2018 10;65(4):224-226

Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA.

High-throughput protein expression platforms are increasingly used to produce proteins for many applications: to support studies in structure/function, regulation and proteomics, as well as for direct use as potential biotherapeutic agents for medical applications. Here we describe a device that we refer to as the flask density reader (FDR) consisting of a through-beam laser and sensor, and a customized culture flask-receiving nest. The FDR has been integrated onto GNF System™'s automated protein expression platform to enable rapid, noninvasive, fully automated spectrophotometric determination of cell densities in suspension mammalian cell cultures. The FDR reduces the risk of culture contamination from frequent flask sampling and greatly reduces the time and effort needed to count cells using off-line methods.
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http://dx.doi.org/10.2144/btn-2018-0059DOI Listing
October 2018

Co-regulatory networks of human serum proteins link genetics to disease.

Science 2018 08 2;361(6404):769-773. Epub 2018 Aug 2.

Icelandic Heart Association, Holtasmari 1, IS-201 Kopavogur, Iceland.

Proteins circulating in the blood are critical for age-related disease processes; however, the serum proteome has remained largely unexplored. To this end, 4137 proteins covering most predicted extracellular proteins were measured in the serum of 5457 Icelanders over 65 years of age. Pairwise correlation between proteins as they varied across individuals revealed 27 different network modules of serum proteins, many of which were associated with cardiovascular and metabolic disease states, as well as overall survival. The protein modules were controlled by cis- and trans-acting genetic variants, which in many cases were also associated with complex disease. This revealed co-regulated groups of circulating proteins that incorporated regulatory control between tissues and demonstrated close relationships to past, current, and future disease states.
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http://dx.doi.org/10.1126/science.aaq1327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6190714PMC
August 2018

A Distinct Type of Pilus from the Human Microbiome.

Cell 2016 Apr 7;165(3):690-703. Epub 2016 Apr 7.

Joint Center for Structural Genomics, http://www.jcsg.org; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

Pili are proteinaceous polymers of linked pilins that protrude from the cell surface of many bacteria and often mediate adherence and virulence. We investigated a set of 20 Bacteroidia pilins from the human microbiome whose structures and mechanism of assembly were unknown. Crystal structures and biochemical data revealed a diverse protein superfamily with a common Greek-key β sandwich fold with two transthyretin-like repeats that polymerize into a pilus through a strand-exchange mechanism. The assembly mechanism of the central, structural pilins involves proteinase-assisted removal of their N-terminal β strand, creating an extended hydrophobic groove that binds the C-terminal donor strands of the incoming pilin. Accessory pilins at the tip and base have unique structural features specific to their location, allowing initiation or termination of the assembly. The Bacteroidia pilus, therefore, has a biogenesis mechanism that is distinct from other known pili and likely represents a different type of bacterial pilus.
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http://dx.doi.org/10.1016/j.cell.2016.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842110PMC
April 2016

UHM-ULM interactions in the RBM39-U2AF65 splicing-factor complex.

Acta Crystallogr D Struct Biol 2016 Apr 24;72(Pt 4):497-511. Epub 2016 Mar 24.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein-protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39-U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemical shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35-U2AF65 and RBM39-SF3b155, the RBM39-UHM-U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM-ULM binding interface, providing a rationale for the known specificity of UHM-ULM interactions. This study therefore establishes a structural basis for specific UHM-ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.
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http://dx.doi.org/10.1107/S2059798316001248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822562PMC
April 2016

Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome.

J Biol Chem 2016 Apr 3;291(18):9482-91. Epub 2016 Mar 3.

From the Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom, the Department of Biology, Centre for Immunology and Infection, University of York, Wentworth Way, Heslington, York YO10 5DD, United Kingdom,

Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys(147), resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys(147) to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca(2+) for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.
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http://dx.doi.org/10.1074/jbc.M115.706143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850288PMC
April 2016

Structure and functional characterization of a bile acid 7α dehydratase BaiE in secondary bile acid synthesis.

Proteins 2016 Mar 18;84(3):316-31. Epub 2016 Jan 18.

Joint Center for Structural Genomics, (http://www.jcsg.org).

Conversion of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) is performed by a few species of intestinal bacteria in the genus Clostridium through a multistep biochemical pathway that removes a 7α-hydroxyl group. The rate-determining enzyme in this pathway is bile acid 7α-dehydratase (baiE). In this study, crystal structures of apo-BaiE and its putative product-bound [3-oxo-Δ(4,6) -lithocholyl-Coenzyme A (CoA)] complex are reported. BaiE is a trimer with a twisted α + β barrel fold with similarity to the Nuclear Transport Factor 2 (NTF2) superfamily. Tyr30, Asp35, and His83 form a catalytic triad that is conserved across this family. Site-directed mutagenesis of BaiE from Clostridium scindens VPI 12708 confirm that these residues are essential for catalysis and also the importance of other conserved residues, Tyr54 and Arg146, which are involved in substrate binding and affect catalytic turnover. Steady-state kinetic studies reveal that the BaiE homologs are able to turn over 3-oxo-Δ(4) -bile acid and CoA-conjugated 3-oxo-Δ(4) -bile acid substrates with comparable efficiency questioning the role of CoA-conjugation in the bile acid metabolism pathway.
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http://dx.doi.org/10.1002/prot.24971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755848PMC
March 2016

Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Phosphopantetheinyl Transferases.

Bioconjug Chem 2015 Dec 4;26(12):2554-62. Epub 2015 Dec 4.

Genomics Institute of the Novartis Research Foundation , 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States.

Post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody-drug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain displayed in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site, almost all ADCs exhibited subnanomolar in vitro cytotoxicity against HER2-positive cell lines. One selected ADC was shown to induce tumor regression in a xenograft model at a single dose of 3 mg/kg, demonstrating that PPTase-mediated conjugation is suitable for the production of highly efficacious and homogeneous ADCs.
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http://dx.doi.org/10.1021/acs.bioconjchem.5b00558DOI Listing
December 2015

Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains.

mBio 2015 Sep 15;6(5):e02327-14. Epub 2015 Sep 15.

Joint Center for Structural Genomics‡ Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA

Unlabelled: Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (or dl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminal l-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

Importance: Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.
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http://dx.doi.org/10.1128/mBio.02327-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600125PMC
September 2015

Estimation of Hydrogen-Exchange Protection Factors from MD Simulation Based on Amide Hydrogen Bonding Analysis.

J Chem Inf Model 2015 Sep 20;55(9):1914-25. Epub 2015 Aug 20.

Genomics Institute of the Novartis Research Foundation , 10675 John Jay Hopkins Drive, San Diego, California 92121, United States.

Hydrogen exchange (HX) studies have provided critical insight into our understanding of protein folding, structure, and dynamics. More recently, hydrogen exchange mass spectrometry (HX-MS) has become a widely applicable tool for HX studies. The interpretation of the wealth of data generated by HX-MS experiments as well as other HX methods would greatly benefit from the availability of exchange predictions derived from structures or models for comparison with experiment. Most reported computational HX modeling studies have employed solvent-accessible-surface-area based metrics in attempts to interpret HX data on the basis of structures or models. In this study, a computational HX-MS prediction method based on classification of the amide hydrogen bonding modes mimicking the local unfolding model is demonstrated. Analysis of the NH bonding configurations from molecular dynamics (MD) simulation snapshots is used to determine partitioning over bonded and nonbonded NH states and is directly mapped into a protection factor (PF) using a logistics growth function. Predicted PFs are then used for calculating deuteration values of peptides and compared with experimental data. Hydrogen exchange MS data for fatty acid synthase thioesterase (FAS-TE) collected for a range of pHs and temperatures was used for detailed evaluation of the approach. High correlation between prediction and experiment for observable fragment peptides is observed in the FAS-TE and additional benchmarking systems that included various apo/holo proteins for which literature data were available. In addition, it is shown that HX modeling can improve experimental resolution through decomposition of in-exchange curves into rate classes, which correlate with prediction from MD. Successful rate class decompositions provide further evidence that the presented approach captures the underlying physical processes correctly at the single residue level. This assessment is further strengthened in a comparison of residue resolved protection factor predictions for staphylococcal nuclease with NMR data, which was also used to compare prediction performance with other algorithms described in the literature. The demonstrated transferable and scalable MD based HX prediction approach adds significantly to the available tools for HX-MS data interpretation based on available structures and models.
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http://dx.doi.org/10.1021/acs.jcim.5b00185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4587094PMC
September 2015

Cofactor-induced reversible folding of Flavodoxin-4 from Lactobacillus acidophilus.

Protein Sci 2015 Oct 30;24(10):1600-8. Epub 2015 Jul 30.

Joint Center for Structural Genomics, La Jolla, California, 92037.

Flavodoxins in combination with the flavin mononucleotide (FMN) cofactor play important roles for electron transport in prokaryotes. Here, novel insights into the FMN-binding mechanism to flavodoxins-4 were obtained from the NMR structures of the apo-protein from Lactobacillus acidophilus (YP_193882.1) and comparison of its complex with FMN. Extensive reversible conformational changes were observed upon FMN binding and release. The NMR structure of the FMN complex is in agreement with the crystal structure (PDB ID: 3EDO) and exhibits the characteristic flavodoxin fold, with a central five-stranded parallel β-sheet and five α-helices forming an α/β-sandwich architecture. The structure differs from other flavoproteins in that helix α2 is oriented perpendicular to the β-sheet and covers the FMN-binding site. This helix reversibly unfolds upon removal of the FMN ligand, which represents a unique structural rearrangement among flavodoxins.
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http://dx.doi.org/10.1002/pro.2743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594659PMC
October 2015

Crystal structure of a two-subunit TrkA octameric gating ring assembly.

PLoS One 2015 31;10(3):e0122512. Epub 2015 Mar 31.

The Joint Center for Structural Genomics, and Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, United States of America; Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.

The TM1088 locus of T. maritima codes for two proteins designated TM1088A and TM1088B, which combine to form the cytosolic portion of a putative Trk K+ transporter. We report the crystal structure of this assembly to a resolution of 3.45 Å. The high resolution crystal structures of the components of the assembly, TM1088A and TM1088B, were also determined independently to 1.50 Å and 1.55 Å, respectively. The TM1088 proteins are structurally homologous to each other and to other K+ transporter proteins, such as TrkA. These proteins form a cytosolic gating ring assembly that controls the flow of K+ ions across the membrane. TM1088 represents the first structure of a two-subunit Trk assembly. Despite the atypical genetics and chain organization of the TM1088 assembly, it shares significant structural homology and an overall quaternary organization with other single-subunit K+ gating ring assemblies. This structure provides the first structural insights into what may be an evolutionary ancestor of more modern single-subunit K+ gating ring assemblies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122512PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380455PMC
March 2016

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells.

Proc Natl Acad Sci U S A 2015 Apr 30;112(15):4666-71. Epub 2015 Mar 30.

Department of Biochemistry and Biophysics, and

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein-DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.
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http://dx.doi.org/10.1073/pnas.1502855112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403148PMC
April 2015

Commensal microbiota is hepatoprotective and prevents liver fibrosis in mice.

FASEB J 2015 Mar 2;29(3):1043-55. Epub 2014 Dec 2.

*Department of Medicine, University of California, San Diego, La Jolla, California, USA; Department of Medicine, VA San Diego Healthcare System, San Diego, California, USA; Genomics Institute of the Novartis Research Foundation, San Diego, California, USA; Systems and Translational Science, RTI International, Research Triangle Park, North Carolina, USA; and Department of Oral Health Sciences, Medical University of South Carolina, Charleston, South Carolina, USA

Translocation of bacteria and their products across the intestinal barrier is common in patients with liver disease, and there is evidence that experimental liver fibrosis depends on bacterial translocation. The purpose of our study was to investigate liver fibrosis in conventional and germ-free (GF) C57BL/6 mice. Chronic liver injury was induced by administration of thioacetamide (TAA) in the drinking water for 21 wk or by repeated intraperitoneal injections of carbon tetrachloride (CCl4). Increased liver fibrosis was observed in GF mice compared with conventional mice. Hepatocytes showed more toxin-induced oxidative stress and cell death. This was accompanied by increased activation of hepatic stellate cells, but hepatic mediators of inflammation were not significantly different. Similarly, a genetic model using Myd88/Trif-deficient mice, which lack downstream innate immunity signaling, had more severe fibrosis than wild-type mice. Isolated Myd88/Trif-deficient hepatocytes were more susceptible to toxin-induced cell death in culture. In conclusion, the commensal microbiota prevents fibrosis upon chronic liver injury in mice. This is the first study describing a beneficial role of the commensal microbiota in maintaining liver homeostasis and preventing liver fibrosis.
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http://dx.doi.org/10.1096/fj.14-259515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4422368PMC
March 2015

Structure-guided functional characterization of DUF1460 reveals a highly specific NlpC/P60 amidase family.

Structure 2014 Dec 20;22(12):1799-1809. Epub 2014 Nov 20.

Joint Center for Structural Genomics (JCSG); Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

GlcNAc-1,6-anhydro-MurNAc-tetrapeptide is a major peptidoglycan degradation intermediate and a cytotoxin. It is generated by lytic transglycosylases and further degraded and recycled by various enzymes. We have identified and characterized a highly specific N-acetylmuramoyl-L-alanine amidase (AmiA) from Bacteroides uniformis, a member of the DUF1460 protein family, that hydrolyzes GlcNAc-1,6-anhydro-MurNAc-peptide into disaccharide and stem peptide. The high-resolution apo structure at 1.15 Å resolution shows that AmiA is related to NlpC/P60 γ-D-Glu-meso-diaminopimelic acid amidases and shares a common catalytic core and cysteine peptidase-like active site. AmiA has evolved structural adaptations that reconfigure the substrate recognition site. The preferred substrates for AmiA were predicted in silico based on structural and bioinformatics data, and subsequently were characterized experimentally. Further crystal structures of AmiA in complexes with GlcNAc-1,6-anhydro-MurNAc and GlcNAc have enabled us to elucidate substrate recognition and specificity. DUF1460 is highly conserved in structure and defines another amidase family.
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http://dx.doi.org/10.1016/j.str.2014.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255150PMC
December 2014

Structural analysis of arabinose-5-phosphate isomerase from Bacteroides fragilis and functional implications.

Acta Crystallogr D Biol Crystallogr 2014 Oct 27;70(Pt 10):2640-51. Epub 2014 Sep 27.

Joint Center for Structural Genomics, http://www.jcsg.org, USA.

The crystal structure of arabinose-5-phosphate isomerase (API) from Bacteroides fragilis (bfAPI) was determined at 1.7 Å resolution and was found to be a tetramer of a single-domain sugar isomerase (SIS) with an endogenous ligand, CMP-Kdo (cytidine 5'-monophosphate-3-deoxy-D-manno-oct-2-ulosonate), bound at the active site. API catalyzes the reversible isomerization of D-ribulose 5-phosphate to D-arabinose 5-phosphate in the first step of the Kdo biosynthetic pathway. Interestingly, the bound CMP-Kdo is neither the substrate nor the product of the reaction catalyzed by API, but corresponds to the end product in the Kdo biosynthetic pathway and presumably acts as a feedback inhibitor for bfAPI. The active site of each monomer is located in a surface cleft at the tetramer interface between three monomers and consists of His79 and His186 from two different adjacent monomers and a Ser/Thr-rich region, all of which are highly conserved across APIs. Structure and sequence analyses indicate that His79 and His186 may play important catalytic roles in the isomerization reaction. CMP-Kdo mimetics could therefore serve as potent and specific inhibitors of API and provide broad protection against many different bacterial infections.
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http://dx.doi.org/10.1107/S1399004714017052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4188006PMC
October 2014

Structural insights into the recognition of phosphopeptide by the FHA domain of kanadaptin.

PLoS One 2014 8;9(9):e107309. Epub 2014 Sep 8.

Joint Center for Structural Genomics, La Jolla, California, United States of America; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, United States of America.

Kanadaptin is a nuclear protein of unknown function that is widely expressed in mammalian tissues. The crystal structure of the forkhead-associated (FHA) domain of human kanadaptin was determined to 1.6 Å resolution. The structure reveals an asymmetric dimer in which one monomer is complexed with a phosphopeptide mimic derived from a peptide segment from the N-terminus of a symmetry-related molecule as well as a sulfate bound to the structurally conserved phosphothreonine recognition cleft. This structure provides insights into the molecular recognition features utilized by this family of proteins and represents the first evidence that kanadaptin is likely involved in a phosphorylation-mediated signaling pathway. These results will be of use for designing experiments to further probe the function of kanadaptin.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107309PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157861PMC
December 2015

Crystal structures of three representatives of a new Pfam family PF14869 (DUF4488) suggest they function in sugar binding/uptake.

Protein Sci 2014 Oct 12;23(10):1380-91. Epub 2014 Aug 12.

Joint Center for Structural Genomics, http://www.jcsg.org; Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California, 94025.

Crystal structures of three members (BACOVA_00364 from Bacteroides ovatus, BACUNI_03039 from Bacteroides uniformis and BACEGG_00036 from Bacteroides eggerthii) of the Pfam domain of unknown function (DUF4488) were determined to 1.95, 1.66, and 1.81 Å resolutions, respectively. The protein structures adopt an eight-stranded, calycin-like, β-barrel fold and bind an endogenous unknown ligand at one end of the β-barrel. The amino acids interacting with the ligand are not conserved in any other protein of known structure with this particular fold. The size and chemical environment of the bound ligand suggest binding or transport of a small polar molecule(s) as a potential function for these proteins. These are the first structural representatives of a newly defined PF14869 (DUF4488) Pfam family.
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http://dx.doi.org/10.1002/pro.2522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287006PMC
October 2014

Molecular characterization of novel pyridoxal-5'-phosphate-dependent enzymes from the human microbiome.

Protein Sci 2014 Aug 14;23(8):1060-76. Epub 2014 Jun 14.

Department of Chemistry, University of California, Davis, California, 95616.

Pyridoxal-5'-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for ∼1.5% of most prokaryotic genomes and are estimated to be involved in ∼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.
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http://dx.doi.org/10.1002/pro.2493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4116655PMC
August 2014

Crystal structure of a putative quorum sensing-regulated protein (PA3611) from the Pseudomonas-specific DUF4146 family.

Proteins 2014 Jun 22;82(6):1086-92. Epub 2013 Nov 22.

Joint Center for Structural Genomics; Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California.

Pseudomonas aeruginosa is an opportunistic pathogen commonly found in humans and other organisms and is an important cause of infection especially in patients with compromised immune defense mechanisms. The PA3611 gene of P. aeruginosa PAO1 encodes a secreted protein of unknown function, which has been recently classified into a small Pseudomonas-specific protein family called DUF4146. As part of our effort to extend structural coverage of novel protein space and provide a structure-based functional insight into new protein families, we report the crystal structure of PA3611, the first structural representative of the DUF4146 protein family.
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http://dx.doi.org/10.1002/prot.24455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4006323PMC
June 2014

Structure and function of a novel LD-carboxypeptidase a involved in peptidoglycan recycling.

J Bacteriol 2013 Dec 11;195(24):5555-66. Epub 2013 Oct 11.

Joint Center for Structural Genomics‡

Approximately 50% of cell wall peptidoglycan in Gram-negative bacteria is recycled with each generation. The primary substrates used for peptidoglycan biosynthesis and recycling in the cytoplasm are GlcNAc-MurNAc(anhydro)-tetrapeptide and its degradation product, the free tetrapeptide. This complex process involves ∼15 proteins, among which the cytoplasmic enzyme ld-carboxypeptidase A (LdcA) catabolizes the bond between the last two l- and d-amino acid residues in the tetrapeptide to form the tripeptide, which is then utilized as a substrate by murein peptide ligase (Mpl). LdcA has been proposed as an antibacterial target. The crystal structure of Novosphingobium aromaticivorans DSM 12444 LdcA (NaLdcA) was determined at 1.89-Å resolution. The enzyme was biochemically characterized and its interactions with the substrate modeled, identifying residues potentially involved in substrate binding. Unaccounted electron density at the dimer interface in the crystal suggested a potential site for disrupting protein-protein interactions should a dimer be required to perform its function in bacteria. Our analysis extends the identification of functional residues to several other homologs, which include enzymes from bacteria that are involved in hydrocarbon degradation and destruction of coral reefs. The NaLdcA crystal structure provides an alternate system for investigating the structure-function relationships of LdcA and increases the structural coverage of the protagonists in bacterial cell wall recycling.
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http://dx.doi.org/10.1128/JB.00900-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3889619PMC
December 2013

Structures of a bifunctional cell wall hydrolase CwlT containing a novel bacterial lysozyme and an NlpC/P60 DL-endopeptidase.

J Mol Biol 2014 Jan 16;426(1):169-84. Epub 2013 Sep 16.

Joint Center for Structural Genomics (http://www.jcsg.org); Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state).
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http://dx.doi.org/10.1016/j.jmb.2013.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3872209PMC
January 2014

Crystal structure of a member of a novel family of dioxygenases (PF10014) reveals a conserved cupin fold and active site.

Proteins 2014 Jan 10;82(1):164-70. Epub 2013 Sep 10.

Joint Center for Structural Genomics, La Jolla, California (http://www.jcsg.org); Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California, 94025.

PF10014 is a novel family of 2-oxyglutarate-Fe(2+) -dependent dioxygenases that are involved in biosynthesis of antibiotics and regulation of biofilm formation, likely by catalyzing hydroxylation of free amino acids or other related ligands. The crystal structure of a PF10014 member from Methylibium petroleiphilum at 1.9 Å resolution shows strong structural similarity to cupin dioxygenases in overall fold and active site, despite very remote homology. However, one of the β-strands of the cupin catalytic core is replaced by a loop that displays conformational isomerism that likely regulates the active site.
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http://dx.doi.org/10.1002/prot.24362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3920835PMC
January 2014

Structural and functional characterization of BaiA, an enzyme involved in secondary bile acid synthesis in human gut microbe.

Proteins 2014 Feb 17;82(2):216-29. Epub 2013 Oct 17.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, 92037; Joint Center for Structural Genomics.

Despite significant influence of secondary bile acids on human health and disease, limited structural and biochemical information is available for the key gut microbial enzymes catalyzing its synthesis. Herein, we report apo- and cofactor bound crystal structures of BaiA2, a short chain dehydrogenase/reductase from Clostridium scindens VPI 12708 that represent the first protein structure of this pathway. The structures elucidated the basis of cofactor specificity and mechanism of proton relay. A conformational restriction involving Glu42 located in the cofactor binding site seems crucial in determining cofactor specificity. Limited flexibility of Glu42 results in imminent steric and electrostatic hindrance with 2'-phosphate group of NADP(H). Consistent with crystal structures, steady state kinetic characterization performed with both BaiA2 and BaiA1, a close homolog with 92% sequence identity, revealed specificity constant (kcat /KM ) of NADP(+) at least an order of magnitude lower than NAD(+) . Substitution of Glu42 with Ala improved specificity toward NADP(+) by 10-fold compared to wild type. The cofactor bound structure uncovered a novel nicotinamide-hydroxyl ion (NAD(+) -OH(-) ) adduct contraposing previously reported adducts. The OH(-) of the adduct in BaiA2 is distal to C4 atom of nicotinamide and proximal to 2'-hydroxyl group of the ribose moiety. Moreover, it is located at intermediary distances between terminal functional groups of active site residues Tyr157 (2.7 Å) and Lys161 (4.5 Å). Based on these observations, we propose an involvement of NAD(+) -OH(-) adduct in proton relay instead of hydride transfer as noted for previous adducts.
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http://dx.doi.org/10.1002/prot.24353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992121PMC
February 2014

Structure and function of the DUF2233 domain in bacteria and in the human mannose 6-phosphate uncovering enzyme.

J Biol Chem 2013 Jun 9;288(23):16789-16799. Epub 2013 Apr 9.

Joint Center for Structural Genomics, Menlo Park, California 94025; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037. Electronic address:

DUF2233, a domain of unknown function (DUF), is present in many bacterial and several viral proteins and was also identified in the mammalian transmembrane glycoprotein N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase ("uncovering enzyme" (UCE)). We report the crystal structure of BACOVA_00430, a 315-residue protein from the human gut bacterium Bacteroides ovatus that is the first structural representative of the DUF2233 protein family. A notable feature of this structure is the presence of a surface cavity that is populated by residues that are highly conserved across the entire family. The crystal structure was used to model the luminal portion of human UCE (hUCE), which is involved in targeting of lysosomal enzymes. Mutational analysis of several residues in a highly conserved surface cavity of hUCE revealed that they are essential for function. The bacterial enzyme (BACOVA_00430) has ∼1% of the catalytic activity of hUCE toward the substrate GlcNAc-P-mannose, the precursor of the Man-6-P lysosomal targeting signal. GlcNAc-1-P is a poor substrate for both enzymes. We conclude that, for at least a subset of proteins in this family, DUF2233 functions as a phosphodiester glycosidase.
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http://dx.doi.org/10.1074/jbc.M112.434977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675612PMC
June 2013

Folate binding site of flavin-dependent thymidylate synthase.

Proc Natl Acad Sci U S A 2012 Sep 10;109(39):15722-7. Epub 2012 Sep 10.

Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.

The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH(2)H(4)folate). Most organisms, including humans, rely on the thyA- or TYMS-encoded classic thymidylate synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent thymidylate synthase (FDTS). Although several crystal structures of FDTSs have been reported, the absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis, kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely facilitate further elucidation of FDTSs' mechanism and the design of structure-based inhibitors as potential leads to new antimicrobial drugs.
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http://dx.doi.org/10.1073/pnas.1206077109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465422PMC
September 2012

Structure of a novel winged-helix like domain from human NFRKB protein.

PLoS One 2012 11;7(9):e43761. Epub 2012 Sep 11.

Joint Center for Structural Genomics, La Jolla, California, United States of America.

The human nuclear factor related to kappa-B-binding protein (NFRKB) is a 1299-residue protein that is a component of the metazoan INO80 complex involved in chromatin remodeling, transcription regulation, DNA replication and DNA repair. Although full length NFRKB is predicted to be around 65% disordered, comparative sequence analysis identified several potentially structured sections in the N-terminal region of the protein. These regions were targeted for crystallographic studies, and the structure of one of these regions spanning residues 370-495 was determined using the JCSG high-throughput structure determination pipeline. The structure reveals a novel, mostly helical domain reminiscent of the winged-helix fold typically involved in DNA binding. However, further analysis shows that this domain does not bind DNA, suggesting it may belong to a small group of winged-helix domains involved in protein-protein interactions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043761PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3439487PMC
March 2013

Identification of unknown protein function using metabolite cocktail screening.

Structure 2012 Oct 30;20(10):1715-25. Epub 2012 Aug 30.

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.

Proteins of unknown function comprise a significant fraction of sequenced genomes. Defining the roles of these proteins is vital to understanding cellular processes. Here, we describe a method to determine a protein function based on the identification of its natural ligand(s) by the crystallographic screening of the binding of a metabolite library, followed by a focused search in the metabolic space. The method was applied to two protein families with unknown function, PF01256 and YjeF_N. The PF01256 proteins, represented by YxkO from Bacillus subtilis and the C-terminal domain of Tm0922 from Thermotoga maritima, were shown to catalyze ADP/ATP-dependent NAD(P)H-hydrate dehydratation, a previously described orphan activity. The YjeF_N proteins, represented by mouse apolipoprotein A-I binding protein and the N-terminal domain of Tm0922, were found to interact with an adenosine diphosphoribose-related substrate and likely serve as ADP-ribosyltransferases. Crystallographic screening of metabolites serves as an efficient tool in functional analyses of uncharacterized proteins.
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http://dx.doi.org/10.1016/j.str.2012.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3472112PMC
October 2012
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