Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses ∼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.

Three structurally and functionally distinct ß-glucuronidases from the human gut microbe Bacteroides uniformis.

The glycoside hydrolases encoded by the human gut microbiome play an integral role in processing a variety of exogenous and endogenous glycoconjugates. Here we present three structurally and functionally distinct ß-glucuronidase (GUS) glycoside hydrolases from a single human gut commensal microbe, Bacteroides uniformis We show using nine crystal structures, biochemical, and biophysical data that whereas these three proteins share similar overall folds, they exhibit different structural features that create three structurally and functionally unique enzyme active sites. Notably, quaternary structure plays an important role in creating distinct active site features that are hard to predict via structural modeling methods. The enzymes display differential processing capabilities toward glucuronic acid-containing polysaccharides and SN-38-glucuronide, a metabolite of the cancer drug irinotecan. We also demonstrate that GUS-specific and nonselective inhibitors exhibit varying potencies toward each enzyme. Together, these data highlight the diversity of GUS enzymes within a single Bacteroides gut commensal and advance our understanding of how structural details impact the specific roles microbial enzymes play in processing drug-glucuronide and glycan substrates.

Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets.

Selection of aptamers from nucleic acid libraries by in vitro evolution represents a powerful method of identifying high-affinity ligands for a broad range of molecular targets. Nevertheless, a sizeable fraction of proteins remain difficult targets due to inherently limited chemical diversity of nucleic acids. We have exploited synthetic nucleotide modifications that confer protein-like diversity on a nucleic acid scaffold, resulting in a new generation of binding reagents called SOMAmers (Slow Off-rate Modified Aptamers). Here we report a unique crystal structure of a SOMAmer bound to its target, platelet-derived growth factor B (PDGF-BB). The SOMAmer folds into a compact structure and exhibits a hydrophobic binding surface that mimics the interface between PDGF-BB and its receptor, contrasting sharply with mainly polar interactions seen in traditional protein-binding aptamers. The modified nucleotides circumvent the intrinsic diversity constraints of natural nucleic acids, thereby greatly expanding the structural vocabulary of nucleic acid ligands and considerably broadening the range of accessible protein targets.

Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase.

There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei.

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series.

De novo design of highly selective miniprotein inhibitors of integrins αvß6 and αvß8.

The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of αvß6 and αvß8 with high selectivity. The αvß6 and αvß8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7Å root-mean-square deviation (RMSD) to the computational design models; the designed αvß6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-αvß6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the αvß8 inhibitor maintains the constitutively fixed extended-closed αvß8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

De novo design of highly selective miniprotein inhibitors of integrins αvß6 and αvß8.

The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvß6 and αvß8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvß6 and αvß8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvß6 and the αvß8 integrins. In a lung fibrosis mouse model, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

Crystal structure of Toxoplasma gondii porphobilinogen synthase: insights on octameric structure and porphobilinogen formation.

Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit ß-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors.

Biological and structural characterization of a host-adapting amino acid in influenza virus.

Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the protein's interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals.

Large-scale design and refinement of stable proteins using sequence-only models.

Engineered proteins generally must possess a stable structure in order to achieve their designed function. Stable designs, however, are astronomically rare within the space of all possible amino acid sequences. As a consequence, many designs must be tested computationally and experimentally in order to find stable ones, which is expensive in terms of time and resources. Here we use a high-throughput, low-fidelity assay to experimentally evaluate the stability of approximately 200,000 novel proteins. These include a wide range of sequence perturbations, providing a baseline for future work in the field. We build a neural network model that predicts protein stability given only sequences of amino acids, and compare its performance to the assayed values. We also report another network model that is able to generate the amino acid sequences of novel stable proteins given requested secondary sequences. Finally, we show that the predictive model-despite weaknesses including a noisy data set-can be used to substantially increase the stability of both expert-designed and model-generated proteins.

SAD phasing using iodide ions in a high-throughput structural genomics environment.

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) focuses on the structure elucidation of potential drug targets from class A, B, and C infectious disease organisms. Many SSGCID targets are selected because they have homologs in other organisms that are validated drug targets with known structures. Thus, many SSGCID targets are expected to be solved by molecular replacement (MR), and reflective of this, all proteins are expressed in native form. However, many community request targets do not have homologs with known structures and not all internally selected targets readily solve by MR, necessitating experimental phase determination. We have adopted the use of iodide ion soaks and single wavelength anomalous dispersion (SAD) experiments as our primary method for de novo phasing. This method uses existing native crystals and in house data collection, resulting in rapid, low cost structure determination. Iodide ions are non-toxic and soluble at molar concentrations, facilitating binding at numerous hydrophobic or positively charged sites. We have used this technique across a wide range of crystallization conditions with successful structure determination in 16 of 17 cases within the first year of use (94% success rate). Here we present a general overview of this method as well as several examples including SAD phasing of proteins with novel folds and the combined use of SAD and MR for targets with weak MR solutions. These cases highlight the straightforward and powerful method of iodide ion SAD phasing in a high-throughput structural genomics environment.

Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei.

As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

Structural basis of the substrate specificity of bifunctional isocitrate dehydrogenase kinase/phosphatase.

Isocitrate dehydrogenase kinase/phosphatase (AceK) regulates entry into the glyoxylate bypass by reversibly phosphorylating isocitrate dehydrogenase (ICDH). On the basis of the recently determined structure of the AceK-ICDH complex from Escherichia coli, we have classified the structures of homodimeric NADP(+)-ICDHs to rationalize and predict which organisms likely contain substrates for AceK. One example is Burkholderia pseudomallei (Bp). Here we report a crystal structure of Bp-ICDH that exhibits the necessary structural elements required for AceK recognition. Kinetic analyses provided further confirmation that Bp-ICDH is a substrate for AceK. We conclude that the highly stringent AceK binding sites on ICDH are maintained only in Gram-negative bacteria.

Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis.

BACKGROUND: Ribose-5-phosphate isomerase is an enzyme that catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate. This family of enzymes naturally occurs in two distinct classes, RpiA and RpiB, which play an important role in the pentose phosphate pathway and nucleotide and co-factor biogenesis. RESULTS: Although RpiB occurs predominantly in bacteria, here we report crystal structures of a putative RpiB from the pathogenic fungus Coccidioides immitis. A 1.9 Å resolution apo structure was solved by combined molecular replacement and single wavelength anomalous dispersion (SAD) phasing using a crystal soaked briefly in a solution containing a high concentration of iodide ions. RpiB from C. immitis contains modest sequence and high structural homology to other known RpiB structures. A 1.8 Å resolution phosphate-bound structure demonstrates phosphate recognition and charge stabilization by a single positively charged residue whereas other members of this family use up to five positively charged residues to contact the phosphate of ribose-5-phosphate. A 1.7 Å resolution structure was obtained in which the catalytic base of C. immitis RpiB, Cys76, appears to form a weakly covalent bond with the central carbon of malonic acid with a bond distance of 2.2 Å. This interaction may mimic that formed by the suicide inhibitor iodoacetic acid with RpiB. CONCLUSION: The C. immitis RpiB contains the same fold and similar features as other members of this class of enzymes such as a highly reactive active site cysteine residue, but utilizes a divergent phosphate recognition strategy and may recognize a different substrate altogether.

Structure of a cyclin-dependent kinase from Giardia lamblia.

Giardia lamblia is the etiologic agent of giardiasis, a water-borne infection that is prevalent throughout the world. The need for new therapeutics for the treatment of giardiasis is of paramount importance. Owing to the ubiquitous nature of kinases and their vital importance in organisms, they are potential drug targets. In this paper, the first structure of a cyclin-dependent kinase (CDK) from G. lamblia (GlCDK; UniProt A8BZ95) is presented. CDKs are cell-cycle-associated kinases that are actively being pursued as targets for anticancer drugs as well as for antiparasitic chemotherapy. Generally, a CDK forms a complex with its associated cyclin. This CDK-cyclin complex is active and acts as a serine/threonine protein kinase. Typically, CDKs are responsible for the transition to the next phase of the cell cycle. Although the structure of GlCDK with its associated cyclin was not solved, the 1.85 Å resolution structure of apo GlCDK and a 2.0 Å resolution structure of GlCDK in complex with adenosine monophosphate are presented and the structural differences from the orthologous human CDK2 and CDK3 are discussed.

Structural genomics of infectious disease drug targets: the SSGCID.

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a consortium of researchers at Seattle BioMed, Emerald BioStructures, the University of Washington and Pacific Northwest National Laboratory that was established to apply structural genomics approaches to drug targets from infectious disease organisms. The SSGCID is currently funded over a five-year period by the National Institute of Allergy and Infectious Diseases (NIAID) to determine the three-dimensional structures of 400 proteins from a variety of Category A, B and C pathogens. Target selection engages the infectious disease research and drug-therapy communities to identify drug targets, essential enzymes, virulence factors and vaccine candidates of biomedical relevance to combat infectious diseases. The protein-expression systems, purified proteins, ligand screens and three-dimensional structures produced by SSGCID constitute a valuable resource for drug-discovery research, all of which is made freely available to the greater scientific community. This issue of Acta Crystallographica Section F, entirely devoted to the work of the SSGCID, covers the details of the high-throughput pipeline and presents a series of structures from a broad array of pathogenic organisms. Here, a background is provided on the structural genomics of infectious disease, the essential components of the SSGCID pipeline are discussed and a survey of progress to date is presented.

High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease.

The establishment of an efficient and reliable protein-purification pipeline is essential for the success of structural genomic projects. The SSGCID Protein Purification Group at the University of Washington (UW-PPG) has established a robust protein-purification pipeline designed to purify 400 proteins per year at a rate of eight purifications per week. The pipeline was implemented using two ÄKTAexplorer 100 s and four ÄKTAprimes to perform immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. Purifications were completed in a period of 5 d and yielded an average of 53 mg highly purified protein. This paper provides a detailed description of the methods used to purify, characterize and store SSGCID proteins. Some of the purified proteins were treated with 3C protease, which was expressed and purified by UW-PPG using a similar protocol, to cleave non-native six-histidine tags. The cleavage was successful in 94% of 214 attempts. Cleaved proteins yielded 2.9% more structures than uncleaved six-histidine-tagged proteins. This 2.9% improvement may seem small, but over the course of the project the structure output from UW-PPG is thus predicted to increase from 260 structures to 318 structures. Therefore, the outlined protocol with 3C cleavage and subtractive IMAC has been shown to be a highly efficient method for the standardized purification of recombinant proteins for structure determination via X-ray crystallography.

Structures of phosphopantetheine adenylyltransferase from Burkholderia pseudomallei.

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the fourth of five steps in the coenzyme A biosynthetic pathway, reversibly transferring an adenylyl group from ATP onto 4'-phosphopantetheine to yield dephospho-coenzyme A and pyrophosphate. Burkholderia pseudomallei is a soil- and water-borne pathogenic bacterium and the etiologic agent of melioidosis, a potentially fatal systemic disease present in southeast Asia. Two crystal structures are presented of the PPAT from B. pseudomallei with the expectation that, because of the importance of the enzyme in coenzyme A biosynthesis, they will aid in the search for defenses against this pathogen. A crystal grown in ammonium sulfate yielded a 2.1 Å resolution structure that contained dephospho-coenzyme A with partial occupancy. The overall structure and ligand-binding interactions are quite similar to other bacterial PPAT crystal structures. A crystal grown at low pH in the presence of coenzyme A yielded a 1.6 Å resolution structure in the same crystal form. However, the experimental electron density was not reflective of fully ordered coenzyme A, but rather was only reflective of an ordered 4'-diphosphopantetheine moiety.

An ensemble of structures of Burkholderia pseudomallei 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase.

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and Northern Australia. Burkholderia is responsible for melioidosis, a serious infection of the skin. The enzyme 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (PGAM) catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate, a key step in the glycolytic pathway. As such it is an extensively studied enzyme and X-ray crystal structures of PGAM enzymes from multiple species have been elucidated. Vanadate is a phosphate mimic that is a powerful tool for studying enzymatic mechanisms in phosphoryl-transfer enzymes such as phosphoglycerate mutase. However, to date no X-ray crystal structures of phosphoglycerate mutase have been solved with vanadate acting as a substrate mimic. Here, two vanadate complexes together with an ensemble of substrate and fragment-bound structures that provide a comprehensive picture of the function of the Burkholderia enzyme are reported.

Structures of a putative ζ-class glutathione S-transferase from the pathogenic fungus Coccidioides immitis.

Coccidioides immitis is a pathogenic fungus populating the southwestern United States and is a causative agent of coccidioidomycosis, sometimes referred to as Valley Fever. Although the genome of this fungus has been sequenced, many operons are not properly annotated. Crystal structures are presented for a putative uncharacterized protein that shares sequence similarity with ζ-class glutathione S-transferases (GSTs) in both apo and glutathione-bound forms. The apo structure reveals a nonsymmetric homodimer with each protomer comprising two subdomains: a C-terminal helical domain and an N-terminal thioredoxin-like domain that is common to all GSTs. Half-site binding is observed in the glutathione-bound form. Considerable movement of some components of the active site relative to the glutathione-free form was observed, indicating an induced-fit mechanism for cofactor binding. The sequence homology, structure and half-site occupancy imply that the protein is a ζ-class glutathione S-transferase, a maleylacetoacetate isomerase (MAAI).