Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli) appear to be ...insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2), proline, xylitol, NDSB 201, CTAB and K(2)PO(4)) solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40%) were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.
The ethanolamine utilization (Eut) microcompartment is a protein‐based metabolic organelle that is strongly associated with pathogenesis in bacteria that inhabit the human gut. The exterior shell of ...this elaborate protein complex is composed from a few thousand copies of BMC‐domain shell proteins, which form a semi‐permeable diffusion barrier that provides the interior enzymes with substrates and cofactors while simultaneously retaining metabolic intermediates. The ability of this protein shell to regulate passage of substrate and cofactor molecules is critical for microcompartment function, but the details of how this diffusion barrier can allow the passage of large cofactors while still retaining small intermediates remain unclear. Previous work has revealed two conformations of the EutL shell protein, providing substantial evidence for a gated pore that might allow the passage of large cofactors. Here we report structural and biophysical evidence to show that ethanolamine, the substrate of the Eut microcompartment, acts as a negative allosteric regulator of EutL pore opening. Specifically, a series of X‐ray crystal structures of EutL from Clostridium perfringens, along with equilibrium binding studies, reveal that ethanolamine binds to EutL at a site that exists in the closed‐pore conformation and which is incompatible with opening of the large pore for cofactor transport. The allosteric mechanism we propose is consistent with the cofactor requirements of the Eut microcompartment, leading to a new model for EutL function. Furthermore, our results suggest the possibility of redox modulation of the allosteric mechanism, opening potentially new lines of investigation.
Applications ranging from synthetic biology to protein crystallization could be advanced by facile systems for connecting multiple proteins together in predefined spatial relationships. One approach ...to this goal is to engineer many distinct assembly forms of a single carrier protein or scaffold, to which other proteins of interest can then be readily attached. In this work we chose GFP as a scaffold and engineered many alternative oligomeric forms, driven by either specific disulfide bond formation or metal ion addition. We generated a wide range of spatial arrangements of GFP subunits from 11 different oligomeric variants, and determined their X-ray structures in a total of 33 distinct crystal forms. Some of the oligomeric GFP variants show geometric polymorphism depending on conditions, while others show considerable geometric rigidity. Potential future applications of this system are discussed.
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•A large suite of GFP mutants has been developed to allow diverse oligomerization•33 distinct crystal structures of designed GFP oligomers have been determined•Target proteins can be oligomerized in diverse ways by attachment to the GFP molecules
Leibly et al. have generated a large suite of GFP oligomers with varying spatial arrangements of subunits, which are elucidated in 33 distinct crystal forms. These new GFP forms have potential applications ranging from synthetic biology to protein crystallization by connecting multiple proteins together in predefined spatial relationships.
Effective control and eradication of malaria will require new tools to prevent transmission. Current antimalarial therapies targeting the asexual stage of Plasmodium do not prevent transmission of ...circulating gametocytes from infected humans to mosquitoes. Here, we describe a new class of transmission-blocking compounds, bumped kinase inhibitors (BKIs), which inhibit microgametocyte exflagellation. Oocyst formation and sporozoite production, necessary for transmission to mammals, were inhibited in mosquitoes fed on either BKI-1-treated human blood or mice treated with BKI-1. BKIs are hypothesized to act via inhibition of Plasmodium calcium-dependent protein kinase 4 and predicted to have little activity against mammalian kinases. Our data show that BKIs do not inhibit proliferation of mammalian cell lines and are well tolerated in mice. Used in combination with drugs active against asexual stages of Plasmodium, BKIs could prove an important tool for malaria control and eradication.
Amyloid protein aggregates are associated with dozens of devastating diseases including Alzheimer's, Parkinson's, ALS, and diabetes type 2. While structure-based discovery of compounds has been ...effective in combating numerous infectious and metabolic diseases, ignorance of amyloid structure has hindered similar approaches to amyloid disease. Here we show that knowledge of the atomic structure of one of the adhesive, steric-zipper segments of the amyloid-beta (Aβ) protein of Alzheimer's disease, when coupled with computational methods, identifies eight diverse but mainly flat compounds and three compound derivatives that reduce Aβ cytotoxicity against mammalian cells by up to 90%. Although these compounds bind to Aβ fibers, they do not reduce fiber formation of Aβ. Structure-activity relationship studies of the fiber-binding compounds and their derivatives suggest that compound binding increases fiber stability and decreases fiber toxicity, perhaps by shifting the equilibrium of Aβ from oligomers to fibers. DOI:http://dx.doi.org/10.7554/eLife.00857.001.
Diseases caused by the apicomplexan protozoans Toxoplasma gondii and Cryptosporidium parvum are a major health concern. The life cycle of these parasites is regulated by a family of calcium-dependent ...protein kinases (CDPKs) that have no direct homologues in the human host. Fortuitously, CDPK1 from both parasites contains a rare glycine gatekeeper residue adjacent to the ATP-binding pocket. This has allowed creation of a series of C3-substituted pyrazolopyrimidine compounds that are potent inhibitors selective for CDPK1 over a panel of human kinases. Here we demonstrate that selectivity is further enhanced by modification of the scaffold at the C1 position. The explanation for this unexpected result is provided by crystal structures of the inhibitors bound to CDPK1 and the human kinase c-SRC. Furthermore, the insight gained from these studies was applied to transform an alternative ATP-competitive scaffold lacking potency and selectivity for CDPK1 into a low nanomolar inhibitor of this enzyme with no activity against SRC.
. Thousands of antimalarial compounds were tested against nine potential
Plasmodium drug targets. Six of the targets, including S-adenosylhomocysteine hydrolase (SAHH), were inhibited by antimalarial ...scaffolds, which may have use in target exploration and validation.
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▶ Inhibitors of six potential
Plasmodium drug targets were identified. ▶ These inhibitors may be used as “tool compounds” to probe the corresponding enzymes. ▶ Most antimalarial compounds’ modes of action remain elusive.
The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for
Plasmodium falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5655 low molecular weight compounds that inhibit growth of
P. falciparum cultures with EC
50 values below 1.25
μM. These compounds were then tested in 384- or 1536-well biochemical assays for activity against nine
Plasmodium enzymes: adenylosuccinate synthetase (AdSS), choline kinase (CK), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), glutamate dehydrogenase (GDH), guanylate kinase (GK), N-myristoyltransferase (NMT), orotidine 5′-monophosphate decarboxylase (OMPDC), farnesyl pyrophosphate synthase (FPPS) and S-adenosylhomocysteine hydrolase (SAHH). These enzymes were selected using TDRtargets.org, and are believed to have excellent potential as drug targets based on criteria such as their likely essentiality, druggability, and amenability to high-throughput biochemical screening. Six of these targets were inhibited by one or more of the antimalarial scaffolds and may have potential use in drug development, further target validation studies and exploration of
P. falciparum biochemistry and biology.
This study sought to determine whether the quality of enzyme preparations can be determined from their melting curves, which may easily be obtained using a fluorescent probe and a standard reverse ...transcription-polymerase chain reaction (RT-PCR) machine. Thermal melt data on 31 recombinant enzymes from
Plasmodium parasites were acquired by incrementally heating them to 90
°C and measuring unfolding with a fluorescent dye. Activity assays specific to each enzyme were also performed. Four of the enzymes were denatured to varying degrees with heat and sodium dodecyl sulfate (SDS) prior to the thermal melt and activity assays. In general, melting curve quality was correlated with enzyme activity; enzymes with high-quality curves were found almost uniformly to be active, whereas those with lower quality curves were more varied in their catalytic performance. Inspection of melting curves of bovine xanthine oxidase and
Entamoeba histolytica cysteine protease 1 allowed active stocks to be distinguished from inactive stocks, implying that a relationship between melting curve quality and activity persists over a wide range of experimental conditions and species. Our data suggest that melting curves can help to distinguish properly folded proteins from denatured ones and, therefore, may be useful in selecting stocks for further study and in optimizing purification procedures for specific proteins.
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 100s 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.
Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and ...domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region.
Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 A resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs.
The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics.
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