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.
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.
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.
Summary High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the ...Mtb proteome. Here we evaluate an intra-genus “homolog-rescue” strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. Of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structures would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1 Å, >85% side chain identity, and ≥80% PSAPF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases.
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.
Thioredoxins are small ubiquitous proteins that participate in a diverse variety of redox reactions via the reversible oxidation of two cysteine thiol groups in a structurally conserved active site. ...Here, the NMR solution structures of a reduced and oxidized thioredoxin from Ehrlichia chaffeensis (Ec‐Trx, ECH_0218), the etiological agent responsible for human monocytic ehrlichiosis, are described. The overall topology of the calculated structures is similar in both redox states and is similar to those of other thioredoxins: a five‐stranded, mixed β‐sheet (β1–β3–β2–β4–β5) surrounded by four α‐helices. Unlike other thioredoxins studied by NMR in both redox states, the 1H–15N HSQC spectrum of reduced Ec‐Trx was missing eight additional amide cross peaks relative to the spectrum of oxidized Ec‐Trx. These missing amides correspond to residues Cys35–Glu39 in the active‐site‐containing helix (α2) and Ser72–Ile75 in a loop near the active site, and suggest a change in backbone dynamics on the millisecond‐to‐microsecond timescale associated with the breakage of an intramolecular Cys32–Cys35 disulfide bond in a thioredoxin. A consequence of the missing amide resonances is the absence of observable or unambiguous NOEs to provide the distance restraints necessary to define the N‐terminal end of the α‐helix containing the CPGC active site in the reduced state. This region adopts a well defined α‐helical structure in other reported reduced thioredoxin structures, is mostly helical in oxidized Ec‐Trx and CD studies of Ec‐Trx in both redox states suggests there is no significant difference in the secondary structure of the protein. The NMR solution structure of reduced Ec‐Trx illustrates that the absence of canonical structure in a region of a protein may be owing to unfavorable dynamics prohibiting NOE observations or unambiguous NOE assignments.
The overall topology of solution NMR structures of the Ehrlichia chaffeensis thioredoxin ECH_0218 (Ec‐Trx) is similar in the reduced and oxidized states and is similar to other reported thioredoxin structures except that the α‐helix (α2) containing the active‐site CGPC motif appears shorter in length in the reduced state than in the oxidized state. This outcome of structure calculations is likely to be owing to missing α2 amide resonances in the 1H–15N HSQC spectrum of reduced Ec‐Trx owing to backbone dynamics on the millisecond‐to‐microsecond timescale that prohibit NOE observations or unambiguous NOE assignments in this region of the protein.
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The fragment FOL7185 (compound 17) was found to be a hit against IspD and IspE enzymes isolated from bacteria, and a series of analogs containing the pyrazolopyrimidine core were ...synthesized. The majority of these compounds inhibited the growth of Burkholderia thailandensis (Bt) and Pseudomonas aeruginosa (Pa) in the Kirby–Bauer disk diffusion susceptibility test. Compound 29 shows inhibitory activity at 0.1mM (32.2μg/mL), which is comparable to the control compound kanamycin (48.5μg/mL). Compound 29 also shows inhibitory activity at 0.5mM against kanamycin resistant P. aeruginosa. Saturation transfer difference NMR (STD-NMR) screening of these compounds against BtIspD and BtIspE indicated that most of these compounds significantly interact with BtIspE, suggesting that the compounds may inhibit the growth of Bt by disrupting isoprenoid biosynthesis. Ligand epitope mapping of compound 29 with BtIspE indicated that hydrogens on 2,4-dichlorophenyl group have higher proximity to the surface of the enzyme than hydrogens on the pyrazolopyrimidine ring.
Macrophage migration inhibitory factor (MIF) is a eukaryotic cytokine that affects a broad spectrum of immune responses and its activation/inactivation is associated with numerous diseases. During ...protozoan infections MIF is not only expressed by the host, but, has also been observed to be expressed by some parasites and released into the host. To better understand the biological role of parasitic MIF proteins, the crystal structure of the MIF protein from
Giardia lamblia
(
Gl
-MIF), the etiological agent responsible for giardiasis, has been determined at 2.30 Å resolution. The 114-residue protein adopts an α/β fold consisting of a four-stranded β-sheet with two anti-parallel α-helices packed against a face of the β-sheet. An additional short β-strand aligns anti-parallel to β4 of the β-sheet in the adjacent protein unit to help stabilize a trimer, the biologically relevant unit observed in all solved MIF crystal structures to date, and form a discontinuous β-barrel. The structure of
Gl
-MIF is compared to the MIF structures from humans (
Hs
-MIF) and three
Plasmodium
species (
falciparum, berghei,
and
yoelii
). The structure of all five MIF proteins are generally similar with the exception of a channel that runs through the center of each trimer complex. Relative to
Hs
-MIF, there are differences in solvent accessibility and electrostatic potential distribution in the channel of
Gl
-MIF and the
Plasmodium
-MIFs due primarily to two “gate-keeper” residues in the parasitic MIFs. For the
Plasmodium
MIFs the gate-keeper residues are at positions 44 (Y⇒R) and 100 (V⇒D) and for
Gl
-MIF it is at position 100 (V⇒R). If these gate-keeper residues have a biological function and contribute to the progression of parasitemia they may also form the basis for structure-based drug design targeting parasitic MIF proteins.
Cryptosporidiosis is an infectious disease caused by protozoan parasites of the Cryptosporidium genus. Infection is associated with mild to severe diarrhea that usually resolves spontaneously in ...healthy human adults, but may lead to severe complications in young children and in immunocompromised patients. The genome of C. parvum contains a gene, CUTA_CRYPI, that may play a role in regulating the intracellular concentration of copper, which is a toxic element in excess. Here, the crystal structure of this CutA1 protein, Cp‐CutA1, is reported at 2.0 Å resolution. As observed for other CutA1 structures, the 117‐residue protein is a trimer with a core ferrodoxin‐like fold. Circular dichroism spectroscopy shows little, in any, unfolding of Cp‐CutA1 up to 353 K. This robustness is corroborated by 1H–15N HSQC spectra at 333 K, which are characteristic of a folded protein, suggesting that NMR spectroscopy may be a useful tool to further probe the function of the CutA1 proteins. While robust, Cp‐CutA1 is not as stable as the homologous protein from a hyperthermophile, perhaps owing to a wide β‐bulge in β2 that protrudes Pro48 and Ser49 outside the β‐sheet.
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