Crystal Soaking Research Articles

A method for high-throughput, low volume soaking of protein crystals in rapid screening fragment libraries

A method for high-throughput, low volume soaking of protein crystals in rapid screening fragment libraries

Crystal structure of Halobacterium salinarum halorhodopsin with a partially depopulated primary chloride-binding site.

The transmembrane pump halorhodopsin in halophilic archaea translocates chloride ions from the extracellular to the cytoplasmic side upon illumination. In the ground state a tightly bound chloride ion occupies the primary chloride-binding site (CBS I) close to the protonated Schiff base that links the retinal chromophore to the protein. The light-triggered trans-cis isomerization of retinal causes structural changes in the protein associated with movement of the chloride ion. In reverse, chemical depletion of CBS I in Natronomonas pharaonis halorhodopsin (NpHR) through deprotonation of the Schiff base results in conformational changes of the protein: a state thought to mimic late stages of the photocycle. Here, crystals of Halobacterium salinarum halorhodopsin (HsHR) were soaked at high pH to provoke deprotonation of the Schiff base and loss of chloride. The crystals changed colour from purple to yellow and the occupancy of CBS I was reduced from 1 to about 0.5. In contrast to NpHR, this chloride depletion did not cause substantial conformational changes in the protein. Nevertheless, two observations indicate that chloride depletion could eventually result in structural changes similar to those found in NpHR. Firstly, the partially chloride-depleted form of HsHR has increased normalized B factors in the region of helix C that is close to CBS I and changes its conformation in NpHR. Secondly, prolonged soaking of HsHR crystals at high pH resulted in loss of diffraction. In conclusion, the conformation of the chloride-free protein may not be compatible with this crystal form of HsHR despite a packing arrangement that hardly restrains helices E and F that presumably move during ion transport.

The cryo-EM structure of the Plasmodium falciparum 20S proteasome and its use in the fight against malaria.

Plasmodium falciparum is the parasite responsible for the most severe form of malaria. Its increasing resistance to existing antimalarials represents a major threat to human health and urges the development of new therapeutic strategies to fight malaria. The proteasome is a protease complex essential in all eukaryotes. Accordingly, inhibition of the Plasmodium 20S proteasome is highly toxic for the parasite at all of its infective and developmental stages. Proteasome inhibitors have antimalarial potential both as curative and transmission blocking agents, but in order to have therapeutic application, they must specifically target the Plasmodium proteasome and not its human counterpart. X-ray crystallography has been widely used to determine structures of yeast and mammalian 20S proteasomes with ligands. However, crystallisation of the Plasmodium proteasome is challenging, as only small quantities of the complex can be directly purified from the parasite. Furthermore, most X-ray structures of proteasome-inhibitor complexes require soaking of crystals with high concentrations of ligand, thus preventing analysis of inhibitor subunit specificity. Instead we chose to determine the Plasmodium falciparum 20S proteasome structure, in the presence of a new rationally designed parasite-specific inhibitor, by high-resolution electron cryo-microscopy and single particle analysis. The resulting map, at a resolution of about 3.6 Å, allows a direct molecular analysis of inhibitor/enzyme interactions. Here we present an overview of this structure, and how it provides valuable information that can be used to assist in the design of improved proteasome inhibitors with the potential to be developed as next-generation antimalarial drugs.

Cleavage of nicotinamide adenine dinucleotide by the ribosome-inactivating protein from Momordica charantia.

The interaction of momordin, a type 1 ribosome-inactivating protein from Momordica charantia, with NADP(+) and NADPH has been investigated by X-ray diffraction analysis of complexes generated by co-crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine-ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide-ribose bond of oxidized NADP(+) is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five-membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors.

Multicomponent mixtures for cryoprotection and ligand solubilization

Mixed cryoprotectants have been developed for the solubilization of ligands for crystallization of protein–ligand complexes and for crystal soaking. Low affinity lead compounds with poor solubility are problematic for structural studies. Complete ligand solubilization is required for co-crystallization and crystal soaking experiments to obtain interpretable electron density maps for the ligand. Mixed cryo-preserving compounds are needed prior to X-ray data collection to reduce radiation damage at synchrotron sources. Here we present dual-use mixes that act as cryoprotectants and also promote the aqueous solubility of hydrophobic ligands. Unlike glycerol that increases protein solubility and can cause crystal melting the mixed solutions of cryo-preserving compounds that include precipitants and solubilizers, allow for worry-free crystal preservation while simultaneously solubilizing relatively hydrophobic ligands, typical of ligands obtained in high-throughput screening. The effectiveness of these mixture has been confirmed on a human transthyretin crystals both during crystallization and in flash freezing of crystals.

Identification of N-ethylmethylamine as a novel scaffold for inhibitors of soluble epoxide hydrolase by crystallographic fragment screening

Soluble epoxide hydrolase (sEH) is a potential target for the treatment of inflammation and hypertension. X-ray crystallographic fragment screening was used to identify fragment hits and their binding modes. Eight fragment hits were identified via soaking of sEH crystals with fragment cocktails, and the co-crystal structures of these hits were determined via individual soaking. Based on the binding mode, N-ethylmethylamine was identified as a promising scaffold that forms hydrogen bonds with the catalytic residues of sEH, Asp335, Tyr383, and Tyr466. Compounds containing this scaffold were selected from an in-house chemical library and assayed. Although the starting fragment had a weak inhibitory activity (IC50: 800μM), we identified potent inhibitors including 2-({[2-(adamantan-1-yl)ethyl]amino}methyl)phenol exhibiting the highest inhibitory activity (IC50: 0.51μM). This corresponded to a more than 1500-fold increase in inhibitory activity compared to the starting fragment. Co-crystal structures of the hit compounds demonstrate that the binding of N-ethylmethylamine to catalytic residues is similar to that of the starting fragment. We therefore consider crystallographic fragment screening to be appropriate for the identification of weak but promising fragment hits.

The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities.

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2-Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

Structure-based drug design against Trypanosoma brucei methionyl-tRNA synthetase

Infection by the protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, commonly known as sleeping sickness. The disease is fatal without treatment; yet, current therapeutic options for the disease are inadequate due to toxicity, difficulty in administration and emerging resistance. Therefore, methionyl-tRNA synthetase of T. brucei (TbMetRS) is targeted for the development of new antitrypanosomal drugs. We have recently completed a high-throughput screening campaign against TbMetRS using a 364,131 compounds library in The Scripps Research Institute Molecular Screening Center. Here we outline our strategy to integrate the power of crystal structures with high-throughput screening in a drug discovery project. We applied the rapid crystal soaking procedure to obtain structures of TbMetRS in complex with inhibitors reported earlier[1] to approximately 70 high-throughput screening hits. This resulted in more than 20 crystal structures of TbMetRS·hit complexes. These hits cover a large diversity of chemical structures with IC50 values between 200 nM and 10 µM. Based on the solved structures and existing knowledge drawn from other in-house inhibitors, the IC50 value of the most promising hit has been improved. Further development of the compounds into potent TbMetRS inhibitors with desirable pharmacokinetic properties is on-going and will continue to benefit from information derived from crystal structures.

Shikimate kinase-like 1 plays an integral role in chloroplast biogenesis

The shikimate pathway is found in plants, fungi, and microbes, but not mammals which makes it an attractive target for antimicrobials and herbicide development. In plants, the shikimate pathway produces precursors of compounds that are crucial for growth, defense and, survival. We focused on the enzyme shikimate kinase (SK) which catalyzes the phosphorylation of shikimate; the fifth reaction of the pathway. Plant SK underwent a gene duplication event yielding shikimate kinase-like genes (SKL1 and SKL2) roughly 400 MYA. Despite the overall similarity in sequence between SK and SKL1, a previous in vitro analysis showed that SKL1 does not catalyze a phosphorylation reaction in the presence of shikimate. When a three-dimensional structural model of SKL1 was compared with the microbial crystal structure of SK, we identified several highly conserved functional domains crucial for the shikimate kinase reaction: the shikimate binding domain (SBD) critical for substrate recognition, the LID domain integral for the transfer of phosphate to bound shikimate, and the walker A and B domains important for ATP binding. Of these domains, only the walker A and B domains are conserved in SKL1. Together with in vitro analysis, it is evident that SKL1 does not interact with shikimate, but the function is still unknown. Sequence alignment and modeled structure also revealed the presence of a novel phosphoglycerate mutase-like (PGML) domain within SKL1.This further suggests neofunctionalization of SKL1 compared to its parent gene SK. Current studies in our lab focus on determining the crystal structure of SKL1. This structure will be used for ligand docking experiments to identify potential substrates of the enzyme. As an additional means of substrate identification, we will also be performing crystal soaking experiments using plant extracts.

I04-1, a future 'fragment screening' beamline facility at Diamond Light Source

I04-1 is one of the six macromolecular crystallography (MX) beamlines at Diamond Light Source (DLS), the third generation synchrotron light source in the UK. It was built and delivered in 2010 as a stable and reliable fixed-wavelength MX station. It is currently preparing to release its user programme for exploiting fragment screening using X-ray crystallography in structural medicinal chemistry projects. For this purpose, the beamline has been going through several upgrades in order to achieve unattended high-throughput ligand crystallography. The new developments are aiming at improving the flux, stability and reliability of the beamline and its auto-alignment. In parallel, a peripheral laboratory is being set up to provide a facility for medium throughput compound soaking. Jointly with the Structural Genomics Consortium (SGC), a semi-automatic crystal soaking and harvesting scheme, which will provide hundreds of MX samples per day, is being tested at DLS. The beamline can currently process 400 crystals per day. However, the recent upgrades and automation should further improve that throughput. In this presentation, we will summarise the current specifications of the beamline and its new features, the development of a peripheral laboratory for compounds soaking and underline the remaining work.

Improved reproducibility of unit-cell parameters in macromolecular cryocrystallography by limiting dehydration during crystal mounting.

In macromolecular cryocrystallography unit-cell parameters can have low reproducibility, limiting the effectiveness of combining data sets from multiple crystals and inhibiting the development of defined repeatable cooling protocols. Here, potential sources of unit-cell variation are investigated and crystal dehydration during loop-mounting is found to be an important factor. The amount of water lost by the unit cell depends on the crystal size, the loop size, the ambient relative humidity and the transfer distance to the cooling medium. To limit water loss during crystal mounting, a threefold strategy has been implemented. Firstly, crystal manipulations are performed in a humid environment similar to the humidity of the crystal-growth or soaking solution. Secondly, the looped crystal is transferred to a vial containing a small amount of the crystal soaking solution. Upon loop transfer, the vial is sealed, which allows transport of the crystal at its equilibrated humidity. Thirdly, the crystal loop is directly mounted from the vial into the cold gas stream. This strategy minimizes the exposure of the crystal to relatively low humidity ambient air, improves the reproducibility of low-temperature unit-cell parameters and offers some new approaches to crystal handling and cryoprotection.

Spatiotemporal development of soaked protein crystal.

Crystal soaking is widely performed in biological crystallography. This paper reports time-resolved X-ray crystallographic and microtomographic analyses of tetragonal crystals of chicken egg-white lysozyme soaked in mother liquor containing potassium hexachloroplatinate. The microtomographic analysis showed that X-ray attenuation spread from the superficial layer of the crystal and then to the crystal core. The crystallographic analyses indicated that platinum sites can be classified into two groups from the temporal development of the electron densities. A soaking process consisting of binding-rate-driven and equilibrium-driven layers is proposed to describe these results. This study suggests that the composition of chemical and structural species resulting from the soaking process varies depending on the position in the crystal.

Identification and Optimization of PDE10A Inhibitors Using Fragment-Based Screening by Nanocalorimetry and X-ray Crystallography

Fragment-based lead discovery (FBLD) is a technique in which small, low-complexity chemical fragments of 6 to 15 heavy atoms are screened for binding to or inhibiting activity of the target. Hits are then linked and/or elaborated into tightly binding ligands, ideally yielding early lead compounds for drug discovery. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we use enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 10A (PDE10A). Two dozen fragments with KI <2 mM were identified and moved to crystal soaking trials. All soak experiments yielded high-resolution diffraction, with two-thirds of the fragments yielding high-resolution co-crystal structures with PDE10A. The structural information was used to elaborate fragment hits, yielding leads with KI <1 µM. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and paired successfully with an X-ray crystallography secondary screen.

Protein crystal harvesting using the RodBot: a wireless mobile microrobot

A new micro-agent is proposed to assist in automated protein crystal harvesting. The microrobot, named the RodBot, is a wireless mobile device driven by rotating magnetic fields (field strength 5–10 mT). When the RodBot rolls on a substrate in a low Reynolds number liquid environment, it generates flows to lift up and trap crystals in a vortex above itself. The gentle fluidic force acting on the crystals is in the range of a few nanonewtons to tens of nanonewtons and is spread over the whole surface of the crystal. The RodBot is capable of trapping protein crystals ranging from a few micrometres to sub-millimetre size. The trapped crystal can be transported to and deposited onto a loop positioned to accept it, obviating the need for more complicated `fishing' systems dependent on particular motions of the loop, the presence of further manipulators or the use of mechanical grippers. The RodBot can be driven in 24- and 96-well plates or in a crystal soaking dish, making the system compatible with existing crystallization hardware.

Structure and function of an insect α-carboxylesterase (αEsterase7) associated with insecticide resistance

Insect carboxylesterases from the αEsterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcαE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of α-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcαE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical α/β-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal α-helix that serves as a membrane anchor. Soaking of LcαE7 crystals in OPs led to the capture of a crystallographic snapshot of LcαE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcαE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants (∼10(6) M(-1) s(-1)) indicative of a natural substrate.

Elucidation of the Structure and Reaction Mechanism of Sorghum Hydroxycinnamoyltransferase and Its Structural Relationship to Other Coenzyme A-Dependent Transferases and Synthases

Hydroxycinnamoyltransferase (HCT) from sorghum (Sorghum bicolor) participates in an early step of the phenylpropanoid pathway, exchanging coenzyme A (CoA) esterified to p-coumaric acid with shikimic or quinic acid as intermediates in the biosynthesis of the monolignols coniferyl alcohol and sinapyl alcohol. In order to elucidate the mode of action of this enzyme, we have determined the crystal structures of SbHCT in its apo-form and ternary complex with shikimate and p-coumaroyl-CoA, which was converted to its product during crystal soaking. The structure revealed the roles of threonine-36, serine-38, tyrosine-40, histidine-162, arginine-371, and threonine-384 in catalysis and specificity. Based on the exact chemistry of p-coumaroyl-CoA and shikimic acid in the active site and an analysis of kinetic and thermodynamic data of the wild type and mutants, we propose a role for histidine-162 and threonine-36 in the catalytic mechanism of HCT. Considering the calorimetric data, substrate binding of SbHCT should occur sequentially, with p-coumaroyl-CoA binding prior to the acyl acceptor molecule. While some HCTs can use both shikimate and quinate as an acyl acceptor, SbHCT displays low activity toward quinate. Comparison of the structure of sorghum HCT with the HCT involved in chlorogenic acid synthesis in coffee (Coffea canephora) revealed many shared features. Taken together, these observations explain how CoA-dependent transferases with similar structural features can participate in different biochemical pathways across species.

Study of the effect of Ca(OAc)2 concentration on the state of the Allosteric Switch in Ras GTPase

Ras GTPase cycles between GDP inactive and GTP active forms and is of great importance in signal transduction that regulates cell proliferation, differentiation and survival. Although signaling is largely attenuated by the presence of GTPase activating proteins (GAPs), our group has recently discovered and allosteric mechanism that may promote hydrolysis of GTP in the presence of Raf and possibly other tight binding effectors, demonstrating subtle levels of regulation of the GTP‐bound protein. The allosteric switch is ON, with complete ordering of the active site, in crystals soaked in 100mM Ca(OAc)2, which binds to an allosteric site remote from the catalytic center. In the presence of CaCl2 the allosteric switch is OFF. Ca(OAc)2 binding is mediated by arginine 97 (R97) through salt bridge interactions with acetate. Here we show that R97A, which cannot bind acetate, is in the OFF state in the presence of 245 mM Ca(OAc)2 but switches to the ON state at 365 mM Ca(OAc)2. The R97F mutant is observed in the OFF state in the presence of 200mM Ca(OAc)2. It is clear that the bulk solvent composition has an important effect in the state of the allosteric switch in R97A. The soaking of crystals in 365 mM Ca(OAc)2 will test whether this is also the case for R97F and other allosteric site mutants.

X-ray structure of the V301L aldo–keto reductase 1B10 complexed with NADP+ and the potent aldose reductase inhibitor fidarestat: Implications for inhibitor binding and selectivity

Only one crystal structure is currently available for tumor marker AKR1B10, complexed with NADP+ and tolrestat, which is an aldose reductase inhibitor (ARI) of the carboxylic acid type. Here, the X-ray structure of the complex of the V301L substituted AKR1B10 holoenzyme with fidarestat, an ARI of the cyclic imide type, was obtained at 1.60Å resolution by replacement soaking of crystals containing tolrestat. Previously, fidarestat was found to be safe in phase III trials for diabetic neuropathy and, consistent with its low in vivo side effects, was highly selective for aldose reductase (AR or AKR1B1) versus aldehyde reductase (AKR1A1). Now, inhibition studies showed that fidarestat was indeed 1300-fold more selective for AR as compared to AKR1B10, while the change of Val to Leu (found in AR) caused a 20-fold decrease in the IC50 value with fidarestat. Structural analysis of the V301L AKR1B10-fidarestat complex displayed enzyme-inhibitor interactions similar to those of the AR-fidarestat complex. However, a close inspection of both the new crystal structure and a computer model of the wild-type AKR1B10 complex with fidarestat revealed subtle changes that could affect fidarestat binding. In the crystal structure, a significant motion of loop A was observed between AR and V301L AKR1B10, linked to a Phe-122/Phe-123 side chain displacement. This was due to the presence of the more voluminous Gln-303 side chain (Ser-302 in AR) and of a water molecule buried in a subpocket located at the base of flexible loop A. In the wild-type AKR1B10 model, a short contact was predicted between the Val-301 side chain and fidarestat, but would not be present in AR or in V301L AKR1B10. Overall, these changes could contribute to the difference in inhibitory potency of fidarestat between AR and AKR1B10.

Vascular Bioactivation of Nitroglycerin by Aldehyde Dehydrogenase-2

Aldehyde dehydrogenase-2 (ALDH2) catalyzes the bioactivation of nitroglycerin (glyceryl trinitrate, GTN) in blood vessels, resulting in vasodilation by nitric oxide (NO) or a related species. Because the mechanism of this reaction is still unclear we determined the three-dimensional structures of wild-type (WT) ALDH2 and of a triple mutant of the protein that exhibits low denitration activity (E268Q/C301S/C303S) in complex with GTN. The structure of the triple mutant showed that GTN binds to the active site via polar contacts to the oxyanion hole and to residues 268 and 301 as well as by van der Waals interactions to hydrophobic residues of the catalytic pocket. The structure of the GTN-soaked wild-type protein revealed a thionitrate adduct to Cys-302 as the first reaction intermediate, which was also found by mass spectrometry (MS) experiments. In addition, the MS data identified sulfinic acid as the irreversibly inactivated enzyme species. Assuming that the structures of the triple mutant and wild-type ALDH2 reflect binding of GTN to the catalytic site and the first reaction step, respectively, superposition of the two structures indicates that denitration of GTN is initiated by nucleophilic attack of Cys-302 at one of the terminal nitrate groups, resulting in formation of the observed thionitrate intermediate and release of 1,2-glyceryl dinitrate. Our results shed light on the molecular mechanism of the GTN denitration reaction and provide useful information on the structural requirements for high affinity binding of organic nitrates to the catalytic site of ALDH2.

Structures of NodZ α1,6-fucosyltransferase in complex with GDP and GDP-fucose.

Rhizobial NodZ α1,6-fucosyltransferase (α1,6-FucT) catalyzes the transfer of the fucose (Fuc) moiety from guanosine 5'-diphosphate-β-L-fucose to the reducing end of the chitin oligosaccharide core during Nod-factor (NF) biosynthesis. NF is a key signalling molecule required for successful symbiosis with a legume host for atmospheric nitrogen fixation. To date, only two α1,6-FucT structures have been determined, both without any donor or acceptor molecule that couldhighlight the structural background of the catalytic mechanism. Here, the first crystal structures of α1,6-FucT in complex with its substrate GDP-Fuc and with GDP, which isabyproduct of the enzymatic reaction, are presented. The crystal of the complex with GDP-Fuc was obtained through soaking of native NodZ crystals with the ligand and its structure has been determined at 2.35 Å resolution. The fucose residue is exposed to solvent and is disordered. The enzyme-product complex crystal was obtained by cocrystallization with GDP and an acceptor molecule, penta-N-acetyl-L-glucosamine (penta-NAG). The structure has been determined at 1.98 Å resolution, showing that only the GDP molecule is present in the complex. In both structures the ligands are located in a cleft formed between the two domains of NodZ and extend towards the C-terminal domain, but their conformations differ significantly. The structures revealed that residues in three regions of the C-terminal domain, which are conserved among α1,2-, α1,6- and protein O-fucosyltransferases, are involved in interactions with the sugar-donor molecule. There is also an interaction with the side chain of Tyr45 in the N-terminal domain, which is very unusual for a GT-B-type glycosyltransferase. Only minor conformational changes of the protein backbone are observed upon ligand binding. The only exception is a movement of the loop located between strand βC2 and helix αC3. In addition, there is a shift of the αC3 helix itself upon GDP-Fuc binding.