Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination

Results

Two different screening methods, which we termed differential scanning fluorimetry (DSF) and differential static light scattering (DSLS), were used to identify solution conditions or ligands that stabilized a protein against thermal denaturation. The DSF screening format has been described, using six proteins as test cases (9). The DSLS format has been described in the patent literature (11); we report on its application (Fig. 3, which is published as supporting information on the PNAS web site). Our first aim was to compare and contrast the two screening methods, as applied to a significant number of different human and parasitic proteins and performed on commercially available systems. Our second aim was to assess whether and how often the preferred solutions or ligands would facilitate protein purification or crystallization.

General Properties of the Two Methods.

Initially, the properties of the two methodologies used were determined based on the analysis of dozens of different proteins on three different, commercially available platforms. For simplicity, we report the results from two representative proteins, citrate synthase and the cytosolic sulfotransferase 1C1 (SULT1C1), to highlight the behavior and dependence of the T _m or temperature of aggregation (T _agg) on the instrumentation and the addition of ligands.

Variability of the instrumentation.

Two different commercial multiwell-format PCR instruments and one fluorescence plate reader were used to determine the observed T _m. Another multiwell-format commercial instrument (Stargazer, Harbinger Biotech, Toronto, Canada) was used to measure the observed T _agg. Pig heart citrate synthase (Roche, Indianapolis, IN), a commercially available protein with well defined properties, was used as a standard to determine the reproducibility of the observed T _m for each instrument (Table 3, which is published as supporting information on the PNAS web site). The reproducibility of the T _m measured with both PCR devices (Mx3005p and iCycler) and the fluorescence plate reader (FluoDia T70) were similar (0.2°C and 0.5°C, respectively). A standard deviation of 0.4°C was determined in the T _agg for the light-scattering method over hundreds of measurements. With the exception of both PCR devices, the observed T _m and T _agg varied slightly between the sides and middle of the plates, likely caused by the uneven heat distribution (Fig. 4, which is published as supporting information on the PNAS web site). Although the variation could be modeled for each individual instrument, we elected not to do so because the variability (<0.3°C) was much smaller than the temperature shift (>2°C) that we observed for the binding of known, specific ligands of micromolar affinity.

The average values for the T _m and T _agg determined for citrate synthase [T _m of 52.4 ± 0.5°C (fluorescence plate reader), T _m of 53.0 ± 0.2°C (PCR), and T _agg of 53.2 ± 0.4°C] compare well with the T _m for citrate synthase determined by either circular dichroism (53.3 ± 0.1°C) or differential scanning calorimetry (53.8 ± 0.3°C) under the same solution conditions. The observed T _m and T _agg values for a given protein were highly reproducible. However, for any given protein, the absolute values of T _m and T _agg often differed by a few degrees depending on the experimental conditions and the instrumentation (see Table 1); for example, the T _agg was more greatly influenced by the protein concentration. In general, under the experimental conditions used here, and with the described instrumentation, the T _m and T _agg values were within 4°C for ≈50% of proteins tested. There were larger variations, sometimes up to 15°; these occurred with proteins that had unusually high initial fluorescence readings. It is likely that these proteins had exposed hydrophobic patches in their initial states, perhaps as a result of partial unfolding.

The effect of known ligands on T_agg and T_m.

We tested the effects of ligands on both T _m and T _agg with the human cytosolic sulfotransferase 1C1 (SULT1C1), which catalyzes the transfer of a sulfate group from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to a variety of substrates. The reaction produces a sulfonated substrate and 3′-phosphoadenosine-5′-phosphate (PAP).

For DSF, human SULT1C1 was aliquoted into each well of a 384-well plate at 100 μg/ml in the presence of SYPRO orange and different concentrations of PAP, and the plate was heated from 27°C to 75°C. The observed T _m of SULT1C1 in the absence of PAP was 48.4 ± 0.2°C. There was a significant increase in the observed T _m in the presence of PAP; the lowest concentrations of PAP that stabilized SULT1C1 >3°C was 87 μM (Fig. 1 A). This concentration of PAP also caused a similar increase in the T _agg (Fig. 1 B). The stability of the protein increased as the concentration of PAP was increased to 9 mM, at which the ΔT _m and ΔT _agg approached plateaus of ≈8°C (Fig. 1 C and D). Our results support the findings of Matulis et al. (6) and Bullock et al. (7) that ligand binding increases protein thermal stability, and that the effect is proportional to the concentration and affinity of the ligand.

Reproducibility of ΔT_agg and ΔT_m.

To use the two methods and the selected hardware as screening platforms, the ΔT _m and ΔT _agg must be reproducible. Accordingly, the T _m and T _agg for SULT1C1 were measured up to 12 times in the presence and absence of 0.5 mM of PAP. The protein consistently showed greater stability in the presence of PAP (T _agg = 53.1 ± 0.2°C; T _m = 53.8 ± 0.5°C) than in its absence (T _agg = 48.4 ± 0.3°C; T _m = 48.4 ± 0.2°C). The resulting ΔT _agg and ΔT _m were 4.7 ± 0.5°C and 5.4 ± 0.7°C, respectively, suggesting that the two methods in these formats are able to measure these parameters reproducibly and can be used to screen proteins for the binding of new ligands.

General Applicability of the Methods.

As anticipated from previous studies on small numbers of proteins (11, 12), we found that increases in both the T _m and T _agg were correlated with binding of ligands. The specific instruments used here could measure the transitions reproducibly within 0.5°C. We were then interested to determine the fraction of proteins to which the two methods could be applied, to assess whether these methods could be applied broadly.

The T _m and T _agg were determined and compared for 61 different proteins (Table 1). For 40 proteins, both a T _m and a T _agg could be measured reproducibly and with thermal envelopes that conformed to the prototypical melting transitions. Neither a T _agg nor a T _m could be measured for 10 proteins, presumably because of high thermal stability or some other property of the protein that was incompatible with the method (e.g., not properly folded). There were 11 proteins that could be analyzed only with one or the other method; for 7 proteins only a T _agg could be measured and for 4 proteins only a T _m could be measured. All proteins that did not display an interpretable T _m displayed aberrantly high initial fluorescence in the presence of SYPRO orange. It is possible that these proteins contain hydrophobic binding pockets/cavities accessible to the dye. Of the 40 proteins for which both a T _m and a T _agg could be measured, the difference between T _agg and T _m varied depending on the protein; for 16 proteins T _agg was lower than T _m, whereas for 24 proteins T _agg was higher than T _m. It is possible that aggregation kinetics or a stabilization effect by the dye account for these differences.

Application of Screening Methods.

We applied the screening platforms to identify ligands or buffer conditions that might stabilize proteins and aid protein purification and/or crystallization. Two types of small-molecule screens were implemented. In the first, the proteins were screened against a set of common solution conditions and sets of physiologically relevant ligands, such as nucleotides and cofactors. In the second, proteins were screened against libraries of small molecules that were designed especially for the protein or protein family being investigated. For example, protein kinases were screened against a set of previously identified and validated inhibitors.

Screening against solutions containing ranges of pH and salt.

A total of 221 proteins were screened by using one of the methods against buffers covering a pH range from 6 to 9 and two different salt concentrations (100 and 500 mM NaCl). In >50% of the cases a condition was identified that stabilized the protein by >4°C against thermal denaturation compared with the original buffer (Hepes buffer, pH 7.5/150 mM NaCl) (Table 4, which is published as supporting information on the PNAS web site). Although it was not possible to extract unifying trends, we did observe that most proteins were stabilized in this assay by the addition of higher concentrations of salt. However, 27% of proteins were more stable in lower concentrations of salt.

In several instances the identification of a stabilizing solution contributed to the ability to purify, concentrate, or crystallize the protein. For example, the E2 ubiquitin-conjugating enzyme from Cryptosporidium parvum was purified and concentrated to 7 mg/ml for crystallization trials in standard buffer (Hepes, pH 7.5 in 500 mM NaCl). A buffer screen using DSLS found that the protein was more stable in low salt at pH 9, and the use of this buffer enabled the protein to be concentrated to 28 mg/ml. Using DSF, more optimal purification conditions for human RGS6 (at pH 6.5), human RGS16 (at pH 9.0), and human RGS17 (at pH 8.5) were identified. None of these RGS proteins could be concentrated under standard conditions, but the use of the optimized conditions allowed them to be concentrated to >10 mg/ml, crystals to be formed, and the structures to be determined [PDB ID codes: 2ES0 (RGS6), 2BT2 (RGS16), and 1ZV4 (RGS17)].

Calpain 1 could be purified and crystallized under standard conditions, but the crystals diffracted poorly (3.0–3.2 Å). A buffer screen showed that the protein was more resistant to aggregation under lower salt conditions, and the use of these conditions during purification led to a different crystal form of higher quality and ease to reproduce, which led to a structure at higher resolution (2.4 Å; PDB ID code 2ARY). Purified Trb2 kinase domain constructs were aggregating and visibly precipitated out of solution before and after gel filtration when using standard buffer conditions (20 mM Hepes, pH 7.5/0.5 M NaCl/2 mM DTT). By diluting the protein and screening in different buffer conditions, the optimal buffer condition was found to be 100 mM NaCl, 20 mM bicine (pH 9.0), and 1 mM DTT; under these conditions, the Trb2 kinase domain was soluble and readily concentrated to 20–30 mg/ml.

Screening against a library of physiologically relevant compounds.

Physiologically relevant small molecules provide a potentially rich source of compounds for stabilizing proteins. Accordingly, we generated libraries that comprised physiologically relevant compounds (PHY library) and other molecules that might be predicted to be “generic” stabilizers of proteins, such as detergents and metals. One representative library comprised 160 compounds that included amino acids, nucleotides, nucleosides, sugars, cofactors, divalent cations, common substrates and products, and some other additives (Table 2). To minimize the number of screens and the protein used, the compounds were combined in different groups of two to six compounds. If a group of compounds was shown to stabilize the proteins, the protein was then rescreened against the individual compounds (deconvolution).

There are several examples in which the use of these libraries contributed directly to a crystal structure. For example, the C2 domain of PrkCh was purified under standard conditions but could not be concentrated beyond 2 mg/ml. We found that the addition of 5% glycerol stabilized the protein and allowed the protein to be concentrated to >4 mg/ml and subsequently crystallized. Interestingly, contrary to our expectation, the addition of glycerol to proteins did not have a general stabilizing effect. Among a subset of 28 proteins tested for the influence of glycerol, only 8 were stabilized by >2°C (at pH 7.5). Pyruvate kinase was purified and crystallized, but the crystals were difficult to optimize. l-phenylalanine was found to stabilize the protein by using DSLS, and the inclusion of l-Phe in the crystallization buffer at 10 mM facilitated the formation of crystals diffracting to 2.2 Å, from which the structure was solved (PDB ID code 1ZJH). Fe-superoxide dismutase was crystallized but the crystals were of poor quality. The inclusion of 5 mM MnCl₂, which was found to stabilize the protein with DSLS, permitted the growth of crystals that diffracted to 2.2 Å, from which the structure was solved (PDB ID code 2AWP). Human Cdc2-like kinase, CLK1, could not be sufficiently concentrated for crystallization. Addition of a mixture of l-arginine and l-glutamic acid (13) enabled concentration to 10 mg/ml, thus providing the means to solve the structure in the presence of a specific inhibitor (PDB ID code 1Z57).

The methodology has also been applied to determine the concentrations of known substrates and cofactors that are optimal for crystallization trials. For example, the bifunctional PAPS synthetase was known to bind ATP. Initial attempts to crystallize PAPS synthetase in the presence of up to 5 mM ATP were unsuccessful. With DSLS, the PAPS synthetase was titrated against higher concentrations of ADP and ATP, and this experiment indicated that much higher concentrations of ADP and ATP were required to saturate the enzyme under the conditions tested (Fig. 2). The protein was then crystallized in the presence of 100 mM ATP, the resulting crystals were diffracted to 2.4 Å, and the structure was subsequently solved (PDB ID code 2AX4). ADP was found in the active site of the crystallized enzyme, suggesting ATP was hydrolyzed in the crystallization trials.

Unbiased small-molecule screens can also guide the experiment in unanticipated directions. Adenosine deaminase was subjected to crystallization trials in the presence and absence of adenine, but no crystals could be obtained. In the screen of physiological compounds, which includes nucleotides and deoxynucleotides, deoxyguanosine was identified as the strongest stabilizer of adenosine deaminase. Although the nucleoside was not found in the structure, crystals of adenosine deaminase that diffracted to 2.0 Å were obtained in the presence of deoxyguanosine (PDB ID code 2AMX).

The components of the library of physiologically relevant compounds were not uniformly found as being active. More than 50% of the compounds were never shown to stabilize any protein. A few additives were frequently identified as stabilizers, raising the possibility of their being false positives. However, among these compounds were those that might be predicted to act as general, nonspecific protein-stabilizing compounds, including n-dodecyl-β-d-maltoside and a mixture of 50 mM l-arginine and 50 mM l-glutamic acid, which stabilized 26% and 16% of all proteins against thermal denaturation (>4°C), respectively. The promiscuous stabilization by l-arginine and l-glutamic acid confirms the report from Golovanov et al. (13). The promiscuity of these ligands suggests that they may prove to be useful additives for crystallization screens.

Focused libraries for specific proteins and protein families.

For some proteins, there may be considerable prior knowledge about the compounds that are likely to bind, and in these instances it may be useful to generate a protein-specific library of compounds. The most direct path for creating such a library is to explore the academic and patent literature, the PDB, and other databases, such as BRENDA (www.brenda.uni-koeln.de), to identify substrates, inhibitors and/or cofactors that have been shown to bind the protein or closely related proteins. In many instances, these compounds are available from commercial suppliers.

Protein family-specific libraries were created for a number of human enzyme families (e.g., deacetylases, sulfotransferases, protein kinases, methyltransferases, and oxidoreductases). In many cases, the use of these libraries identified compounds that facilitated protein crystallization. In one example, purified NAD-dependent deacetylase sirtuin 5 (SIRT5) was screened against a set of compounds known to bind deacetylases, and suramin was shown to stabilize the protein. SIRT5 was then cocrystallized with suramin, and crystals that diffracted to 2 Å were obtained, and the structure was solved (PDB ID code 2FZQ).

For protein kinases, the use of a library of inhibitors proved to be a very effective strategy for increasing the success rate in producing well diffracting crystals. Our ≈500-compound library comprised mostly compounds that mimic the binding mode of adenine (14, 15). To date, this library has been used to screen 32 serine-threonine protein kinases, and for 84% of them at least one compound was identified that caused a T _m shift of >4°C (O.Y.F., A. Bullock, F.H.N., B.M., and S. Knapp, unpublished work). In 9 of 12 cases in which we determined the structure of the catalytic domain, the use of the inhibitor in crystallization trials directly contributed to obtaining the crystal structure (Table 5, which is published as supporting information on the PNAS web site). For example, Cdc2-like kinase, CLK1, was cocrystallized with 10Z-hymenialdisine (PDB ID code 1Z57). Among the different examples, electron density corresponding to the ligand could be seen in all proteins except PDB ID codes 2FK9, 1ZJH, and 2AMX.

Correlation of protein stabilization and affinity of binding.

The thermal stabilization assays were used primarily to identify compounds that could promote protein purification or crystallization. We observed that the increase of the transition temperatures, ΔT _m and ΔT _agg, were highly reproducible when measured with DSF and DSLS, respectively [using the proteins SULT1C1, PIM-1 (7), and CLK1]. We have not undertaken a systematic effort to correlate the degree of temperature shift with binding affinities, although inhibition data of selected compounds for several proteins, including three protein kinases (PIM-1, CLK1, and CLK3) showed that T _m shifts >4°C translate into values for IC₅₀ <1 μM. At least in one instance, the degree of stabilization was correlated with the relative affinity; in studies of a set of compounds derived from one scaffold, there was a correlation between T _m and binding affinities (7). Operationally, we have observed that temperature shifts >2°C are experimentally reproducible, but that higher temperature shifts (>4°C) are better correlated with positive outcomes in protein crystallization.