Chemical modification of surface and buried sites

The two constructs of AurA used for this work (T288C and F275C) contain a number of mutations introduced by site-directed mutagenesis (Figure 2). As described previously [18], both constructs contain C290A and C393A mutations to prevent undesired chemical modification from occurring at exposed cysteine residues. Two cysteine residues (C247 and C319) remain; however, our previous work confirmed that these residues are buried within the protein and not accessible to chemical modification. The T288C construct has an additional alanine substitution at the adjacent T287 phosphorylation site because this construct was originally designed to investigate the effect of AurA phosphorylation exclusively at position 288 [18].

Under identical reaction conditions, C288 and C275 both react to form dehydroalanine (Dha) from cysteine (Figure S1). Formation of C288^Dha proceeds to completion within 2 hours consistent with the location of C288 on a flexible surface-exposed loop; however reaction at C275, which is located deep in the kinase ATP binding pocket, does not proceed to completion in 2 hours, resulting in a small amount of residual cysteine at this position which is carried through to the second step of the reaction. We then reacted a wide range of thiol nucleophiles with C288^Dha and C275^Dha (Figure 3 and Table 1) and determined the success of each reaction by intact protein mass spectrometry (LC-MS) (Figures S2–S3). Consistent with previous reports [18], PTMs corresponding to lysine and N-terminal methionine gluconoylation or phospho-gluconoylation were observed resulting in additional peaks of +178 Da or +258 Da alongside that of the expected mass. Pleasingly, we observed no difference in the success of thiol addition reactions at the C275 site compared to the surface exposed C288 site, with the notable exception of the small amount of unreacted C275 cysteine that is carried through each C275 reaction (Table 1). We propose that the high level of modification achieved at C275 highlights the flexibility and accessibility of this region in the absence of a bound ligand, and suggests that in the apo form of AurA, both C275 and C275^Dha can adopt a chemically-accessible ‘DFG-out’ conformation in which the side-chain of cysteine and the sp² carbon of dehydroalanine point outwards into the ATP binding site. This is in contrast to published crystal structures that show an inaccessible ‘DFG-in’ conformation of F275 when bound to ATP [24]. Indeed, the modification of F275 to either cysteine or dehydroalanine may make a ‘DFG-out’ conformation more prevalent, as the favorable interactions of the lipophilic phenylalanine side-chain with the hydrophobic pocket observed in the ‘DFG-in’ conformation are reduced. The success of chemical modification at this residue under non-denaturing conditions differs from other methods in which the protein is either subjected to harsher conditions to gain access to the hindered cysteine [25], or is unfolded using guanidinium hydrochloride [9]–[11], both of which are undesirable when working with larger, more complex proteins such as AurA. Additionally, we have previously shown that the reaction conditions applied here are not detrimental to the catalytic activity of AurA thereby demonstrating proof of protein viability under these conditions [18].

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Figure 3. Reagents used for addition at C288^Dha and C275^Dha.

Reagents are colored as indicated in Figure 1.

https://doi.org/10.1371/journal.pone.0103935.g003

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Table 1. LC-MS results of chemical modification of Aurora-A C288^Dha and C275^Dha.

https://doi.org/10.1371/journal.pone.0103935.t001

We were able to determine general reactivity trends for thiol addition reactions. Polar uncharged and polar aromatic thiols all react efficiently with Dha at both C288 and C275 (Table 1, entries 1–9). We used 5000 eq. of each thiol reagent as standard conditions, however for one example, 4-methoxybenzenethiol (MOBZ), we determined the minimum excess required to drive full conversion. We found AurA 288^Dha reacted completely to the desired MOBZ product when >250 eq. of MOBZ were used; whereas reactions with up to 250 eq. MOBZ still contained unreacted Dha after 2 hours (Figure S4). Hydrophobic aromatic thiols also react to completion under our standard conditions (10–12), with the exception of 4-fluorobenzenethiol (FBZS, 13), which did not react at either site presumably as a result of lower nucleophilicity. Representative examples of different thiol reactions that showed excellent conversion at both sites are provided in the Supporting Information, including LC-MS/MS confirmation of the site of modification and quantification of protein recovery after the reaction, which is typically around 75% (Figure S5).

We observed that thiol addition reactions using charged reagents were less successful than those using uncharged or aromatic thiols (Table 1, 14–19). Negatively charged reagents either did not react, or did not react to completion (14–16) consistent with previous work in which thiophosphate [(SPO₃)³⁻] addition to Dha at C288 was incomplete and required purification to obtain the desired product [18]. Furthermore, additional unidentified peaks were observed by LC-MS for reactions with 3-mercaptopropanoic acid (MPA, 14) and 2-mercaptoacetic acid (TGA, 16). Using positively charged thiol reagents gave mixed results (17–19). Reactions using 2-(4-methylpiperazin-1-yl)ethanethiol (MPES, 17) were unsuccessful whilst reactions with 2-(dimethylamino)ethanethiol (MNES, 18) worked efficiently to give the desired product, however adducts of 102–103 Da were observed on both C288 and C275 samples (vide infra); and reaction of C275 with 2-aminoethanethiol (NES, 19) resulted in multiple unidentified peaks observed by LC-MS with only a small amount of the C275^Dha starting material detected. A thermal denaturation screen, conducted to optimize buffer conditions, revealed that 2-aminoethanol completely unfolds AurA when used as a buffer component at a concentration of >5% (v/v) (Figure S6). It is therefore likely that NES has a similar effect and, although used at a lower concentration, acts to denature the protein.

Synthesis of N-acetyl-lysine and N, N-dimethyl-lysine mimics

In addition to generating a range of unnatural amino acids, we produced a chemical mimic of N-acetyl-lysine using N-acetylcysteamine (ACCN, 5). This reaction was successful at both the C288 and F275C sites (Figure 4), reacted to completion and yielded a protein recovery of ∼80%. This demonstrates the potential of this method to incorporate mimics of PTM-residues in a site-specific manner for applications where selective lysine N-acetylation is desired, such as for in vitro studies of protein epigenetic modification and analogous to our previous work describing the selective installation of phospho-serine mimics to facilitate studies of kinase activation [18]. Li and co-workers have described an elegant alternative method to generate an N-acetyl-lysine mimic via radical-mediated thiol-ene addition [11], however their substrate proteins were significantly smaller; namely: Ubiquitin (8.5 kDa), Histone H3 (15 kDa) and Histone H4 (11 kDa). Furthermore, their reactions on histones were carried out under denaturing conditions. Additionally Chalker and co-workers previously applied the synthetic method described here to incorporate an N-acetyl-lysine mimic into Histone H3 [6]. The work described here extends the methodology to a much larger protein, AurA kinase, and proceeds under native conditions that are more likely to maintain the integrity of larger protein substrates.

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Figure 4. Generation of N-acetyl-lysine mimic at exposed and hindered sites on AurA.

LC-MS indicates complete conversion of Dha to ACCN product at C288 and C275, with a small amount of unreacted cysteine present at C275. LC-MS/MS confirms the sites of modification.

https://doi.org/10.1371/journal.pone.0103935.g004

We reacted C288^Dha and C275^Dha with MNES to specifically generate an N, N-dimethyl-lysine mimic, a procedure of potential general use in the study of lysine methylation states under epigenetic control. Both C288^Dha and C275^Dha reacted to completion to give the desired product (18), however, extra mass additions of 102–103 Da were also observed on both samples (Figures S2.R and S3.Q). The extra mass was indicative of disulfide-linked adducts of the 105 Da MNES reagent with one of the internal cysteines within AurA (Figure 5). The constructs of AurA used in this study contain two internal buried cysteines, C247 and C319, which were predicted to be inaccessible to chemical modification (Figure 2). However, we postulate that the positively charged MNES reagent causes partial unfolding of AurA to expose C247 and facilitate formation of a disulfide bond with MNES (Figure 5). Chalker and co-workers have previously demonstrated the successful reaction of MNES with Histone H3 to produce an N, N-dimethyl-lysine mimic [6]; however, in this case, the construct did not contain any bystander cysteines that would be prone to side reaction. An alternative method described by Simon and co-workers for the selective installation of methylated lysine analogues via cysteine alkylation uses denaturing conditions that would be unsuitable for application to larger proteins such as AurA [10]. Although both methods are appropriate for the modification of small protein substrates containing a single cysteine residue, the conditions we demonstrate here are applicable to larger protein substrates with the proviso that caution should be exercised when using positively charged thiol reagents that may have the potential to elicit protein unfolding and potential side reactions at bystander cysteine residues.

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Figure 5. Identification of MNES adducts on AurA.

Extra mass adducts of 102 Da were observed on AurA C288 after reaction with MNES in addition to the expected C288 MNES product. This mass corresponds (within the error associated with intact protein LC-MS) to the MNES reagent, which was confirmed by LC-MS/MS as forming a disulfide-linked adduct with C247, which is close to the surface of AurA and could become accessible with slight protein unfolding.

https://doi.org/10.1371/journal.pone.0103935.g005

AurA mutagenesis, expression and purification

Site-directed mutagenesis of AurA kinase domain (122–403) in a pET30-based vector was carried out using a QuikChange protocol (Agilent). The mutations were C290A, C393A, and either F275C for the 275 construct or T288C and T287A for the 288 construct. Proteins were expressed in E. coli BL21-CodonPlus -RIL (Agilent) or Rosetta 2 (Merck) DE3 cells in LB medium, with initial growth at 37°C followed by overnight incubation at 21°C after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside. Bacterial pellets were resuspended in lysis buffer (50 mM Tris pH 7.5, 300 mM NaCl, 5 mM MgCl₂, 10% glycerol, 40 mM imidazole), supplemented with protease inhibitors and DNase (Roche), lysed by sonication and clarified by centrifugation. Lysates were filtered then subjected to Ni²⁺ affinity chromatography (GE Healthcare). Proteins were eluted in lysis buffer containing 250 mM imidazole, purified to homogeneity by S75 size exclusion chromatography (GE Healthcare) (gel filtration buffer: 50 mM Tris pH 7.5, 200 mM NaCl, 5 mM MgCl₂, 10% glycerol, 10 mM β-mercaptoethanol (BME)), then concentrated to ∼10 mg/mL and flash frozen for future use. Protein concentrations were measured in triplicate with a ND-1000 Spectrophotometer (NanoDrop) using molecular weights and extinction coefficients calculated by ProtParam (ExPASy).

Chemical modification of AurA C288 and C275

α,α′-Di-bromo-adipoyl(bis)amide (DBAA) was synthesized and characterized as described [26]. An aliquot of protein at ∼10 mg/mL* was shaken for 15 min at room temperature (RT) with 10 mM dithiothreitol then exchanged using a PD MiniTrap G-25 column (GE Healthcare) into 50 mM bicine pH 8.0. A 500× molar excess of DBAA dissolved in dimethylformamide (DMF) was added to the eluted protein (final DMF concentration was 5%; additional bicine buffer was added if necessary), and the reaction was shaken at RT for 2 hours to form the dehydroalanine. Protein was desalted and exchanged into 50 mM Tris pH 7.5 over either a PD MidiTrap G-25 or PD-10 column (GE Healthcare), depending on sample volume. Protein concentrations were typically 0.2–0.5 mg/mL by this stage due to dilution. For thiol addition reactions, 5000 eq. (as standard, although some reactions were carried out using 1500 eq.) of thiol nucleophile reagent (various suppliers) were added to 500 µL aliquots of the desalted protein and shaken for 2 hours at RT. The reaction mixture was either analyzed directly by LC-MS, or cleaned up by exchanging into gel filtration buffer without BME for protein quantitation and LC-MS/MS.

*Typically 12.5–40 µL protein was used for each anticipated thiol addition reaction. 125 µL of protein was used for each reaction where final clean-up and quantitation were intended, in order to maintain high enough protein concentration for accurate quantitation (i.e. >0.5 mg/mL).

Mass spectrometry

Mass spectrometry methods are available as Protocol S1.