Observed surface lysine acetylation of human carbonic anhydrase II expressed in Escherichia coli

Abstract

Acetylation of surface lysine residues of proteins has been observed in Escherichia coli (E. coli), an organism that has been extensively utilized for recombinant protein expression. This post-translational modification is shown to be important in various processes such as metabolism, stress-response, transcription, and translation. As such, utilization of E. coli expression systems for protein production may yield non-native acetylation events of surface lysine residues. Here we present the crystal structures of wild-type and a variant of human carbonic anhydrase II (hCA II) that have been expressed in E. coli and exhibit surface lysine acetylation and we speculate on the effect this has on the conformational stability of each enzyme. Both structures were determined to 1.6 Å resolution and show clear electron density for lysine acetylation. The lysine acetylation does not distort the structure and the surface lysine acetylation events most likely do not interfere with the biological interpretation. However, there is a reduction in conformational stability in the hCA II variant compared to wild type (∼4°C decrease). This may be due to other lysine acetylation events that have occurred but are not visible in the crystal structure due to intrinsic disorder. Therefore, surface lysine acetylation events may affect overall protein stability and crystallization, and should be considered when using E. coli expression systems.

Introduction

Post-translational modifications of surface lysine residues in proteins are frequently associated with intrinsically important physiological processes of both eukaryotic and prokaryotic organisms.^1–4 These processes include acetylation, methylation, succinylation, ubquitination, sumoylation, and more recently, pyrrolation.^5,6 One of the most important lysine modifications is acetylation, which has been shown to play key roles in various cellular pathways in mammals, such as cell survival, apoptosis, cell differentiation, and metabolism.^7,8 In particular, there is an increasing interest in deciphering the mechanisms of chromatin-based transcriptional control and epigenetic programming through understanding lysine modifications of histones.^5,7 Interestingly, a majority of mammalian proteins that undergo lysine acetylation are localized to the mitochondria, so the evolutionary lineage of eukaryotic mitochondria to bacteria suggests this modification is widespread in prokaryotes.⁹ As such, this post-translational modification has been observed in Escherichia coli (E. coli), an organism that has been extensively utilized for recombinant protein expression, and has been shown to be important in various processes including metabolism, stress response, transcription, and translation.^8,10 This observation further implies the possibility of non-biological lysine acetylation in recombinantly produced proteins from an E. coli expression system. A possible mechanism for surface lysine acetylation by acetyltransferase is presented in Figure 1.

Figure 1.

Proposed mechanism for acetylation of surface lysine residues. Acetyl-CoA acts as a substrate for acetyltransferase (Enz) to attach acetyl groups to the amide of the side-chains of surface lysine residues.

Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the interconversion of CO₂ and water to bicarbonate and a proton.^11–13 Humans express 14 hCA isoforms of which 11 are catalytically active and have been shown to be involved in multiple physiological processes such as respiration, pH regulation, bone resorption, and the formation of gastric acid and aqueous humor. In addition, isoforms hCA IX and XII have been shown to play significant roles in tumor biology and have become anticancer drug targets, with structure-based drug design being one method used toward drug development.^14–23 As such, large quantities of hCA II and a variant termed hCA IX-mimic (constructed for CA inhibitor design) are expressed in E. coli. Here we present the X-ray crystal structures of hCA II and hCA IX-mimic that display surface lysine acetylation and we discuss the implications of these modifications.

Results and Discussion

The crystal structures of hCA II and hCA IX-mimic were both studied in complex with an acetylated glucosyl sulfamate inhibitor and refined to a maximum resolution of 1.6 Å (Table1, hCA IX-mimic structure has been published elsewhere; PDB ID: 4R5A¹⁶). The inhibitor was bound in the active site similarly to previously observed sulfamate compounds, interacting directly with the active site zinc and displacing the ordered water network.¹⁶ Additional density in the omit (Fo–Fc) electron density map was observed, indicating acetylation of lysine residues at positions 112 and 154 for hCA II and hCA IX-mimic, respectively [Fig. 2(A,B)]. These lysines were modified with terminal acetyl groups and refined, producing B-factors of 33.2 and 35.6 Ų for lysine 112 (hCA II) and 154 (hCA IX-mimic), respectively. These values are comparable to the B-factors for other side chain residues [Fig. 2(C,D)]. The positions of these lysine residues are clearly accessible for enzymatic acetylation as they both reside on the enzyme surface (Fig. 3). This observation also implies that acetylation of these surface lysine residues does not perturb crystallization.

Table 1.

X-Ray Crystallography Statistics

	hCA II PDB: 4ZWI
Space group Cell dimensions (Å;°)	P21 a = 42.2, b = 42.1, c = 72.3; β = 104.3
Resolution (Å)	20.0–1.60 (1.65–1.60)
Total reflections	28,923
Rsyma(%)	6.0 (43.5)
I/Iσ	20.5 (2.7)
Redundancy	3.5 (3.4)
Completeness (%)	97.8 (97.1)
Rcrystb(%)	17.9 (23.9)
Rfreec(%)	22.5 (27.6)
# of protein atoms	2145
# of water molecules	271
# of ligand molecules (Zn, inhibitor, DMSO)	1, 1, 7
Ramachandran stats (%): favored, allowed, generously allowed	96.5, 3.53, 0.00
Avg. B factors (Å2): main-chain, side-chain, solvent, ligand(s)	17.1, 21.2, 30.2, 43.6
rmsd for bond lengths, angles (Å,°)	0.006, 1.191

^aR_sym = (∑|I - |/∑ ) x 100.

^bR_cryst = (∑|F_o - F_c|/∑ |F_o|) x 100.

^cR_free is calculated in the same way as R_cryst except it is for data omitted from refinement (5% of reflections for all data sets).

Values in parenthesis correspond to the highest resolution shell.

Figure 2.

Electron density for acetylated lysine residues in hCA II and hCA IX-mimic (PDB ID: 4R5A¹⁶). Omit (Fo – Fc) difference electron density map (green, contoured at 3.0 σ), for acetyl group on (A) Lys112 (cyan) and (B) Lys154 (gray). 2Fo–Fc electron density (blue, contoured at 1.2 σ), for acetylated (C) Lys 112 (cyan) and (D) Lys154 (gray). Figure made using PyMol.²⁹.

Figure 3.

Surface lysines of hCA II (pink; depicted as sticks). Labeled (arrows) indicate the observed acetylation sites. Figure made using PyMol.²⁹

Global analysis of surface lysine residues of hCA II indicates there are a total of 24 possible acetylation sites, including positions 112 and 154 that were observed (Fig. 3). Of the 24 residues, 8 are disordered with incomplete electron density. Previous analysis of lysine acetylation sites recognized by acetyltransferases in E. coli (termed the acetylome, Fig. 1) has shown that surface lysines directly adjacent to tyrosine or histidine residues (N- to C-terminus) are highly favorable recognition sites.⁸ Based on this analysis, it would be predicted that surface lysines at positions 9, 39, 113, and 127 have the highest chance of being acetylated, but as previously indicated, acetylation was only observed at residues 112 and 154. The lack of observed acetylation events can be justified by structural analysis of residues surrounding the lysine in question and identifying possible interactions. Many of the surface lysine residues are predicted to form intrahydrogen bonds with nearby residues within the structure, making them inaccessible for acetylation by an acetyltransferase (Table2). Specifically, residue 127 is expected to form an intrahydrogen bond with Asp139 (bond distance = 3.2 Å). Analysis of both crystal structures also indicates there are several surface lysine residues (specifically residues 39, 111, 113, and 225) that likely form intermolecular crystal contacts (Table2). Therefore, the process of crystallization may act as a selective pressure that eliminates the observation of acetylated lysine residues- since crystal contact points are required for crystal formation, acetylation would impede protein crystallization. In addition, it is possible that more acetylation events occurred but are simply not observed in the crystal structure depending on the thermal motion of the lysine residue. Several surface lysine residues, including residue 9, are disordered and considered free in solution due to the lack of observable electron density for side-chain atoms. Interestingly, the observed acetylated lysine residues in hCA II and hCA IX-mimic are predicted to be unfavorable lysine recognition sites relative to the aforementioned positions. However, both surface residues 112 and 154 are ordered and do not have any predicted interactions in their respective structures, leaving them accessible for acetylation independent of adjacent residues.

Table 2.

Summary of Surface Lysine Interactions and Susceptibility to Acetylation

			hCA II		hCA IX-mimic
Res. #	Seq.	Propensitya	Ordered	Interactions	Ordered	Interactions
9	YGKHN	H	✗	–	✗	–
18	WHKDF	L	✓	His 15	✓	His 15
24	IAKGE	L	✓	H2O 125, His 17	✓	H20 46, His 17
39	TAKYD	H	✓	bGln 137	✓	bGln 137
45c	SLKPL	L	✗	–	✗	–
76	QDKAV	L	✓	Thr 155	✓	Thr 155
80	VLKGG	L	✗	–	✗	–
111	VDKKK	L	✓	bLys 257	✓	–
112	DKKKY	O1	✓	Acetylated	✓	bLys 127 carbonyl
113	KKKYA	H	✓	bH2O 157, His 133	✓	–
127	NTKYG	H	✓	Asp 139	✓	Asp 139
133c	FGKAV	L	✗	–	✗	–
149d	FLKVG	L	✗	–	✗	–
154d	SAKPG	O2	✓	H2O 141, Ser 217	✓	Acetylated
159c	LQKVV	L	✓	Glu 221, H2O 49	✗	–
168	SIKTK	L	✓	Glu 238	✗	–
170	KTKGK	L	✓	–	N/A	Mutated to Glu
172	KGKSA	L	✓	Glu 234	✗	–
213	VLKEP	L	✓	Asp 190	✓	Asp 190
225	VLKFR	L	✓	bAsn 178	✗	–
228c	FRKLN	L	✗	–	✗	–
252	PLKNR	L	✓	Lys 24 carbonyl	✓	Gly 25 carbonyl
257	QIKAS	L	✗	–	✗	–
261	SFK	L	✗	–	✗	–

^aH, high; L, low; O¹, observed in hCA II; O², observed in hCA IX-mimic.

^bCrystal contact.

^cLysine residue acetylated in hCA IX-mimic mass spec analysis.

^dLysine residue acetylated in hCA II mass spec analysis.

Mass spectrometry was also performed in order to analyze the acetylation of lysine residues for the purified hCA II and hCA IX-mimic protein samples (Supporting Information Table S1 and Fig. S1). For hCA IX-mimic, 38 unique peptides were identified with approximately 96% coverage, while hCA II had 39 unique peptides with 85% coverage. MS/MS data analysis indicated six acetylated lysine residues in hCA IX-mimic sample (45, 133, 149, 154, 159, and 228), signified by a +42 Da modification. In the hCA II sample, two acetylated lysine residues were identified (149 and 154) (Supporting Information Table S1). Acetylation of lysine 154 observed in the crystal structure of hCA IX-mimic was confirmed by mass spectrometry. However, acetylation of lysine 112 seen in the crystal structure of hCA II was not confirmed. The protein samples used to set up crystal trays were purified separately from those used for the mass spec analysis, so the acetylation of lysine residues may be dependent on preparation and possibly be affected by variable steps in the expression process, such as the time of E. coli growth pre- and post-induction.

The acetylation of surface lysine residues is predicted to have an effect on the isoelectric point (pI) and overall net charge of the enzyme. The pI of hCA IX-mimic is calculated to be 6.9 in the non-acetylated form and decreases to an estimated value of 5.3 with the six acetylated lysine residues (ExPASy Compute pI/Mw). Comparison of the electrostatic potential surface images shows a significant increase in negative electrostatic surface potential upon the acetylation of the six lysine residues indicated by mass spec (Fig. 4). In comparison, wild type hCA II has a predicted pI of 6.6 and is reduced in the presence of two acetylated lysine residues to an estimated pI of 6.0. Similarly, the acetylated electrostatic surface shows a decrease in positive potential upon acetylation (Fig. 4). The calculated pI values for wild type hCA II and hCA IX-mimic are similar in value, which is to be expected since these two proteins differ by seven active site mutations and only one mutation results in a change of charge (K170E).

Figure 4.

Electrostatic surface potential of (A) hCA II wild type (B) hCA IX-mimic (C) hCA II with two acetylated lysine residues observed in mass spec (D) hCA IX-mimic with six acetylated lysine residues observed in mass spec.

Previous studies have been performed analyzing this effect of lysine acetylation on enzyme surface charge and protein stability. Analysis of bovine CA II (bCA II) with acetylated surface lysine residues showed that a decrease in positive surface potential upon lysine acetylation destabilized bCA II in the presence of urea. Each individual acetylated lysine residue was observed to destabilize the enzyme by ∼0.2–0.5 kcal mol⁻¹, depending on the ionic strength of the buffer and extent of charged residue shielding by solvent ions. Acetylated bCA II was also more destabilized in guanidium chloride in comparison to wild type, but exhibited increased conformational stability in sodium dodecyl sulfate.¹⁷ Although the hCA II and hCA IX-mimic samples were not acetylated to the same extent as bCA II, the loss of positive surface charge upon lysine acetylation is predicted to have a similar effect on the stability of each isoform in the aforementioned denaturants.

We predict that the effect of surface lysine acetylation on conformational stability of hCA II is minimal based on Differential Scanning Fluorimetry (DSF) data that produces an overall melting temperature (T_M) that is close to previously published values (Supporting Information Fig. S2A,B).^18,19 Alternatively, we observe a ∼4°C decrease in T_M in hCA IX-mimic compared to hCA II, indicating a decrease in conformational stability (Supporting Information Fig. S2). The reason for this is unclear, especially since hCA IX-mimic is identical to hCA II except for seven active site substitutions (see Ref.16); however, according to the mass spec data, hCA IX-mimic has more acetylated surface lysines that are likely not detected in the crystal structure due to intrinsic disorder. This would explain the decrease in conformational stability in hCA IX-mimic compared to hCA II, similar to what was observed in the surface acetylated bCA II.¹⁷

The phenomenon of surface lysine acetylation of proteins expressed in E. coli has been observed previously and has been determined to be important for many physiological processes.^5,6,8 Therefore, when non-native proteins, including hCAs, are recombinantly produced in E. coli, there is a probability that they may undergo non-natural acetylation of surface lysines, such as those observed at positions 112 and 154 of hCA II and hCA IX-mimic, respectively (Fig. 3). It is unknown, however, if the same surface lysine acetylation occurs when the enzyme resides in human tissues or if the acetylation of surface lysines on hCAs has any biological significance. Therefore, it can be concluded that non-natural surface lysine acetylation can occur in proteins produced in E. coli, including hCAs. Furthermore, the acetylation of single surface lysine residues is predicted to decrease the pI and surface charge of the enzyme, decreasing its stability in the presence of multiple denaturants.

Materials and Methods

Protein expression and purification

The design and engineering of hCA IX-mimic has been discussed previously.^16,20 In brief, hCA IX-mimic is hCA II with amino acid substitutions A65S, N67Q, E69T, I91L, F131V, K170E, and L204A to “mimic” the active site of hCA IX; the surface of hCA IX-mimic is identical to hCA II. hCA II and hCA IX-mimic were expressed in BL21(DE3) E. coli cells as previously reported.^16,20 Briefly, E. coli cells containing the plasmid encoding for either hCA II or hCA IX-mimic were grown in Luria broth supplemented with 100 µg/mL ampicillin to an OD₆₀₀ of 0.6 at which point the expression of either hCA II or hCA IX-mimic was induced by addition of isopropyl β-d−1-thiogalactopyranoside for ∼4 h at 37°C. Cells were then harvested and enzymatically lysed. Both hCA II and hCA IX-mimic were purified in one step by affinity chromatography using an agarose resin coupled to the inhibitor p-(aminomethyl)benzenesulfonamide (p-AMBS; Sigma). Elution of each enzyme was done using sodium azide, followed by buffer-exchange against 50 mM Tris-HCl pH 7.8, and concentration using centrifugation. The final protein concentration was determined by UV/Vis spectroscopy at 280 nm, and measured 55 and 43 mg mL⁻¹ for hCA II and hCA IX-mimic, respectively. Purity was estimated by SDS-PAGE and Coomassie-blue staining.

Crystallization and X-ray data collection

Purified hCA II and hCA IX-mimic were crystallized in 1.6 M Na-Citrate, 50 mM Tris, pH 7.8 using hanging drop vapor diffusion.^20,21 Crystals were observed after 5 days for both hCA II and hCA IX-mimic. Crystals were then soaked with ∼25 mM stock solution of the inhibitor for 24 h prior to data collection. X-ray diffraction data was collected “in-house” using an RU-H3R rotating Cu anode (λ = 1.5418 Å) operating at 50 kV and 22 mA utilizing an R-Axis IV⁺⁺ image plate detector (Rigaku) with a crystal-to-detector distance of 100 mm, a 1° oscillation angle, and an exposure time of 5 min per image. A total of 180 images were collected. The data were indexed, integrated, and scaled using HKL2000.²² Data for both hCA II and hCA IX-mimic were scaled to the monoclinic P2₁ space group to ∼1.6 Å resolution (Table1, hCA IX-mimic structure has been published elsewhere¹⁶).

Structure determination and refinement

The structures of hCA II and hCA IX-mimic were determined using molecular replacement (MR) with the crystal structure of hCA II (PDB ID: 3KS3)²³ as a search model. MR solutions were calculated using PHENIX.^24,25 The starting phases of both hCA II and hCA IX-mimic yielded a unique solution comprising of monomeric asymmetric units. Refinement and generation of chemical restraints files were also completed using PHENIX.²⁵ Each refinement was completed with 5% of the unique reflections selected at random and excluded to calculate R_free.²⁶ Generation of acetylated lysine residue coordinate files and manual refitting of the models between each refinement were completed using Coot.^27,28 Restraints files for the acetylated lysine residues were generated using PHENIX by simply treating the acetylated lysines as ligands.²⁵ The final model of hCA II was refined to an R_cryst of 17.9% and R_free of 22.5% (Table1). The model of hCA IX-mimic was previously refined to R_cryst of 15.5% and R_free of 18.5%.¹⁶ All figures were made using PyMOL.²⁹

Mass spectrometry

Bands were extracted for hCA II and hCA IX-mimic from an SDS-PAGE gel after purification and sequenced using mass spectrometry analysis. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.4.1). Mascot was set up to search for hCA with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 10.0 ppm. Carbamidomethyl of cysteine was specified in Mascot as a fixed modification. Gln->pyro-Glu of the N-terminus, deamidated of asparagine and glutamine, and oxidation of methionine were specified in Mascot as variable modifications.

Scaffold (version Scaffold_4.3.2, Proteome Software, Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 85.0% probability by the Peptide Prophet algorithm with Scaffold delta-mass correction.³⁰ Protein identifications were accepted if they could be established at greater than 85.0% probability and contained at least one identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm.³¹ Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters.

Differential scanning fluorimetry

DSF was used to assess protein conformational stability in terms of melting temperature (T_M). Samples of purified hCA II and hCA IX-mimic at a concentration of 0.25 mg/mL were incubated with 0.01% Sypro-Orange dye (no. S6651; Invitrogen) for ∼30 min on ice prior to data collection. Both hCA II and hCA IX-mimic were in 50 mM Tris-HCl, pH 7.8. Melting curve assays were conducted in a quantitative PCR (qPCR) instrument (RG-3000; Corbett Research) with temperature ramping from 30 to 99°C, increasing at a rate of 0.1°C every 6 s. Runs containing only buffer were also conducted in the same manner to be used to subtract background signal during data processing. The melting temperature (T_M) was defined as the maximum value of the first derivative (dRFU/dT; change in fluorescence/change in temperature) of the signal which is produced in terms of relative fluorescent units (RFU). Results are summarized in Supporting Information Figure S1.

Supporting Information

Additional Supporting Information may be found in the online version of this article.

Supporting Information