Structural basis for the hyperthermostability of an archaeal enzyme induced by succinimide formation

Abstract

Stability of proteins from hyperthermophiles (organisms existing under boiling water conditions) enabled by a reduction of conformational flexibility is realized through various mechanisms. A succinimide (SNN) arising from the post-translational cyclization of the side chains of aspartyl/asparaginyl residues with the backbone amide -NH of the succeeding residue would restrain the torsion angle Ψ and can serve as a new route for hyperthermostability. However, such a succinimide is typically prone to hydrolysis, transforming to either an aspartyl or β-isoaspartyl residue. Here, we present the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase and, using enhanced sampling molecular dynamics simulations, address the mechanism of its increased thermostability, up to 100°C, imparted by an unexpectedly stable succinimidyl residue at position 109. The stability of SNN109 to hydrolysis is seen to arise from its electrostatic shielding by the side-chain carboxylate group of its succeeding residue Asp110, as well as through n → π^∗ interactions between SNN109 and its preceding residue Glu108, both of which prevent water access to SNN. The stable succinimidyl residue induces the formation of an α-turn structure involving 13-atom hydrogen bonding, which locks the local conformation, reducing protein flexibility. The destabilization of the protein upon replacement of SNN with a Φ-restricted prolyl residue highlights the specificity of the succinimidyl residue in imparting hyperthermostability to the enzyme. The conservation of the succinimide-forming tripeptide sequence (E(N/D)(E/D)) in several archaeal GATases strongly suggests an adaptation of this otherwise detrimental post-translational modification as a harbinger of thermostability.

Significance

Succinimidyl (SNN) residues, formed as intermediates during the post-translational deamidation and dehydration of asparaginyl and aspartyl residues in proteins, are prone to hydrolysis and are thus generally unstable. This intermediate is restricted in conformational freedom and, if stabilized, can considerably impact protein stability. Here, we present the structure of a stable, succinimide-harboring glutamine amidotransferase (GATase) from Methanocaldococcus jannaschii, an archaeon surviving under high pressure (200 atm) and high temperature (85°C) conditions. Using molecular dynamics simulations, we identify the key interactions that guard the succinimide from hydrolysis, which in turn imparts thermostability to the enzyme. The conservation of the SNN-harboring sequence motif in GATases of thermophiles hint that stable succinimides could be a widespread mechanism of thermostability.

Introduction

Succinimide formation by intramolecular cyclization at Asx/Xxx (Asp/Asn, Gly) segments in proteins is an intermediate step in the process of deamidation, which often accompanies aging (1). Succinimidyl/aminosuccinyl (Asu, succinimide, or SNN) residues are unstable, readily hydrolyzing under aqueous conditions to Asp and β-Asp (isoAsp) residues (2, 3, 4). Facile stereo inversion at the C^α atom of the succinimide results in the formation of D-Asp, often used as a marker of aging (4). Stable succinimides are uncommon in proteins, with relatively few-well characterized examples (5, 6, 7, 8, 9). We recently established mass spectrometrically the presence of a stable succinimide in the hyperthermophilic protein glutamine amidotransferase, a subunit of the enzyme GMP synthetase from the archaeon, Methanocaldococcus jannaschii. This post-translational modification (PTM) at Asn109 confers remarkable thermal stability on the WT protein, which resists unfolding up to 100°C. Mutants lacking the succinimide melted at appreciably lower temperatures (10). Many structural features have been implicated in conferring stability to proteins from thermophilic organisms. These include shortening or deletion of loops (11), increased occurrence of proline residues (12, 13, 14), increased number of hydrogen bonds and disulphide bridges (14, 15, 16, 17), and post-translational modifications such as glycosylation (18) and methylation (19). The observation of succinimide-induced hyperthermostability in M. jannaschii glutamine amidotransferase (MjGATase) prompted us to investigate the structural origins of this phenomenon. Toward this end, we determined the crystal structure of MjGATase in both apo and ligand-bound forms, revealing that the succinimide residue (SNN109) lies at the solvent-exposed tip of a loop in the protein, with the succeeding residue Asp(D)110 shielding its one face from hydrolytic attack by water while its other face is shielded by the backbone carbonyl group of the preceding residue Glu(E)108 through n → π^∗ interaction. Structure determination of the D110G mutant showed an absence of electron density for residues 108–110, suggesting enhanced flexibility in the absence of the succinimide constraint. In contrast, the structure of N109P mutant showed that the absence of key SNN-mediated interactions present in the WT protein could be the cause for impaired thermostability of the mutant. To probe the origins of protein stabilization by the succinimidyl residue, we turned to molecular dynamics (MD) simulation, which has been widely used to delineate functional dynamics of proteins (20, 21, 22, 23, 24, 25, 26, 27), mechanisms of protein folding/unfolding pathways (28, 29, 30, 31, 32, 33, 34, 35) and protein stability mechanisms (31, 32, 33, 34,36, 37, 38, 39, 40). MD simulations coupled with replica exchange with solute (enzyme) scaling (REST2) (41), a protocol that mitigates the problem of insufficient sampling of conformational space (42,43), and well-tempered metadynamics (WT-MTD) (44) simulations have been used herein to study the difference in the thermal stabilities of the WT enzyme and the single-site mutants N109S and N109P (referred to as SNN109S and SNN109P hereafter), which lack the succinimide. REST2 simulations highlight the role of a series of backbone hydrogen bonds facilitated by the succinimide constraint, which imposes a local conformational lock. Additional long-range side-chain hydrogen bonds and van der Waals interactions involving residues 109 and 110 contribute to succinimide-induced stabilization. Furthermore, free energy calculations using the WT-MTD technique on WT and the SNN109S mutant reinforce our observation on the mechanism of succinimide-induced conformational lock in imparting a higher thermostability.

Materials and methods

Generation of SNN109P mutant of MjGATase

The SNN109P mutant was generated using standard procedures as described in the Supporting materials and methods and confirmed by DNA sequencing.

Purification of MjGATase_WT and mutant proteins

The genes for WT and mutant enzymes were cloned in pST39 expression vector, and proteins were expressed in Rosetta (DE3) pLysS Escherichia coli overexpression strain. Proteins were purified following protocols similar to that used for MjGATase WT (10), described in detail in the Supporting materials and methods.

Mass spectrometric analysis of MjGATase_SNN109P and its tryptic digest

Mass spectra of purified MjGATase_SNN109P were acquired using a Q Exactive HF orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Details of the method are provided in the Supporting materials and methods.

CD

Circular dichroism (CD) was performed to compare the structure and stability of WT and SNN109S with SNN109P following the standard protocol detailed in the Supporting materials and methods.

Crystallization, data collection, structure solution, and refinement

Crystallization of WT and mutants was set up under a 1:1 mixture of silicon and paraffin oil using the microbatch method (45), and crystals were obtained only at 4°C. X-ray diffraction data on all crystals were collected using a Rigaku RU200 x-ray diffractometer (Tokyo, Japan) equipped with a rotating anode-type light source with an osmic mirror that gives a monochromatic light source of wavelength 1.54179 Å. Diffraction images were processed using iMOSFLM (46). Structure solutions were obtained using a molecular replacement method using the Phaser module of CCP4 (47). For the WT crystal, the structure of GATase from Pyrococcus horikoshii (Protein Data Bank (PDB): 1WL8) was used as template. For refinement of the structures, Refmac (48) module of CCP4 and AutoBuild module of Phenix (49) were used. Details of crystallization, data collection, and structure solution and refinement are provided in the Supporting materials and methods.

Interaction potential, or force field, of succinimidyl residue

The force field parameters of succinimidyl residue were extracted from existing database of structurally similar molecules and benchmarked against crystal structures as well as quantum chemical and classical mechanical simulations. Details of force field parameterization and the developed parameters are provided in the Supporting materials and methods.

Regular MD simulation

MD simulations for WT MjGATase, WT P. horikoshii GATase (PhGATase), and SNN109S, SNN109P, and D110G mutants of MjGATase were performed at 27°C for 300 ns each. Details of the simulation protocol are provided in the Supporting materials and methods.

REST2 followed by regular MD simulation

REST2 (41) simulations were performed separately for the WT MjGATase and its SNN109S and SNN109P mutants (starting from the last frame of the corresponding regular MD simulation) with 28 replicas for each system in the effective temperature range 47–327°C for the tempered region (protein) while the solvent was still in reference temperature of 47°C for all replicas. The replicas were allowed to exchange every 10 ps. The average exchange probability between the successive replicas was found to be 35%.

After 250-ns-long REST2 simulation, the last frame of the fourth and sixth replicas (with REST2 “effective” temperatures of 70 and 87°C, respectively) were warmed up gradually to 70 and 87°C, respectively. Regular MD was then performed for 300 ns for these systems at these temperatures. The analyses presented here are from these 200–300 ns portion of the normal MD trajectories, after the REST2 run. All the figures presented as snapshots from MD simulation were taken from the conformation of the central member of the most predominant cluster obtained from cluster analysis. Details of the REST2 simulation protocol and validation are provided in the Supporting materials and methods.

WT-MTD simulations

The free energy profile for WT MjGATase and its SNN109S mutant with respect to the radius of gyration (collective variable) of the residues from the succinimide-containing loop (residues 108–115) was reconstructed from 2200-ns-long WT-MTD (44) at 27°C. Details of the WT-MTD simulation protocol and validations are provided in the Supporting materials and methods.

NBO analysis

Energetics of the n → π^∗ interactions and n → σ^∗ interactions (hydrogen bond) contributing toward the stabilization of succinimidyl residue were obtained through natural bond orbital (NBO) analysis (50) on the quantum-optimized (only electronic level, keeping the atomic position fixed) structure of the polypeptide sequence 108–112 extracted from the crystal structure of WT MjGATase and from the structure obtained from the cluster analysis over the post-REST2 equilibrium MD run at 87°C. Details of this analysis are provided in the Supporting materials and methods.

Structure analysis and figure rendering

Figures of three-dimensional structures were drawn with PyMol (DeLano Scientific, https://pymol.org/2/) (51).

Results

Analysis of the structure of MjGATase and site-specific mutants

Crystals of MjGATase (space group P32, 2 molecules/asymmetric unit) yielded structures of both apo and ligand (acivicin)-bound forms, to a resolution of 1.67 Å, by molecular replacement, using PhGATase (PDB: 1WL8) as a model. Data collection and refinement statistics are reported in Table 1. The overall fold of the molecule and the position of the segment with respect to the succinimidyl residue along with the electron density corresponding to this post-translational modification are shown in Fig. 1 a. A common feature of the class I amidotransferases is the central region comprising seven β-strands surrounded by α-helices and loops (52). This structural feature is conserved across eukaryotes, prokaryotes, and archaea, as seen from the superposition of the structure of MjGATase with GATases of GMP synthetase from seven representative organisms (Fig. S1 a). The succinimide-containing segment, residues 108–110 (Glu-Asu(SNN)-Asp), lies at the solvent-exposed tip of the loop (residues 106–114) connecting the two antiparallel strands (residues 97–105 and 115–124) of a β-hairpin (Fig. 1 c). The segment 108–110 forms a local α-turn structure stabilized by a 13-atom hydrogen bond, Glu108 CO-HN Phe112 (Fig. 1 b) (53, 54, 55). The catalytic residues Cys76, His163, and Glu165 are proximate to the succinimide-containing loop. Active site occupancy is without any effect on the succinimide-containing loop as established by the determination of the structure of an acivicin (5CS)-modified enzyme (Fig. S1 b). Acivicin is a glutamine analog that irreversibly modifies the active site cysteine residue (56,57).

Table 1.

Summary of x-ray crystallographic data collection and refinement statistics

Property		MjGATase	MjGATase with acivicin		MjGATase D110G	MjGATase N109P
Data collection statistics
Resolution range (Å)		56.53–1.67	56.59–1.67	50.99–2.29		35.49–1.89
Wavelength (Å)		1.54179	1.54179	1.54179		1.54179
Space group	P 32		P 32	P 21 21 21		P1
Unit cell parameters
a, b, c	65.28 Å, 65.28 Å, 97.14 Å		65.35 Å, 65.35 Å, 97.37 Å	36.18 Å, 66.65 Å, 79.18 Å		35.50 Å, 68.05 Å, 75.35 Å
α, β, γ	90.00°, 90.00°, 120.00°		90.00°, 90.00°, 120.00°	90.00°, 90.00°, 90.00°		90.95°, 90.47°, 91.45°
% completeness	99.7 (83.6)		99.7 (89.5)	91.2 (97.8)		96.8 (73.3)
Average mosaicity (°)	0.60		0.80	0.50		0.80
Total reflections	11,997 (42,843)		11,242 (41,922)	1248 (5936)		3362 (11,939)
Unique reflections	1730 (6644)		1737 (7137)	299 (1258)		1628 (5717)
Multiplicity	6.9 (6.4)		6.5 (5.9)	4.3 (4.7)		2.1 (2.1)
Number molecules/asymmetric unit	2		2	1		4
(I/σ(I))	20.7 (6.0)		19.9 (4.1)	10.2 (4.1)		5.3 (3.4)
CC1/2	0.973 (0.766)		0.981 (0.858)	0.970 (0.881)		0.988 (0.702)
Rmeas	0.134 (0.303)		0.094 (0.371)	0.143 (0.396)		0.089 (0.321)
Rmerge (%)	12.4 (27.8)		8.6 (33.8)	12.6 (35.5)		6.5 (23.5)
Wilson B factor (Å2)	18.5		21.4	19.1		12.4
Refinement statistics
Rwork/Rfree	0.1610/0.2144		0.1993/0.2350	0.2797/0.2884		0.2041/0.2640
Average B, all atoms (Å2)	23.0		25.0	18.0		14.0
Number of atoms
Protein	3482		3206	1495		6253
Ligand	0		0	0		0
Number of non-H atoms
Protein	2895		2940	1421		5668
Ligand	0		0	0		0
Water	524		266	54		585
Root mean square deviation
Bond angle (°)	2.2051		2.0224	1.6129		1.6245
Bond length (Å)	0.0240		0.0124	0.0128		0.0082
Residues in Ramachandran plot
Favored	358 (98%)		360 (99%)	176 (97%)		705 (96%)
Allowed	7 (2%)		4 (1%)	5 (3%)		27 (4%)
Outliers	1 (0%)		0	0		6 (1%)
PDB	7D40		7D95	7D96		7D97

Values for the outer shell are given in parentheses.

Figure 1.

Interactions of SNN109 in MjGATase. (a) Structure of MjGATase. Inset: Succinimidyl residue with electron density (2F₀-F_c, contoured to 1σ). (b) SNN-harboring loop bends to form overlapping β-turn (3.8 Å hydrogen bond) and α-turn (3.1 Å hydrogen bond). (c) SNN is connected to active site and distal β-sheet residues through tertiary contacts. SNN-containing β-hairpin residues, green; active site residues, purple; and residues mediating tertiary contacts, cyan. (d) Residues in the vicinity of SNN (4 Å distance cutoff). To see this figure in color, go online.

The network of contacts illustrated in Fig. 1 c highlight close interactions between residues flanking the succinimide moiety and the segment 151–165 containing two active site residues. Key intersegment contacts are the hydrogen bonds between the side chains of Asp110 and Tyr158 and the van der Waals contacts between SNN109 and the side chains of Lys151, Leu111 and Val160, and Phe112 and Phe162 (Table S1). The stabilizing effect of the succinimide on the temperature dependence of MjGATase activity has been previously noted, with the WT enzyme showing stability and activity up to ∼100°C, whereas mutants lacking this PTM lose activity by ∼85°C (10). The unusual stability of the succinimide residue may be rationalized by the network of contacts shown in Fig. 1 d. The carboxymethyl side chain of Asp110 folds over one face of the five-membered succinimide ring, whereas the carbonyl group of Glu108 protects the opposite face (Figs. 1 d and 3 a). Interestingly, mutation of Asp110 to Gly increases the flexibility of this tripeptide segment, resulting in the absence of electron density for the residues E108-G110, whereas the rest of the structure overlaps well with that of WT (Fig. S1, c and d). The Glu108 CO-SNN109 CO contacts of 2.7 and 3.3 Å lie within the ranges established by Raines and co-workers for stabilizing n → π^∗ interactions in biomolecules (58, 59, 60). An overall stabilization energy of 11.7 kJ/mol for the interaction of Glu108 CO with the CO groups of the succinimide is estimated from NBO analysis (50) (Figs. 3, b and c, and S2).

Figure 3.

Figure360 Shielding of SNN from hydrolysis.

For a Figure360 author Presentation of this Figure, see https://doi.org/10.1016/j.bpj.2021.07.014

(a) Crystal structure of MjGATase: the side chain of Asp110 shields the front end of SNN from hydrolysis. SNN is shielded from the rear end through n → π^∗ interactions between the backbone carbonyl oxygen of Glu108 to backbone (b) and side-chain (c) carbonyl carbons of SNN 109. Except for SNN, only backbone atoms (NH-C_α-CO) for the rest of the residues are shown (63). (d) Three water molecules in the vicinity of SNN in the crystal structure of MjGATase are present in the structure of PhGATase. (e) Schematic representation of the distances and angles used in (f) and (g), which describe results of MD simulations. θ₁ is the angle between the side-chain carboxylate plane of Asp 110 (C_γ-O_δ1-O_δ2 plane) and succinimide plane (C-N-C_γ plane). r₁ is the distance between the N and C_γ atoms of Asp110 (i.e., distance between these two planes). r₂ represents distance of oxygen of water molecules within 4 Å of C (or C_γ) of SNN, and θ₂ is the angle between SNN-plane and the line joining this C (or C_γ) atom to such oxygen atoms. Probability distributions P(r₂, cosθ₂) showing the approach of water toward C (f) and C_γ (g) atoms of SNN are given. The black dotted rectangles highlight the regions favorable for water attack on C or C_γ (θ₂ around 90°). To see this figure in color, go online.

Removal of the succinimide-induced conformational restraint in the SNN109S mutant results in a reduction of stability. Introduction of a different restraint in the form of a prolyl residue at position 109 in the SNN109P mutant resulted in greater loss of stability. As expected, the WT and mutants showed similar far-UV CD spectra at 25°C (Fig. 2 a), but a variation in thermal stability was clearly evident, WT being stable at 100°C and SNN109P exhibiting a T_m of 78°C, the lowest among all the mutants studied (Fig. 2 b). Notably, a search of the sequence database yielded 84 nonredundant archaeal sequences, belonging to either thermococci or methanococci, in which a very high degree of conservation was noted for residues 108–110, 139, 151, and 158 (numbering follows MjGATase) (Fig. S4, a and b). Although the overall structure of MjGATase_SNN109P mutant overlays well on the WT structure with a root mean square deviation (RMSD) of 0.390 Å (Fig. S3 a), the SNN loop shows a movement of the backbone that is reflected as a large change in the Φ, Ψ values of residues 107, 108, and 109 (Figs. 2, c and d, and S3 b). As a consequence, hydrogen bonds involving Glu108 CO are absent in MjGATase_SNN109P, leading to the loss of α- and β-turns (Fig. 2 e), but the polypeptide chain in MjGATase_SNN109P bends in a manner leading to hydrogen bonds between Pro109 CO-Phe112 NH (3.0 Å, β-turn) and Asp110 CO-Lys113 NH (3.1 Å, β-turn), resulting in the formation of two consecutive β-turns (Fig. 2 f). Furthermore, because of the different positioning of the side chain of Glu108, the salt bridge with Arg117 is broken in the mutant (Fig. S3 c).

Figure 2.

Impact of replacement of SNN with prolyl residue. (a) Far-ultraviolet CD spectra show secondary structure conservation at 25°C. (b) Molar ellipticity illustrates loss of secondary structure at higher temperatures for mutants. (c) Structural differences between WT (green) and SNN109P (purple) in loop region (residues 105–116). (d) Different paths traced by polypeptide 107–111. (e) Larger difference observed in conformation of side chains of residues 108–110. (f) α- and β-turns in WT (Fig. 1b) replaced with two consecutive β-turns formed by hydrogen bonding between Pro109 CO-Phe112 NH and Asp110 CO-K113 NH in MjGATase_SNN109P. (g) MjGATase_SNN109P structure revealed loss of specific SNN-mediated contacts. To see this figure in color, go online.

Pro109 shows overall fewer interatomic interactions compared to SNN109 (Fig. 2 g), including the loss of an hydrogen bond between K107 CO-NH of N109 (SNN) present in the WT, owing to the imino nitrogen of the prolyl residue. Apart from these local changes, the movement of the backbone in MjGATase_SNN109P has also resulted in loss of tertiary interactions of the SNN loop with the protein core. In the mutant, the electron density for the side chain of Lys107 is absent, whereas the backbone geometry in the WT permits the formation of a salt bridge between Lys107 and Glu137. The interaction between Lys107 NH-Leu139 CO (3.1 Å in the WT) and van der Waals interactions of SNN with K151 and Y158 are lost in the mutant. In addition, the movement of Asp110 side chain COOH has resulted in a weakening of the interaction with Tyr158 OH (Fig. S3 d). Taken together, the absence of these interactions has dramatically reduced the thermal stability of the mutant.

Hydrolytic stability of the succinimide

Hydrolysis of the succinimide requires approach of a water molecule to either of its carbonyl group in a direction perpendicular to the plane of the ring (61,62). Both faces of the ring are sterically shielded from solvent approach, with the Asp110 side-chain carboxylate lying parallel to one face while the other is shielded by the Glu108 backbone carbonyl group (Fig. 3 a). However, at elevated temperatures, movement of the Asp110 side chain due to the population of alternative rotamers may permit the close approach of a water molecule. Three water molecules are observed in the vicinity of the succinimide in the crystal structure (Fig. 3 d). Notably, all the three molecules are conserved in the structure of the P. horikoshii enzyme (PhGATase, PDB: 1WL8). None of the water molecules meet the orientational requirements for hydrolytic attack. We therefore turned to MD simulations as a function of temperature to examine the possibility of side-chain and water reorientation in the vicinity of the succinimide. Fig. 3 e defines the angular parameters θ₁ and θ₂ and the distances r₁ and r₂, which describe the Asp110 side-chain orientation and the approach of the nucleophile to either of the succinimide carbonyl groups, respectively. MD simulation shows retention of the parallel orientation of Asp110 side-chain carboxylate group over the succinimide plane even at elevated temperature (Fig. S5 b; Video S1).

Video S1. Succinimide stability WT 27

Reduced flexibility of the side chain carboxylate of D110 and CO of E108 as visualized from MD simulation5

This rigid orientation of the side-chain carboxylate group of D110 arises from its strong hydrogen bond with the side-chain hydroxyl oxygen of Tyr158 from one of the core β-strands of MjGATase (Figs. 1 c and 5 a). Thus, irrespective of temperature, water hardly approaches either of the succinimide carbonyl groups in a direction perpendicular to the SNN ring plane (Fig. 3, f and g). In MD simulations of the homologous enzyme PhGATase, the parallel orientation of the side-chain carboxylate plane of the succeeding Glu (E113) residue shields the succinimide from the hydrolytic attack in a plane perpendicular to one face of the succinimide ring (Fig. S5, c and d). However, in MjGATase_D110G, the absence of the Asp side chain following the succinimide residue allows the approach of water in a direction perpendicular to the succinimide plane (Fig. S5 e), leading to the facile hydrolysis of the succinimide in this mutant. These findings reaffirm our experimental observation that Asp110 plays an important role in the stabilization of succinimide (10). Taken together, the experiments and MD simulations point to a key role for the Asp110 residue in stabilizing the succinimide modification at position 109.

Figure 5.

Bar graph of average number of hydrogen bond(s) (<N_{Hbond}>) from crystal structure and MD simulations. (a) <N_{Hbond}> between residues Asp110 and Tyr158 controlling the stability of succinimide. (b) <N_{Hbond}> describing the conformational lock in the SNN-loop region. (c) Average number of backbone hydrogen bonds between two β-strands forming the distal β-sheet connected to the β-hairpin containing succinimide. A donor-acceptor distance cutoff of 3.5 Å and donor-acceptor-hydrogen angle cutoff of 40° has been used to define a hydrogen bond. The number of hydrogen bonds in the x-ray crystal structure of WT MjGATase was calculated based on only donor-acceptor distance criterion. For crystal structure, it is not an average number. The numerical values of the bar graphs are provided in Table S2. To see this figure in color, go online.

Succinimide induces formation of a conformational lock, limiting loop flexibility

MD simulations provide an opportunity to examine the impact of side chain-backbone cyclization on the conformational flexibility of loop containing the succinimide modification. Experimental studies have established that the succinimide-lacking MjGATase mutants SNN109S (10) and SNN109P are much less stable than the WT enzyme, precipitating at significantly lower temperatures (Fig. 2 b). Diffraction quality crystals could not be obtained for the SNN109S mutant despite considerable effort. The structure of SNN109S was therefore modeled in PyMol using the MjGATase structure as a template and energy minimized. The WT, SNN109S, and SNN109P mutant structures were then subjected to REST2 MD simulations with replicas ranging over effective temperatures between 47 and 327°C, followed by normal MD at temperatures of 27, 70, and 87°C (see the Supporting materials and methods for details, including the necessity of REST2, which provides enhanced sampling over normal MD). In the WT enzyme, a strong 13-atom (C₁₃, 5 → 1) hydrogen bond observed between Glu108 CO and Phe112 NH is maintained at temperatures up to 70°C, indicative of the role of succinimide in stabilizing the α-turn motif. Interestingly, at 87°C, there is a local conformational transition with population of type II′ β-turn structures, stabilized by a 10-atom (C₁₀, 4 → 1) hydrogen bond between the backbone CO of the succinimide ring and the Phe112 NH groups (Figs. 4 a and 5 b). At 87°C, both Asp110 and Leu111 populate a second distinct conformational state, corresponding to the formation of a type II′ β-turn centered at residues 110–111 (Fig. 4 d). The succinimide-containing segment is thus conformationally constrained, restricting loop flexibility, even at the highest temperature examined through MD simulation. In the mutant SNN109S, the dominant conformations of the loop segment involve a β-turn hydrogen bond between S109 CO and Phe112 NH groups. This interaction is almost completely lost at 87°C (Figs. 4 b and 5 b). We refer to these hydrogen bonds resulting in α-turn and β-turn motifs as the “conformational lock.” In SNN109P mutant as well, the dominant conformation at 27°C involves β-turn hydrogen bonds between P109 CO and Phe112 NH and D110 CO and K113 NH groups, and these interactions are completely lost at higher temperatures (Figs. 4 c and 5 b).

Figure 4.

The contributions of conformational lock as seen from simulations. (a) MD simulations show that the conformational lock formed by the cyclization of residue at position 109 in MjGATase_WT is maintained at all temperatures although in different forms. β-turn exists in the structure at 87°C. (b) The hydrogen bonds that form α- and β-turns in the WT protein are replaced by a new set of hydrogen bonds in the SNN109S mutant. At 27°C, the hydrogen bonds between Ser109 CO-Phe112 NH and Asp110 CO-Lys113 NH constitute two β-turn structural motifs. However, these bonds are weakened at 70°C and are lost at 87°C. (c) For SNN109P mutant, the hydrogen bond between Pro109 CO-Phe112 NH constitutes a β-turn structural motif at 27°C, which disappears at higher temperatures. The percentile strength of the conformational lock measured as the probability of its occurrence in the corresponding simulation (Fig. 5b) is mentioned in (a)–(c). (d) The Φ, Ψ values (at three different temperatures) of residues around succinimidyl (WT), seryl (MjGATase_SNN109S), and prolyl (MjGATase_SNN109P) residues are plotted on Ramachandran map for comparison. Fig. S8 provides a comparison of the degree of dynamics with respect to crystal structure for residues 107–111 in WT, SNN109S, and SNN109P mutants at 27°C. To see this figure in color, go online.

Fig. 4d compares the effect of temperature on the backbone torsion angles of the residues in the pentapeptide segment of the succinimide-containing loop, residues 107–111, for the WT enzyme and the mutants. It is evident that in case of SNN109S and SNN109P, even at 70°C, the Glu108, Ser109/Pro109, and Asp110 show considerable conformational excursions compared to the WT enzyme, as seen from the Ramachandran plots. The stability of the folded form of the loop consisting of residues 105-117 is considerably diminished when the backbone conformational constraint at position 109 is through a prolyl residue, in contrast to that achieved via the formation of a succinimidyl residue. The role of the succinimide-induced “conformational lock” was further probed using WT-MTD simulations at 27°C, by applying a biasing potential on the radius of gyration (Rg) of residues in the segment 108–115, containing the succinimide. This procedure promotes the breaking and forming of the “conformational lock” several times, thereby boosting the sampling of conformational space of this region of the polypeptide chain to an extent greater than that through the REST2 protocol. However, this calculation has not been performed for the SNN109P mutant, as the complete loss of conformational lock at higher temperatures suggests an efficient conformational sampling for this system through REST2 itself (Fig. 5 b). Free energy profiles obtained from WT-MTD simulations show that the absence of the succinimide constraint in the SNN109S mutant allows this loop to exist in a locally unfolded conformation at a higher free energy (at the minimal corresponding to Rg-value ∼7 Å). In contrast, there is little effect on the local conformation of this segment in the WT enzyme (Fig. 6 a; Video S2). The free energy profiles in Fig. 6 a establish two distinct minima in the SNN109S mutant corresponding to the folded and less ordered conformations of the loop (Video S3). This observation is further supported by gas phase folding simulations of the unfolded 105–117 peptide segment with SNN at position 109, wherein it is seen to fold in a more compact manner with a β-turn hydrogen bond compared to that in the absence of succinimide (Fig. S6). Additionally, the MD simulations reveal that succinimide formation also has a long-range effect that limits the conformational lability of segments of the polypeptide chain distal to the site of modification.

Figure 6.

Short-range and long-range impacts of SNN on MjGATase structure, unraveled by simulations. (a) Free energy profiles with respect to the radius of gyration of residues near SNN (residues 108–115) calculated from WT-MTD simulations. Conformations at each of the minima are shown. Free energy minimal conformation of WT (green) shows a significantly good overlap with crystal structure (cyan). The SNN109S mutant can cross the free energy barrier to exist in a higher energy state in a completely unfolded conformation. (b) Overlay of the extended β-hairpin region of WT at 27 (green), 70 (pale brown), and 87°C (brown). (c) Overlay of the extended β-hairpin region of WT (green), MjGATase_SNN109S (blue), and MjGATase_SNN109P (magenta) mutants. To see this figure in color, go online.

Video S2. SNNloop metaD WT 27

The movie shows that the succinimide containing loop (residues 108-115) remains folded in MjGATase_WT during the well-tempered metadynamics simulation with the radius of gyration of succinimide loop as the reaction coordinate at 27 °C.6

Video S3. SNNloop metaD SNN109S 27

The movie shows that the loop region from 108-115 in MjGATase_SNN109S can switch between folded and unfolded states during the well-tempered metadynamics simulation with the radius of gyration of residues 108-115 as the reaction coordinate at 27 °C. This indicates that the loop in the mutant protein can unfold at higher temperatures.7

Fig. 6, b and c show the effect of increasing temperature on the polypeptide chain spanning residues 86–136. The hairpin formed by the flanking antiparallel β-strand segments residues 97–105 and 115–124, connected by the loop 106–114, is further connected by three and two residue loops, respectively, to a second antiparallel β-sheet segment encompassing the β-strands spanning residues 86–93 and 127–136 (distal β-sheets). The disruption of the antiparallel β-sheet distal to the succinimide loop segment is evident in the SNN109S and SNN109P mutants at 70 and 87°C (Figs. 5 c and 6 c). In contrast, the overall fold of the polypeptide chain is maintained at high temperatures in the WT enzyme (Figs. 5 c and 6, b and c; Videos S4, S5, S6, S7, S8, and S9). A similar trend is reflected in the root mean-square fluctuation of residues and fraction of native contacts with respect to crystal structure obtained from simulations (Fig. S7). Inspection of Fig. 1 c suggests that long-range tertiary contacts involving the SNN109-Phe112 segment with Leu139, Lys151, Tyr158, and Phe162 residues from the core β-sheets may be the contributing factors in the transmission of the structure-stabilizing effect of the succinimide (Table S1).

Video S4. Distal β-sheet WT 27

200 - 300 ns timeframe from REST2 simulation on the extended β-hairpin (residues 86-136) of MjGATase_WT at 27 °C.8

Video S5. Distal β-sheet WT 87

200 - 300 ns timeframe from REST2 simulation on the extended β-hairpin (residues 86-136) of MjGATase_WT at 87 °C.9

Video S6. Distal β-sheet SNN109S 27

200 - 300 ns timeframe from REST2 simulation on the extended β-hairpin (residues 86-136) of MjGATase_SNN109S at 27 °C.10

Video S7. Distal β-sheet SNN109S 87

200 - 300 ns timeframe from REST2 simulation on the extended β-hairpin (residues 86-136) of MjGATase_SNN109S at 87 °C.1

Video S8. Distal β-sheet SNN109P 27

200 - 300 ns timeframe from REST2 simulation on the extended β-hairpin (residues 86-136) of MjGATase_SNN109P at 27 °C.2

Video S9. Distal β-sheet SNN109P 87

200 - 300 ns timeframe from REST2 simulation on the extended β-hairpin (residues 86-136) of MjGATase_SNN109P at 87 °C.3

Discussion

Proteins from thermophiles have been shaped by the selective pressures of evolution to acquire structural features that determine their stability at temperatures as high as 80–120°C. Despite the accumulation of a large body of structural and mutational data on proteins from hyperthermophiles, unambiguous correlates between sequence and conformational motifs contributing to thermal stability remain elusive (64, 65, 66). In the case of MjGATase being analyzed in this report, succinimide formation by side-chain backbone cyclization in an exposed loop segment contributes to hyperthermostability. In the absence of the backbone constraint, the SNN109S mutant is stable up to a temperature of 85°C. Locking residue 109 into the five-membered succinimide ring freezes the backbone torsion angle ψ to a value of −128.8°, placing it in the bottom right quadrant of the Ramachandran Map, a region of conformational space energetically unfavorable for L-amino acid residues in proteins. The promotion of intramolecular backbone hydrogen bonds at the loop tip (residues 106–114) is clearly a factor limiting loop dynamics as evidenced from the REST2 and WT-MTD simulations. Additionally, an important tertiary nonbonded contact is established between the Leu139 side-chain methyl group and the CβH₂ of the Asn109 residue, which is now rigidly held within the succinimide ring. Long-range tertiary contacts involving the SNN109-Phe112 segment with Leu139, Lys151, Y158, and F162 residues serve to transmit the structural stabilization, imposed by post-translational succinimide formation, to the distal active site segment. In proteins, the L-Pro residue is the sole amino acid in which a backbone cyclic constraint limits the N-C^α torsion angle (ϕ) to values close to −60°, a feature that promotes the formation of locally folded β-turn conformations (67). The SNN109P mutant harboring a prolyl residue in place of succinimide that was generated in this study showed remarkably lower thermal stability than the WT enzyme. Although the segment corresponding to SNN loop in the crystal structure of the mutant displayed the presence of two consecutive β-turns, at higher temperatures, it was unable to retain the conformational lock stabilizing the peptide segment in a folded state, as evidenced from the MD simulations. Furthermore, many contacts of the SNN loop with the core of the protein that play a key structure-stabilizing role in the WT are absent in MjGATase_SNN109P. This observation indicates that although proline can decrease the conformational entropy of unfolding (68), this entropic contribution is masked by decreased enthalpic contributions as a consequence of loss of van der Waals and electrostatic interactions. Together, these results emphasize that the presence of a succinimidyl modification provides a superior constraint both entropically and enthalpically and that the structure-stabilizing role of a specific amino acid residue is dependent on the location of that residue and its environment in the protein structure. Structural studies of model peptides reveal that β II′ conformations are favored in L-Asu peptides, with the Asu residue occupying the i + 1 position of the turn motif (69,70). In the MjGATase structure, the Asu (SNN)109 residue occupies the i + 1 position of an α-turn, a motif formed by the local conformations adopted by the succeeding residues Asp110 and Leu111. The recent demonstration of the effectiveness of a succinimidyl peptide as an inhibitor of taspase, a leukemia-related threonine protease (71), may stimulate further conformational analysis of succinimide-containing peptides. A very recent publication has also demonstrated the structure-stabilizing effect of an iso-aspartyl residue in MurA from Enterobacter cloacae, highlighting the emerging role of backbone protein modifications (72,73). As mentioned earlier, the observation of stable succinimides in proteins is uncommon and typically a slow process, with only a few examples available in the PDB (5, 6, 7, 8). In MjGATase, the crystal structure and the MD simulations taken together establish that steric and electrostatic shielding by the Asp110 residue on one face of the succinimide ring and by the backbone Glu108 CO (through potential n → π^∗ interactions with carbonyl groups of succinimide) on the other face preclude the approach of solvent water, thereby preventing hydrolysis (however, at 87°C, along with the electrostatic shielding by the side-chain carboxylate group of Asp110, the n → π^∗ interaction between the backbone CO of Glu108 and the CO groups of SNN109, which was ∼11.7 kJ/mol in the crystal structure, reduces to just 1.3 kJ/mol, providing a weak protection to the front end of the succinimide plane from water attack, whereas the water attack from the rear end of the succinimide plane is prevented by the protein itself). The hydrolytic instability of the D110G mutant demonstrated earlier (10) lends support to this interpretation. The key residues involved in interacting with the succinimide at position 109 are highly conserved in sequences of GATase from archaeal thermophiles, suggesting an evolutionary adaptation of sequence to meet the demands of thermal stability. The results presented above emphasize the utility of REST2 simulations in enhanced sampling of polypeptide conformational space, permitting examination of relatively minor structural changes on the unfolding of inherently stable proteins. A comparison of the simulations carried out on the WT enzyme and the mutants SNN109S and SNN109P reveals complete unfolding of the distal β-sheet formed by antiparallel strands, 86–93 and 127–136 in the mutant proteins. Interestingly, in the structure of MjGATase_SNN109P, the electron density corresponding to residues 93–95 (the loop region connecting distal β-sheets) is missing. Thus, both simulation and experiments suggest that the effects of changes in the SNN-harboring loop segment are not localized but are cascaded further into the protein. In contrast, in the WT enzyme, minor conformational changes are observed at 87°C, but the integrity of the structure over the core region 86–136 is maintained, affirming the role of the succinimide. As a caveat, it should be noted that although REST2 and WT-MTD simulations offer enhanced conformational sampling, a detailed view of the complete pathway of unfolding is beyond the scope of these simulations. Deamidation of Asn residues is a commonly encountered phenomenon contributing to “wear and tear” of proteins (74). The facile hydrolysis of the succinimide intermediate can lead to Asp or iso-Asp formation and stereochemical inversion, modifications that can be detrimental to activity (1, 2, 3, 4). The MjGATase example illustrates how protein sequences can adapt an apparently detrimental process to serve the function of enhancing thermal stability, under an evolutionary imperative.

Author contributions

A.V.D. and S.K. purified protein and obtained crystals. A.V.D., A.B., and S.K. collected data and solved structures. A.C. generated mutant, purified protein, and performed spectroscopic studies. S.D., T.K., and S.B. generated the force field for the succinimidyl residue. S.D. and S.B. carried out all simulation studies. A.V.D., P.B., and H.B. analyzed structures. A.V.D., S.D., P.B., S.B., and H.B. wrote the manuscript.

Editor: Alexandr Kornev.

Supporting citations

References (75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109) can be found in the Supporting material.

Supporting material

Table S1. Tertiary contacts measured by the average minimal distance between nonhydrogen atoms of corresponding residue pairs from MD simulations

Tertiary contacts are van der Waals (vdW) contacts and hydrogen bonds (H-bonds). These network of tertiary contacts correlates distal β-sheet from extended β-hairpin and active site residues with the residues present in succinimide containing loop. For WT-MTD, the position of free energy minimum with respect to radius of gyration (Rg) of residues 108-115 are mentioned within parenthesis and the average minimum distances are calculated over configurations around the corresponding free energy minimum (for WT: 5.36-5.56 Å; for SNN109S mutant, lower free energy minimum: 5.52-5.72 Å; for SNN109S mutant, higher free energy minimum: 6.90-7.10 Å). X denotes SNN, Ser and Pro, respectively for WT, SNN109S and SNN109P. ^∗For crystal structure, it is not an average number. The weakening of tertiary contact for SNN109S mutant compared to that of WT may not be found in WT-MTD for those contacts which does not involve residues within 108-115, as biasing force had been applied only to residues 108-115.

Table S2. Average number of hydrogen bonds controlling the stability of succinimide and describing the conformational lock in the SNN-loop region from MD simulations

Average number of backbone hydrogen bonds between two β-strands forming the distal β-sheet connected to the β-hairpin containing succinimide are also reported. For WT-MTD, the position of the free energy minimum with respect to radius of gyration (Rg) of residues 108-115 are mentioned within parenthesis and the average number of hydrogen bonds are calculated over configurations around the corresponding free energy minimum (for WT: 5.36-5.56 Å; for SNN109S mutant, lower free energy minimum: 5.52-5.72 Å; for SNN109S mutant, higher free energy minimum: 6.90-7.10 Å). A donor-acceptor distance cut-off of 3.5 Å and donor-acceptor-hydrogen angle cut-off of 40° has been used to define a hydrogen bond. Number of hydrogen bonds in the X-ray crystal structure of WT MjGATase was calculated based on only donor-acceptor distance criterion. For crystal structure, it is not an average number. This table has been represented as bar graphs in Figure 5 of the main article.

Figure360. An Author Presentation of Fig. 3

4