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Article

Temperature-Dependent Structural Changes of the Active Site and Substrate-Binding Cleft in Hen Egg White Lysozyme

by
Ki Hyun Nam
College of General Education, Kookmin University, Seoul 02707, Republic of Korea
Crystals 2025, 15(2), 111; https://doi.org/10.3390/cryst15020111
Submission received: 31 December 2024 / Revised: 18 January 2025 / Accepted: 20 January 2025 / Published: 22 January 2025
(This article belongs to the Section Biomolecular Crystals)
Browse Figures
Versions Notes

Abstract

:
Lysozyme plays a crucial role in the natural immune system, protecting against invading bacteria or viruses. The room-temperature (RT) structure of lysozymes is important for understanding accurate structural information compared to the crystal structure determined at cryogenic temperature. Several RT structures of lysozymes are determined by serial crystallography, but their temperature-dependent structural properties are not fully elucidated. To better understand the temperature-dependent structural change, the RT and cryogenic temperature structures of hen egg white lysozyme (HEWL) were determined by serial synchrotron crystallography (SSX) and macromolecular crystallography (MX), respectively. Structural comparisons of HEWLRT and HEWLCryo showed that the positions of the loops above the substrate-binding cleft of HEWL differed. The width of the substrate-binding cleft between the α- and β-domains of HEWLRT was wider than that of HEWLCryo. The distance between the two catalytic residues Glu53 and Asp70 and their interaction with neighbor residues and water molecules showed the distant between HEWLRT and HEWLCryo. Due to temperature, the subtle movements of the active site and substrate-binding cleft of HEWL led to different docking results for N-acetylglucosamine and N,N′,N″-triacetylchitotriose. These results will provide useful information to more accurately understand the molecular function of HEWL and insights into the temperature effects for ligand design.

1. Introduction

Lysozyme plays an important role in innate immunity and is found in animal tissues, saliva, tears, and egg whites, providing protection against bacteria, viruses, and fungi [1,2]. Lysozyme decomposes peptidoglycan, a major component of bacterial cell walls, with an alternating structure of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) [3]. Lysozyme hydrolyzes the β-1,4-glycosidic bond between NAM and NAG sugars [4], destroying the cell wall structure and causing the bacteria to die [5]. Lysozyme is involved in antibacterial activity, immune function, and inflammation regulation [6] and is utilized in the food industry as a preservative to inhibit bacterial growth [7,8]. In medicine, lysozyme is an antibacterial, anti-inflammatory, and wound treatment agent [6].
Hen egg white lysozyme (HEWL), one of the best-known forms of lysozyme, is abundant in chicken egg whites [9,10]. The structural and biochemical properties of HEWL, such as its catalytic site and substrate-binding cleft, have been well studied to understand how it enzymatically disassembles bacterial cell walls [11,12].
Many crystal structures of HEWL provide useful information to understand the molecular function of lysozyme [11,12]. Still, most crystal structures have been determined at cryogenic temperatures, which causes the biologically less relevant molecular flexibility [13,14]. Several room-temperature (RT) structures of lysozyme have been determined by macromolecular crystallography (MX), but they may contain inaccurate structural information due to potential radiation damage [15]. These experimental limitations can be overcome by serial crystallography (SX) using X-ray free electron laser or synchrotron X-ray [14]. SX determines the RT structure while minimizing radiation damage [13,16,17,18]. Structures determined by SX are more biologically relevant than those determined by conventional cryocrystallography techniques and offer more accurate structural information on molecular flexibility. Several RT structures of HEWL have been determined by SX [19,20,21,22,23,24,25,26], but these studies primarily utilized HEWL crystals and structures as model samples to demonstrate the development of SX experimental techniques. Accordingly, the RT structural properties of HEWL using SX, which provides biologically relevant structural information, have not been fully elucidated. There are no detailed analyses of whether there are structural differences between RT HEWL determined by SX and cryogenic structures determined by traditional cryocrystallography.
To better understand the structural characteristics of HEWL at RT, the crystal structure of HEWL was determined at RT using serial synchrotron crystallography (SSX) and at cryogenic temperatures using MX. The substrate-binding cleft and active site of the room-temperature (RT) structure of HEWL (HEWLRT) were compared with those of the cryogenic structure of HEWL (HEWLCryo). Docking studies of substrates and inhibitors were performed on both the HEWLRT and HEWLCryo structures to investigate the effects of temperature-dependent structural changes. These results expand our understanding of the structural changes of HEWL depending on temperature and provide insights into the importance of RT structures for subsequent research, such as ligand design.

2. Materials and Methods

2.1. Sample Preparation

Lyophilized lysozyme powder from chicken white egg (Catalog no. L6876) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The lysozyme powder was dissolved in a buffer containing 10 mM Tris-HCl (pH 8.0) and 200 mM NaCl to a concentration of 100 mg/mL (for SSX) or 20 mg/mL (for MX). The crystallization solution was composed of 0.1 mM sodium acetate (pH 4.2), 3.5 M NaCl, and 5% (w/v) polyethylene glycol 8000.

2.2. Crystallization for SSX

For SSX experiments, the large-scale batch crystallization method was performed as reported previously [27]. The HEWL solution (100 mg/mL, 100 μL) was mixed with the crystallization solution (100 μL) in a 1.5 mL microcentrifuge tube. This mixture was vortexed for 30 s and incubated at 22 °C. For MX experiments, HEWL crystallization was performed using the hanging drop vapor diffusion method at 22 °C. The HEWL solution (20 mg/mL, 2 μL) was mixed with the crystallization solution (2 μL) and equilibrated against the reservoir solution (500 μL). The crystal size of HEWL for SSX and MX was ~30 to 40 and 150 to 200 μm, respectively.

2.3. X-Ray Data Collection

SSX and MX data were collected at the 11C beamline at Pohang Light Source II (Pohang, Republic of Korea) [28]. The X-ray energy was 12.54 KeV. The photon flux was 5 × 1011 for 1 s. The X-ray beam size was ~3.5 × 8 µm (full width at half-maximum). Diffraction data were recorded on a Piluatus 6M detector (Dectris, Baden-Dättwil, Switzerland).

2.3.1. SSX Data Collection

HEWL crystals were embedded in a polyacrylamide (PAM) injection matrix using a dual-syringe setup [24]. A syringe containing HEWL crystals embedded in PAM was installed into the Fusion Touch 100 syringe pump (CHEMYX, Stafford, TX, USA). PAM containing HEWL crystals were extruded from the syringe needle using a syringe pump [29]. The samples were delivered to the X-ray interaction point at a flow rate of 200 nL/min. The X-ray exposure time for the crystal samples was 100 ms per image. Data collection was performed at 25 °C. Hit images containing Braggs peaks (>20, SNR = 5) were filtered using the Cheetah [30] program. The hit images were indexed and processed using the CrystFEL [31] program with the XGANDALF [32] indexing algorithm.

2.3.2. MX Data Collection

A single lysozyme crystal was soaked into a cryoprotectant solution containing the reservoir solution supplemented with 20% (v/v) ethylene glycol for 5 s. The cryoprotected crystal was mounted on the goniometer under a 100 K liquid nitrogen stream. Diffraction data were indexed, integrated, and scaled with the Xia2 [33] program.

2.4. Structure Determination

The phasing problem was solved by the molecular replacement method using MOLREP [34] implemented in CCP4 [35]. The crystal structure of HEWL (Protein Data Bank (PDB) code 7WUC) [25] was used as a search model. The manual model building was performed with COOT [36]. The final structure was refined with phenix, refine in PHENIX [37]. Water molecules were added during the structural refinement with default parameters. The quality of the final structures was verified by MolProbity [37]. The structure figures were generated by PyMOL (http://www.pymol.org; accessed on 22 November 2024).

2.5. Ligand Docking

The substrate-binding cavity of the HEWL and the docking of N-acetyl-α-d-glucosamine (GlcNAc) and the N,N′,N″-triacetylchitotriose (NAG3) to HEWL were performed using CB-Dock2 [38]. The ideal structure data file for NAG was obtained from the PDB website (https://www.rcsb.org/ligand/NAG; accessed on 19 January 2025). The GlcNAc molecule was docked into the HEWL based on the crystal structure of lysozyme complexed with GlcNAc (PDB code 9LYZ) as a template structure. The coordinate of NAG3 was obtained from crystal structure lysozyme complexed with HEWL complexed with NAG3 (PDB code 5NJQ). The NAG3 molecule was docked into the HEWL based on the crystal structure of lysozyme complexed with NAG3 (PDB code 5NJQ) as a template structure. Docking simulations were carried out in this study without any optimization of the side-chain conformation. The binding affinity between HEWLs and ligands was calculated with PRODIGY-LIGAND [39].

3. Results

3.1. Structure Determination of HEWLs

Protein structure can be affected by crystallization conditions and the data collection environment. Accordingly, previously determined crystal structures of HEWL at RT by SX and at cryogenic temperature by MX could not be compared directly due to differences in their crystallization conditions and data collection environments. To minimize these experimental effects, in this study, data collection of HEWL using SSX and MX was performed using identical crystallization solutions, and diffraction data were collected at the same synchrotron beamline.
For the SSX experiment, many crystal samples were obtained using the batch crystallization method in a microtube. To minimize sample consumption, HEWL crystals were embedded with the PAM injection matrix as a sample delivery medium and delivered to the X-ray interaction point at RT. A total of 10,000 images were collected, of which 9848 contained the Braggs peaks as hit images. Then, 22,963 crystal diffraction patterns were obtained from 9316 indexed images. The hit, indexing, and multicrystal hit rates were 98.48%, 94.59%, and 246.48%, respectively. HEWLRT data were processed up to a resolution of 1.55 Å. The overall completeness, redundancy, I/σ, CC*, and Rsplit were 100%, 956.5, 8.99, 0.999, and 6.34, respectively (Table 1).
For the MX experiment, diffraction data can be collected using small-sized HEWL crystals obtained from the SSX crystallization experiment. However, unlike SSX, where the crystal sample is exposed to X-rays only once, the center of the crystal aligned with the X-rays is continuously exposed to X-rays during data collection in MX. This can potentially cause radiation damage to the crystal structure and result in inaccurate structural information. To minimize radiation damage, large crystal samples can be used to provide new crystal volume during crystal rotation, and data collection can reduce the average dose [40]. To grow large-sized crystals, the protein concentration was lowered to reduce crystal nucleation. Crystals with dimensions of ~200 to 300 μm were obtained from HEWL at a concentration of 20 mg/mL and used for MX data collection. In MX data collection, the crystal was immersed in a cryoprotectant solution, and data were collected at 100 K and processed up to 1.55 Å. Overall, the conditions of the crystallization solution collected from SSX and MX data and the characteristics of the X-ray beam used in the experiment were the same, but the difference was the inclusion of 20% (v/v) glycerol for cryoprotection and the longer X-ray exposure to crystal than SSX in MX. The electron density maps of HEWLRT and HEWLCryo were observed to build whole amino acids. The Rwork/Rfree of the final HEWLRT and HEWLCryo structures were 18.24/21.96 and 17.46/18.86, respectively (Table 1).

3.2. Structural Comparison of HEWLRT and HEWLCryo

The crystal structures of HEWLRT and HEWLCryo consisted of α- and β-domains (Figure 1A). The substrate-binding cleft is situated at the interface between the α- and β-domains, with the catalytic residue Glu53 located in the α-domain and Asp70 in the β-domain (Figure 1A). Superimposition of HEWLRT and HEWLCryo revealed structural similarity with a root mean square deviation of 0.146 Å and showed the positional differences between the two domains (Figure 1B). In the α-domain, between HEWLRT and HEWLCryo, the positions of the Cα atoms of Asn121 and Val127 differ by approximately 0.43 and 0.27 Å, respectively (Figure 1B). In the β-domain, between HEWLRT and HEWLCryo, the positions of the Cα atoms of Thr65, Arg79, and Gly89 differ by approximately 0.85, 0.37, and 0.56 Å, respectively (Figure 1B). Among these subtly moved positions, the side chains of Arg79 and Asn121 between HEWLRT and HEWLCryo exhibited different conformations.
Meanwhile, electron density analysis revealed partial negative Fo-Fc electron density corresponding to the side chain of the Asn77 residue in HEWLCryo (Supplementary Figure S1), suggesting that this side chain adopts multiple conformations, whereas no such density was observed in HEWLRT. For structural analysis, the major side-chain conformation of Asn77 in HEWLCryo was used.
The distance between the Cα atoms of catalytic residues Glu53 and Asp70 of HEWLRT and HEWLCryo were 9.51 and 9.28 Å, respectively. The distances between the OE1 atom of Glu53 and the OE2 atom of Asp70 in HEWLRT and HEWLCryo were 5.61 and 5.08 Å, respectively (Figure 1C,D). In HEWLRT, the OE2 atom of Glu53 was 3.61 Å from the N atom of Val127 (Figure 1C). The OD1 atom of Asp70 interacted with the ND2 atom of Asn77 and the OD1 atom of Asn64 at distances of 2.58 and 3.08 Å, respectively. The OD2 atom of Asp70 interacted with the ND2 atom of Asn64 at a distance of 3.30 Å. The distance between the ND2 atom of Asn77 and the OD1 atom of Asn64 was 2.72 Å (Figure 1C).
In HEWLCryo, the OE2 atom of Glu53 was 3.80 Å from the N atom of Val127 (Figure 1D). The OD1 and OD2 atoms of Asp70 interacted with the OD1 and ND2 atoms of Asn64 at distances of 2.99 and 3.47 Å, respectively. The distance between the ND2 atom of Asn77 and the OD1 atom of Asn64 was 3.18 Å (Figure 1D). Meanwhile, the OD1 atom of Asp70 was 3.82 Å away from the ND2 atom of Asn77 (Figure 1D).
Superposition of HEWLRT and HEWLCryo revealed significant conformational and positional changes in the vicinity of the residue involved in the catalytic residue Asp70. The rotation angles of the Glu53 and Asp70 side chains of HEWLRT and HEWLCryo were approximately 4° and 15°, respectively (Figure 1E). The position of the side chain of Asn64 of HEWLRT and HEWLCryo were apart by 0.75 Å. The temperature induced the conformation and position change of the active site region on the β-domain in HEWL. The distance between the substrate-binding cleft and the distance between catalytic residues of HEWLRT were wider than those of HEWLCryo.

3.3. Comparison of the Structural Flexibility of HEWLRT and HEWLCryo

Flexibility, rigidity, and/or internal motion in proteins are crucial to understanding the molecular function and provide insights into protein engineering [41]. Theoretically, RT structures provide relatively biologically relevant structural flexibility compared to crystal structures obtained by the cryogenic environment [13,14]. To understand temperature-dependent structural flexibility, the B-factors of HEWLRT and HEWLCryo were analyzed. The overall B-factor value of HEWLRT and HEWLCryo was 28.91 and 20.29 Å2, respectively. B-factor putty presentation showed that the β1-β2 loop, β3-η1 loop, α3-η2 loop, and C-terminus in HEWLRT and HEWLCryo are relatively more flexible than other regions (Figure 2A). In particular, the flexibility of the loop region above the substrate-binding cleft of HEWLRT was significantly higher than HEWLCryo, indicating that the flexibility of the substrate-binding cleft region of HEWL may increase with temperature, which could affect substrate recognition and catalytic activity. The B-factor plot showed an overall similar trend, but the B-factor values of most residues in HEWLRT were higher than those in HEWLCryo (Figure 2B). Normalized B-factor analysis showed that the α1-α2 loop of HEWLRT showed a relatively high B-factor compared to HEWLCryo, whereas the α3-η2 loop of HEWLRT was lower than that of HEWLCryo (Figure 2C), indicating that the relative flexibility region in HEWL can be changed depending on the temperature.

3.4. Comparison of Water Molecules on HEWLRT and HEWLCryo

Water molecules are important in many biological processes in stabilizing protein structures, assisting protein folding, and improving binding affinity [42]. During structural refinement, water molecules are automatically added to avoid bias in model building. The number of water molecules in HEWLRT and HEWLCryo was 82 and 181, respectively. These water molecules were distributed more throughout the protein surface than in specific locations (Figure 3A). The B-factor value of the water molecules in HEWLRT and HEWLCryo was 37.28 and 32.61, respectively, indicating that water molecules in HEWLRT are more dynamic than HEWLCryo, indicating that the rigidity and mobility of water molecules on the surface of HEWL can be changed depending on temperature.
In the substrate-binding cleft, the observed number of water molecules on HEWLRT and HEWLCryo was 10 and 19, respectively (Figure 3B). The position and distribution of these water molecules on the substrate-binding cleft between HEWLRT and HEWLCryo slightly differed (Figure 3B). In HEWLRT and HEWLCryo, two water molecules (W1 and W2) were located at similar positions between the two catalytic residues, Glu53 and Asp70, and interacted with these catalytic residues. The W1 molecule interacted with the OE1 atom of Glu53 in HEWLRT and HEWLCryo at distances of 2.52 and 2.49 Å, respectively. The W2 molecule interacted with the OD2 atom of Asp70 in HEWLRT and HEWLCryo at distances of 3.19 and 3.20 Å, respectively. The W1 and W2 molecules formed a water bridge at the +1 subsite in HEWLRT and HEWLCryo, with distances of 2.70 and 2.77 Å, respectively. These results indicated that the water interaction distance of Glu53 and Asp70 is mostly similar between HEWLRT and HEWLCryo. In the superimposition of HEWLRT and HEWLCryo, the positional difference of W1 and W2 from HEWLRT and HEWLCryo was 0.19 and 0.09 Å, respectively. In contrast, the interaction angle of Glu53-W1-W2 in HEWLRT and HEWLCryo was 95.9° and 94.5°, respectively, indicating similar coordination of the W1 molecule. In contrast, the interaction angle of W1-W2-Asp70 in HEWLRT and HEWLCryo was 82.3° and 68.8°, respectively, indicating distinct water coordination for the W2 molecule. This difference in angle was due to conformational changes in the Asp70 side chain and its neighboring residues on the β-domain between HEWLRT and HEWLCryo. Meanwhile, in HEWLCryo, W1 and W2 molecules further formed water bridge interactions with other water molecules in the substrate-binding cleft. In contrast, direct interactions of W1 and W2 molecules with other water molecules in the substrate-binding cleft were not observed in HEWLRT. The positional and distribution differences of other water molecules, excluding W1 and W2, observed in the substrate-binding cleft of HEWLRT and HEWLCryo may be caused by different residue conformations at the active site region due to temperature changes.

3.5. Ligand Docking into HEWLRT and HEWLCryo

The subtle structural changes in the active sites of HEWLRT and HEWLCryo were considered to influence subsequent studies, such as ligand docking and inhibitor design. To understand how these structural differences affect such studies, the substrate-binding cavity was analyzed, and docking experiments were performed for HEWLRT and HEWLCryo.
The substrate-binding cavity of HEWLRT consisted of 15 amino acids (Glu53, Asn62, Asp70, Qln75, Ile76, Asn77, Trp80, Trp81, Ile116, Asp119, Asn121, Ala125, Trp126, and Val127). The substrate-binding cavity of HEWLCryo was composed of Asn64 in addition to the substrate-binding cavity of HEWLRT. Structure-based cavity detection showed that the cavity volume of the HEWLRT and HEWLCryo was 193 and 210 Å3, respectively.
GlcNAc, a fundamental component of the HEWL substrate, and NAG3, a HEWL inhibitor, were docked into the substrate-binding cleft of HEWLs. To avoid bias in ligand docking to HEWL, an automatic docking program was used. Docking results showed that the GlcNAc molecule was located at subsite D in HEWLRT and HEWLCryo. The 6-hydroxyl group of GlcNAc was oriented toward the active site region, whereas the acetyl group of GlcNAc was oriented toward the solvent region. The Vina docking score of GlcNAc to HEWLRT and HEWLCryo was −3.4 and −3.3, respectively. The calculated binding affinities (ΔG) of GlcNAc to HEWLRT and HEWLCryo were −4.96 and −5.13 kcal/mol, respectively.
In HEWLRT, the O5 and O6 atoms of GlcNAc interacted with Asp70 (atom: OD2) and Glu53 (OE1/OE2) at distances of 3.38 and 3.32/2.76 Å, respectively (Figure 4A). In HEWLCryo, the O5 and O6 atoms of GlcNAc interacted with Asp70 (OD2) and Glu53 (OE1/OE2) at distances of 3.19 and 3.54/2.97 Å, respectively (Figure 4B). Superimposition of GlcNAc-docked HEWLRT and HEWLCryo showed that the positions of atoms in GlcNAc shifted by 0.30 to 0.73 Å (Figure 4C).
In NAG3 docking to HEWL, the three NAG units from the NAG3 molecule were commonly located at subsites A to C in HEWLRT and HEWLCryo (Figure 4D–F). The orientation of NAG units at subsites B and C on HEWLRT and HEWLCryo was similar, but the orientation of the NAG unit at subsite A on HEWLRT and HEWLCryo differed. At subsite A, the distinct conformation of Arg91 and Asn121 residues between HEWLRT and HEWLCryo was observed, which caused different docking results for the NAG molecule at subsite A. The Vina docking scores of NAG3 to HEWLRT and HEWLCryo were −5.4 and −5.8, respectively. The calculated binding affinities (ΔG) of NAG3 to HEWLRT and HEWLCryo were −4.95 and −4.91 kcal/mol, respectively.
For HEWLRT, the O1 atom of NAG at subsite C interacted with Asp70 (OD1) and Asn77 (ND2) at distances of 4.05 and 4.00 Å, respectively (Figure 4D). The O3, O6, and O7 atoms of NAG at subsite C interacted with Trp81 (NE1), Trp62, and Trp81 (NE1) at distances of 3.36, 2.99, and 3.19 Å, respectively. The O6 and O7 atoms of NAG at subsite B interacted with Asp119 (OD2) and Asn121 (ND2) at distances of 2.83 and 5.17 Å, respectively (Figure 4D). For HEWLCryo, the O1 atom of NAG at subsite C interacted with Asp70 (OD1) and Asn77 (ND2) at distances of 3.93 and 3.15 Å, respectively (Figure 4E). The O3, O6, and O7 atoms of NAG at subsite C interacted with Trp81 (NE1), Trp62, and Trp81 (NE1) at distances of 3.10, 2.86, and 3.13 Å, respectively. The O6 and O7 atoms of NAG at subsite B interacted with Asp119 (OD2) and Asn121 (ND2) at distances of 3.00 and 3.85 Å, respectively (Figure 4E). Superimposition of NAG3-docked HEWLRT and HEWLCryo showed that the positions of atoms in NAG at subsite A shifted by 0.25 to 3.78 Å (Figure 4F). Although the substrate-binding cavity, Vina docking scores, and calculated binding affinity for GlcNAc and NAG3 to HEWL were not significantly different, the detailed molecular interactions differed between HEWLRT and HEWLCryo.
Meanwhile, in the recalculated electron density map in the PDB, the quality of the electron density corresponding to the side chains of Arg91 and Asn121 in both HEWLRT and HEWLCryo showed partial disorder, indicating that multiple conformations of these residues were possible, which could affect the docking simulation. Accordingly, docking simulations with NAG3 were performed using HEWLRT and HEWLCryo after omitting the side chains of Arg91 and Asn121. The results showed that the position of the docked NAG3 molecule in HEWLRT and HEWLCryo was slightly different, and the distance between NAG3 and HEWL also differed (Supplementary Figure S2). The Vina docking scores of NAG3 to HEWLRT and HEWLCryo were −5.3 and −5.8, respectively. The calculated binding affinities (ΔG) of NAG3 to HEWLRT and HEWLCryo were −5.08 and −5.15 kcal/mol, respectively, which showed a differing trend from the previous analysis conducted without omitting the side chains of Arg91 and Asn121. These results also indicate that the changes in the substrate binding site due to temperature affect the docking results.

4. Discussion

Lysozyme is an innate immune protein widely used in various industries, including medicine and food [6,7,8]. To understand temperature-dependent structural changes, the crystal structures of HEWLRT and HEWLCryo were determined. The position and conformation of the residues involved in the catalytic site and substrate-binding cleft between HEWLRT and HEWLCryo and the water molecules on HEWL were subtly different due to temperature. These results indicated that temperature affects the conformation of the active site and substrate-binding cleft of HEWL, causing a difference in the width of the substrate-binding cleft between the α- and β-domains. B-factor analysis showed that HEWLRT exhibited higher structural flexibility overall than HEWLCryo, which followed the trend that higher temperatures increase protein flexibility. In particular, the B-factors of the loops above the substrate-binding cleft significantly increased, which affected the substrate recognition of HEWL. A previous biochemical study showed that the lytic activity of HEWL at RT (22 °C) was higher than at low temperatures (5 °C) [43]. This indicated that the HEWLRT structure has relatively more optimal active structural conformation and flexibility than the low temperature HEWL structure. This analysis will provide important information for understanding enzyme activity at different temperatures, along with the fact that enzymatic activity decreases at low temperatures, mostly due to the thermodynamic effects of the chemical reaction [44,45]. Theoretically, HEWLRT determined by SSX provides a more reliable structure in terms of temperature and radiation damage than HEWLCryo. Taken together, the crystal structure of HEWLRT determined by SSX offered more accurate structural information on molecular flexibility, active site conformation, and the hydration environment of the active site of HEWL.
In contrast, in this study, GlcNAc and NAG3 ligands were docked into HEWL to understand how subtle structural differences in the active and substrate-binding sites, depending on the temperature, may affect subsequent studies. GlcNAc and NAG3 bound to HEWLRT and HEWLCryo showed positional similarity but subtle differences in the substrate-binding cleft and distinct protein-ligand interactions. In particular, NAG at subsite A unit in the NAG3 ligand showed distinct conformations and positions between HEWLRT and HEWLCryo. As a result, subtle structural changes in amino acids observed at RT and cryogenic temperature can affect the outcomes of ligand docking studies. Moreover, considering that the flexibility of the molecule and water molecules are factors that influence ligand docking, structural information at RT can provide more reliable docking results for subsequent research compared to cryogenic temperature [46,47]. These results provided insights into the structural changes in HEWL with temperature and the importance of temperature and structural changes in other protein structures. To better understand the flexibility of HEWLRT and HEWLCryo, a molecular dynamics simulation study combined with binding energy analysis using MM-PBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) or MM-GBSA (Molecular Mechanics Generalized Born Surface Area) protocols would be valuable for future research.
In particular, the accuracy of the protein structure is critical for downstream applications such as structure-based drug design (SBDD) [48,49]. The high accuracy of the experimental structure used in SBDD can contribute to rational ligand design in subsequent studies. Conversely, less reliable experimental structures can lead to inefficient ligand design in SBDD studies, consuming time and resources. Consequently, the biological reliability of the experimental structure has a significant impact on the design and results of subsequent structure-based studies.
As demonstrated by HEWLRT and HEWLCryo in this study, temperature is an important factor that can influence the structural information of the active and substrate-binding sites. For proteins targeted by SBDD, using structural information in a biologically relevant temperature environment will contribute to rational design and increase the success rate of ligand binding. In contrast, even for crystal structures determined at RT, when diffraction data are collected using general cryocrystallography techniques, they may exhibit less biologically relevant molecular flexibility due to radiation damage [15]. Therefore, to minimize radiation damage in a biological temperature environment, it is important to determine the RT structure using SX and apply it to structural analysis and subsequent research.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cryst15020111/s1, Figure S1: 2Fo-Fc and Fo-Fc electron density map for Asp70 and Asn77 in HEWLRT and HEWLCryo; Figure S2: Computational docking of NAG3 to HEWLRT and HEWLCryo after omitting the side chains of Arg91 and Asn121.

Funding

This work was funded by the National Research Foundation of Korea (NRF) (NRF-2021R1I1A1A01050838).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The structure factors and coordinates were deposited in PDB (http://rcsb.org) under accession codes 8YBG (HEWLRT) and 8YBH (HEWLCryo).

Acknowledgments

I would like to thank the beamline staff at the 11C beamline at the Pohang Accelerator Laboratory for their assistance with data collection. The author thanks the Global Science experimental Data hub Center (GSDC) at the Korea Institute of Science and Technology Information (KISTI) for providing computing resources and technical support.

Conflicts of Interest

The author declares no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
SXserial crystallography
SSXserial synchrotron crystallography
MXmacromolecualr crystallography
RTroom temperature
HEWLhen egg white lysozyme
PAMpolyacrylamide
NAGN-acetylglucosamine
NAMN-acetylmuramic acid
SBDDstructure-based drug design

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Crystals 15 00111 g001
Figure 1. Crystal structure of HEWL determined by SSX at RT and MX at cryogenic temperature. (A) Overall structure of HEWLRT comprising α- and β-domains. (B) Superimposition of HEWLRT (red) and HEWLCryo (blue). The positional difference of the loop regions is indicated by sticks. Close-up view of the active site of (C) HEWLRT and (D) HEWLCryo. (E) Superimposition of the active site of HEWLRT (green) and HEWLCryo (cyan).
Figure 1. Crystal structure of HEWL determined by SSX at RT and MX at cryogenic temperature. (A) Overall structure of HEWLRT comprising α- and β-domains. (B) Superimposition of HEWLRT (red) and HEWLCryo (blue). The positional difference of the loop regions is indicated by sticks. Close-up view of the active site of (C) HEWLRT and (D) HEWLCryo. (E) Superimposition of the active site of HEWLRT (green) and HEWLCryo (cyan).
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Figure 2. Comparison of the structural flexibility of HEWLRT and HEWLCryo. (A) B-factor putty representation of (A) HEWLRT and (B) HEWLCryo. (C) B-factor and normalized B-factor plots of HEWLRT (red) and HEWLCryo (blue).
Figure 2. Comparison of the structural flexibility of HEWLRT and HEWLCryo. (A) B-factor putty representation of (A) HEWLRT and (B) HEWLCryo. (C) B-factor and normalized B-factor plots of HEWLRT (red) and HEWLCryo (blue).
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Figure 3. Comparison of water molecules on HEWLRT and HEWLCryo. (A) Defined water molecules on the surface structure of HEWLRT and HEWLCryo. (B) Superimposition of water molecules on the substrate-binding cleft of HEWLRT and HEWLCryo. Water molecule interactions with the catalytic Glu53 and Asp70 residues of (C) HEWLRT and (D) HEWLCryo.
Figure 3. Comparison of water molecules on HEWLRT and HEWLCryo. (A) Defined water molecules on the surface structure of HEWLRT and HEWLCryo. (B) Superimposition of water molecules on the substrate-binding cleft of HEWLRT and HEWLCryo. Water molecule interactions with the catalytic Glu53 and Asp70 residues of (C) HEWLRT and (D) HEWLCryo.
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Figure 4. Computational docking of GlcNAc and NAG3 to HEWLRT and HEWLCryo. GlcNAc docking to (A) HEWLRT and (B) HEWLCryo. (C) Superimposition of GlcNAc docking structures of HEWLRT (red stick) and HEWLCryo (blue stick). NAG3 docking to (D) HEWLRT and (E) HEWLCryo. (F) Superimposition of NAG3 docking structures of HEWLRT (red stick) and HEWLCryo (blue stick).
Figure 4. Computational docking of GlcNAc and NAG3 to HEWLRT and HEWLCryo. GlcNAc docking to (A) HEWLRT and (B) HEWLCryo. (C) Superimposition of GlcNAc docking structures of HEWLRT (red stick) and HEWLCryo (blue stick). NAG3 docking to (D) HEWLRT and (E) HEWLCryo. (F) Superimposition of NAG3 docking structures of HEWLRT (red stick) and HEWLCryo (blue stick).
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Table 1. Data processing and structure refinement statistics.
Table 1. Data processing and structure refinement statistics.
Data CollectionHEWLRTHEWLCryo
X-ray SourceBeamline 11C, PLS IIBeamline 11C, PLS II
Space groupP43212P43212
Cell dimension
a, b, c (Å)78.50, 78.50, 38.3077.21, 77.21, 37.95
Resolution (Å)79.36–1.55 (1.60–1.55)50.00–1.55 (1.58–1.55)
Unique reflections17,968 (1755)16,999 (836)
Completeness (%)100 (100)99.9 (99.9)
Redundancy956.5 (529.6)21.5 (17.7)
<I/sigma>8.99 (1.20)35.16 (2.2)
CC1/20.9960 (0.3343)0.988 (0.813)
CC*0.9990 (0.7079)0.997 (0.947)
Rsplit6.34 (93.55)
Refinement
Resolution (Å)55.51–1.5527.30–1.55
Rwork0.18240.1746
Rfree0.21960.1886
No. of non-H atoms
Protein10011001
Water82181
R.m.s.deviation
Bonds (Å)0.0060.006
Angles (°)0.8850.838
Average B factors (Å2)
Protein28.9120.29
Water37.2832.61
Ramachandran plot
Favored (%)99.2198.41
Allowed (%)0.791.59
PDB code8YBG8YBH
Values for the outer shell are given in parentheses.
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Nam, K.H. Temperature-Dependent Structural Changes of the Active Site and Substrate-Binding Cleft in Hen Egg White Lysozyme. Crystals 2025, 15, 111. https://doi.org/10.3390/cryst15020111

AMA Style

Nam KH. Temperature-Dependent Structural Changes of the Active Site and Substrate-Binding Cleft in Hen Egg White Lysozyme. Crystals. 2025; 15(2):111. https://doi.org/10.3390/cryst15020111

Chicago/Turabian Style

Nam, Ki Hyun. 2025. "Temperature-Dependent Structural Changes of the Active Site and Substrate-Binding Cleft in Hen Egg White Lysozyme" Crystals 15, no. 2: 111. https://doi.org/10.3390/cryst15020111

APA Style

Nam, K. H. (2025). Temperature-Dependent Structural Changes of the Active Site and Substrate-Binding Cleft in Hen Egg White Lysozyme. Crystals, 15(2), 111. https://doi.org/10.3390/cryst15020111

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