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Original Research Article

DTT 2022; 1(1): 40-44

Published online July 31, 2022 https://doi.org/10.58502/DTT.22.007

Copyright © The Pharmaceutical Society of Korea.

Characterization of Bacterial Proteasome Component HslV from Staphylococcus aureus

Kang Mu Kwon1,2 , Hyo Jung Kim1,2

1College of Pharmacy, Woosuk University, Wanju, Korea
2Research Institute of Pharmaceutical Sciences, Woosuk University, Wanju, Korea

Correspondence to:Hyo Jung Kim, hyojungkim@woosuk.ac.kr

Received: April 15, 2022; Revised: May 26, 2022; Accepted: June 11, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Proteins are constantly synthesized and degraded to maintain protein homeostasis. The ubiquitin-proteasome system is well known as a targeted protein breakdown process in eukaryotic cells. Although the archaea and actinobacterial families have a relevant system, many prokaryotic cells lack proteasomes. HslUV, a bacterial heat shock protein, is known to perform a proteasomal activity. HslU functions as ATPase, HslV is responsible for protease, and both activities are tightly connected in the two-component HslUV system. Here, we identified the characteristics of HslV from Staphylococcus aureus. Although only multimeric HslUV shows its activity, monomer S. aureus HslV remains stable and successfully crystallized. This study will provide a detailed understanding of the bacterial proteasomal system.

KeywordsHslU, HslV, bacterial proteasomal system, ubiquitin-proteasome, Staphylococcus aureus

Cells continuously create and destroy proteins to maintain protein homeostasis; this cycle is referred to as protein turnover. The protein turnover rate can change in response to internal or external stimuli, including hormones, inflammation, or low energy status (Rolfs et al. 2021). Consequently, misfolded, damaged, or aged proteins are removed from the cellular protein pool and replaced with new proteins. Protein degradation occurs in eukaryotes via two types of proteolytic machineries: the lysosome and proteasome (Ciechanover 2005). In lysosomal proteolysis, proteins are engulfed by membrane-enclosed vesicles, such as autophagosomes or endocytic vesicles. Conversely, the ubiquitin-proteasome system uses a diverse collection of E1, E2, and E3 ubiquitin ligases to selectively target proteins for degradation by the proteasome (Park et al. 2020). The proteasome is a 2,500-kDa cylindrical protein complex containing a 20S catalytic core particle capped by either one or two 19S regulatory particles (Tanaka 2009; Choi et al. 2021). The 20S core particle is responsible for the proteasome’s proteolytic activities using its central chamber, while the 19S regulatory particle recognizes polyubiquitnated substrates. All 20S core particles are composed of the outer (α subunits) and inner (β subunits) heptameric rings (Kunjappu and Hochstrasser 2014). A major function of the α subunit ring is to regulate entry into the central proteolytic chamber, while the β subunit cleaves targeted proteins. However, most bacteria do not have proteasomes. Instead, molecular architecture HslUV (originally discovered in Escherichia coli) and CodWY (originally discovered in Bacillus subtilis) are considered bacterial counterparts of the eukaryotic proteasome (Kavalchuk et al. 2022). HslV and CodW harbor the protease activity that shows a similar primary sequence with the eukaryotic β subunit of the 20S core particle, while HslU and CodY provide ATPase activity. Furthermore, the protease and ATPase function together during protein degradation (Rohrwild et al. 1996; Sousa et al. 2000; Jeong et al. 2020) (Fig. 1).

Figure 1.E. coli HslUV complex. The HslUV complex consists of two HslU hexamers and the dodecameric HslV, “double-donut” hexameric rings (PDB ID 1G3I). The proteolytic active sites are located in the inner chamber of HslV. HslU identifies and translocates target proteins into this chamber using ATP.

In this research, a preliminary study of HslV from Staphylococcus aureus was conducted. S. aureus causes mild to severe infectious diseases, ranging from skin infection to life-threatening conditions. However, current management regimens are limited because of their ability to develop resistance to almost all antibiotics (Tong et al. 2015). Therefore, understanding the bacterial adaptation mechanism to environmental changes is indispensable for developing better therapeutics. S. aureus has adapted to stresses by developing response mechanisms, which include heat shock proteins that are produced to refold or destroy damaged proteins (Anderson et al. 2006). Furthermore, S. aureus HslUV is a “prokaryotic proteasome” that degrades specific proteins in an ATP-dependent reaction (Sousa et al. 2000). Here, we overexpressed, crystallized, and studied the properties of S. aureus HslV. As the first step of structural determination, our research will promote a better understanding of the HslUV mechanism on a molecular basis.

Expression and purification of S. aureus HslV

The SAV1253 gene coding for HslV was amplified from the genomic DNA of S. aureus Mu50 by polymerase chain reaction. NdeI and XhoI restriction sites were used for cloning using a pET-21a(+) vector (Novagen, USA). The resulting construct has eight additional residues (LEHHHHHH) that encode the C-terminal hexahistidine tag. The sequences of cloned genes were confirmed by DNA sequencing. The recombinant plasmid was transformed into E. coli BL21 (DE3). Cells were grown in LB medium supplemented with ampicillin (50 μg/mL). Recombinant protein HslV expression was induced by adding isopropyl β-D-1-thiogalactopyranoside to 0.5 mM when the OD600 reached 0.5. Cells were harvested following centrifugation at 4,000 × g at 4℃ after an additional 4 h of growth. The cell pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, and 500 mM NaCl) and disrupted using an Ultrasonic processor (Cole-Parmer, USA). Cell lysate was centrifuged at 20,000 × g for 1 h at 4℃. The cleared supernatant was purified by binding to a Ni-NTA (Ni-nitrilotriacetate) affinity column (Qiagen, Germany) and eluted with an elution buffer containing 200 mM imidazole. Furthermore, purification and buffer exchanges were achieved by size-exclusion chromatography. The HslV was prepared with a 10 mg/mL concentration in 50 mM Tris-HCl, pH 7.5, and 200 mM NaCl and loaded onto a Superdex 75 (100/300GL) column (GE Healthcare Life Science, USA) that was previously equilibrated with the same buffer. The purified HslV was concentrated at 10 mg/ml by ultrafiltration in 10,000 Da molecular mass cut-off spin columns (Millipore, USA).

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was conducted according to the Laemmli method using 15% (w/v) polyacrylamide gel (Laemmli 1970). The samples were treated with 1% (w/v) SDS and 5% (v/v) 2-mercaptoethanol before electrophoresis in a vertical Mini Gel system (Bio-Rad Laboratories, USA). The proteins were stained with Coomassie Brilliant Blue R250 (Thermo Scientific, USA).

Oligomeric state of S. aureus HslV

Size-exclusion chromatography was employed to determine the oligomeric state of S. aureus HslV. A major peak appeared at a molecular mass of approximately 20 kDa, indicating that the protein is predominately monomeric. The corresponding SDS-PAGE indicates the main band at approximately 20 kDa with > 95% purity (Fig. 2). Considering the calculated mass of the S. aureus HslV (19.8 kDa), the size-exclusion chromatography result is consistent with the monomeric state. Although HslV only functions when it forms multiple complexes, monomeric S. aureus HslV shows stable properties. The S. aureus HslV monomer is stable overnight at room temperature and does not indicate any sign of degradation. This finding does not agree with the previous study, which revealed a trimeric state of S. aureus HslV in solution (Jeong et al. 2020). This discrepancy would be due to the different final buffer conditions that affect electrostatic and hydrophobic inter-residue interactions (pH, metal, and salt concentration). Additionally, the hexahistidine affinity tag was attached to the N-terminal in the previous study, but here, we used the C-terminal hexahistidine tagged construct. Since S. aureus HslV undergoes N-terminal cleavage for activation, an additional tag would alter the oligomerization state. The monomer state shows a structurally stable sandwich fold, certifying steady monomeric characterization in solution.

Figure 2.Monomeric state of S. aureus HslV. The size-exclusion chromatography of S. aureus HslV and corresponding SDS-PAGE are shown. The major peak at the molecular weight of approximately 20 kDa indicates a monomeric state.

Crystallization of S. aureus HslV

Crystals of S. aureus HslV were grown using sitting-drop vapor diffusion at 20℃. Initial crystallization conditions were established using screening kits from Hampton Research (Crystal screen I and II, Index, PEG/Ion, and MembFac) and Emerald Biosystems (Wizard I, II, III, and IV). For the optimal growth of HslV crystals, 1 μL (10 mg/mL) of the protein was mixed with 1 μL of precipitant solution (30% PEG400, 100 mM CAPS, and pH 10.5) and was equilibrated against a 1-mL reservoir of the precipitant solution. This condition yields short rod-shaped crystals that grew to dimensions 0.1 × 0.05 × 0.05 mm in 5 days (Fig. 3). Crystals were flash frozen in a stream of nitrogen at 100 K, and diffraction data were collected at the BL-5C beamline of the Pohang Accelerator Laboratory, Republic of Korea.

Figure 3.Morphology of S. aureus HslV crystals. Rod-shaped S. aureus HslV crystals are obtained in 5 days.

Data collection and processing

For data collection under cryogenic conditions, the crystals were transferred to a cryoprotectant solution with 20% (v/v) glycerol under crystallization conditions for several minutes before being flash frozen in a stream of nitrogen gas at 100 K. Data were obtained from ADSC Quantum 270 CCD in beamline 5C of Pohang Accelerator Laboratory, Republic of Korea (Fig. 4). The peaks obtained from consecutive diffraction images were indexed and integrated using the Scalepack package of HKL2000 software (Otwinowski and Minor 1997). The integrated data were processed to refine the electron density by applying a scale factor. The unit cell of S. aureus HslV crystals is a C-centered monoclinic in the Bravais lattice, and its space group was C2, with a large cell parameter: a = 221.407, b = 209,523, and c = 6309.842. This indicates that many monomers are packed in an asymmetric unit (Table 1).

Table 1 Crystallographic data collection

S. aureus HslV
Data collection
BeamlinePAL-5C
Wavelength (Å)0.97
Rotation range per image (°)1
Total rotation range (°)180
Exposure time per image (s)1
Space groupC2
Unit cell parameters (Å)a = 221.407
b = 209.523
c = 6309.842
Resolution (Å)40.0−3.5
Total no. of reflections31556
Rmerge0.76 (0.80)a)
CC1/20.335
I/sigma14.9 (13.0)

a)Numbers in parentheses indicate the statistics for the last resolution shell.


Figure 4.A representative diffraction image. A diffraction image from S. aureus HslV was obtained at the BL-5C beamline of the Pohang Accelerator Laboratory. The resolution circle for 3.0 Å is shown.

The HslUV is a large complex with > 600 kDa molecular weight, 125 Å width, and 150 Å height. Two hexamers of HslV and 1-2 hexamers of HslU consist the HslUV arrangement. The HslV forms an internal cavity to cleave target proteins, and HslU activates HslV by N-terminal cleavage. S. aureus HslV utilizes Thr9 as a nucleophile after its exposure by removal of the N-terminal loop (Jeong et al. 2020). Herein, we showed that S. aureus HslV forms a stable monomer and can crystallize. To solve the structure, molecular replacement methods were applied with the program Phaser within the CCP4 suite using the available template HslV (PDB ID 6KR1) as the search model. Since the protein is expected to have an intact N-terminal structure, identifying the differences from the N-terminal-cleaved structure (PDB ID 6KR1) will improve the understanding of the HslUV activation mechanism.

No potential conflict of interest relevant to this article was reported.

We thank the beamline staff members at Pohang Light Source (BL-5C), Republic of Korea. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) (No. 2022R1F1A1065273) and “Leaders in INdustry-university Cooperation 3.0” Project, supported by the Ministry of Education and National Research Foundation of Korea.

  1. Anderson KL, Roberts C, Disz T, Vonstein V, Hwang K, Overbeek R, Olson PD, Projan SJ, Dunman PM (2006) Characterization of the Staphylococcus aureus heat shock, cold shock, stringent, and SOS responses and their effects on log-phase mRNA turnover. J Bacteriol 188:6739-6756. doi: 10.1128/JB.00609-06.
    Pubmed KoreaMed CrossRef
  2. Choi WH, Kim S, Park S, Lee MJ (2021) Concept and application of circulating proteasomes. Exp Mol Med 53:1539-1546. doi: 10.1038/s12276-021-00692-x.
    Pubmed KoreaMed CrossRef
  3. Ciechanover A (2005) Proteolysis: from the lysosome to ubiquitin and the proteasome. Nat Rev Mol Cell Biol 6:79-87. doi: 10.1038/nrm1552.
    Pubmed CrossRef
  4. Jeong S, Ahn J, Kwon AR, Ha NC (2020) Cleavage-dependent activation of ATP-dependent protease HslUV from Staphylococcus aureus. Mol Cells 43:694-704. doi: 10.14348/molcells.2020.0074.
  5. Kavalchuk M, Jomaa A, Müller AU, Weber-Ban E (2022) Structural basis of prokaryotic ubiquitin-like protein engagement and translocation by the mycobacterial Mpa-proteasome complex. Nat Commun 13:276. doi: 10.1038/s41467-021-27787-3.
    Pubmed KoreaMed CrossRef
  6. Kunjappu MJ, Hochstrasser M (2014) Assembly of the 20S proteasome. Biochim Biophys Acta 1843:2-12. doi: 10.1016/j.bbamcr.2013.03.008.
    Pubmed KoreaMed CrossRef
  7. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. doi: 10.1038/227680a0.
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  8. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307-326. doi: 10.1016/S0076-6879(97)76066-X.
    Pubmed CrossRef
  9. Park J, Cho J, Song EJ (2020) Ubiquitin-proteasome system (UPS) as a target for anticancer treatment. Arch Pharm Res 43:1144-1161. doi: 10.1007/s12272-020-01281-8.
    Pubmed KoreaMed CrossRef
  10. Rohrwild M, Coux O, Huang HC, Moerschell RP, Yoo SJ, Seol JH, Chung CH, Goldberg AL (1996) HslV-HslU: a novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. Proc Natl Acad Sci U S A 93:5808-5813. doi: 10.1073/pnas.93.12.5808.
    Pubmed KoreaMed CrossRef
  11. Rolfs Z, Frey BL, Shi X, Kawai Y, Smith LM, Welham NV (2021) An atlas of protein turnover rates in mouse tissues. Nat Commun 12:6778. doi: 10.1038/s41467-021-26842-3.
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  12. Sousa MC, Trame CB, Tsuruta H, Wilbanks SM, Reddy VS, McKay DB (2000) Crystal and solution structures of an HslUV protease-chaperone complex. Cell 103:633-643. doi: 10.1016/s0092-8674(00)00166-5.
    Pubmed CrossRef
  13. Tanaka K (2009) The proteasome: overview of structure and functions. Proc Jpn Acad Ser B Phys Biol Sci 85:12-36. doi: 10.2183/pjab.85.12.
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  14. Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr (2015) Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 28:603-661. doi: 10.1128/CMR.00134-14.
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Article

Original Research Article

DTT 2022; 1(1): 40-44

Published online July 31, 2022 https://doi.org/10.58502/DTT.22.007

Copyright © The Pharmaceutical Society of Korea.

Characterization of Bacterial Proteasome Component HslV from Staphylococcus aureus

Kang Mu Kwon1,2 , Hyo Jung Kim1,2

1College of Pharmacy, Woosuk University, Wanju, Korea
2Research Institute of Pharmaceutical Sciences, Woosuk University, Wanju, Korea

Correspondence to:Hyo Jung Kim, hyojungkim@woosuk.ac.kr

Received: April 15, 2022; Revised: May 26, 2022; Accepted: June 11, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Proteins are constantly synthesized and degraded to maintain protein homeostasis. The ubiquitin-proteasome system is well known as a targeted protein breakdown process in eukaryotic cells. Although the archaea and actinobacterial families have a relevant system, many prokaryotic cells lack proteasomes. HslUV, a bacterial heat shock protein, is known to perform a proteasomal activity. HslU functions as ATPase, HslV is responsible for protease, and both activities are tightly connected in the two-component HslUV system. Here, we identified the characteristics of HslV from Staphylococcus aureus. Although only multimeric HslUV shows its activity, monomer S. aureus HslV remains stable and successfully crystallized. This study will provide a detailed understanding of the bacterial proteasomal system.

Keywords: HslU, HslV, bacterial proteasomal system, ubiquitin-proteasome, Staphylococcus aureus

Introduction

Cells continuously create and destroy proteins to maintain protein homeostasis; this cycle is referred to as protein turnover. The protein turnover rate can change in response to internal or external stimuli, including hormones, inflammation, or low energy status (Rolfs et al. 2021). Consequently, misfolded, damaged, or aged proteins are removed from the cellular protein pool and replaced with new proteins. Protein degradation occurs in eukaryotes via two types of proteolytic machineries: the lysosome and proteasome (Ciechanover 2005). In lysosomal proteolysis, proteins are engulfed by membrane-enclosed vesicles, such as autophagosomes or endocytic vesicles. Conversely, the ubiquitin-proteasome system uses a diverse collection of E1, E2, and E3 ubiquitin ligases to selectively target proteins for degradation by the proteasome (Park et al. 2020). The proteasome is a 2,500-kDa cylindrical protein complex containing a 20S catalytic core particle capped by either one or two 19S regulatory particles (Tanaka 2009; Choi et al. 2021). The 20S core particle is responsible for the proteasome’s proteolytic activities using its central chamber, while the 19S regulatory particle recognizes polyubiquitnated substrates. All 20S core particles are composed of the outer (α subunits) and inner (β subunits) heptameric rings (Kunjappu and Hochstrasser 2014). A major function of the α subunit ring is to regulate entry into the central proteolytic chamber, while the β subunit cleaves targeted proteins. However, most bacteria do not have proteasomes. Instead, molecular architecture HslUV (originally discovered in Escherichia coli) and CodWY (originally discovered in Bacillus subtilis) are considered bacterial counterparts of the eukaryotic proteasome (Kavalchuk et al. 2022). HslV and CodW harbor the protease activity that shows a similar primary sequence with the eukaryotic β subunit of the 20S core particle, while HslU and CodY provide ATPase activity. Furthermore, the protease and ATPase function together during protein degradation (Rohrwild et al. 1996; Sousa et al. 2000; Jeong et al. 2020) (Fig. 1).

Figure 1. E. coli HslUV complex. The HslUV complex consists of two HslU hexamers and the dodecameric HslV, “double-donut” hexameric rings (PDB ID 1G3I). The proteolytic active sites are located in the inner chamber of HslV. HslU identifies and translocates target proteins into this chamber using ATP.

In this research, a preliminary study of HslV from Staphylococcus aureus was conducted. S. aureus causes mild to severe infectious diseases, ranging from skin infection to life-threatening conditions. However, current management regimens are limited because of their ability to develop resistance to almost all antibiotics (Tong et al. 2015). Therefore, understanding the bacterial adaptation mechanism to environmental changes is indispensable for developing better therapeutics. S. aureus has adapted to stresses by developing response mechanisms, which include heat shock proteins that are produced to refold or destroy damaged proteins (Anderson et al. 2006). Furthermore, S. aureus HslUV is a “prokaryotic proteasome” that degrades specific proteins in an ATP-dependent reaction (Sousa et al. 2000). Here, we overexpressed, crystallized, and studied the properties of S. aureus HslV. As the first step of structural determination, our research will promote a better understanding of the HslUV mechanism on a molecular basis.

Materials and Methods

Expression and purification of S. aureus HslV

The SAV1253 gene coding for HslV was amplified from the genomic DNA of S. aureus Mu50 by polymerase chain reaction. NdeI and XhoI restriction sites were used for cloning using a pET-21a(+) vector (Novagen, USA). The resulting construct has eight additional residues (LEHHHHHH) that encode the C-terminal hexahistidine tag. The sequences of cloned genes were confirmed by DNA sequencing. The recombinant plasmid was transformed into E. coli BL21 (DE3). Cells were grown in LB medium supplemented with ampicillin (50 μg/mL). Recombinant protein HslV expression was induced by adding isopropyl β-D-1-thiogalactopyranoside to 0.5 mM when the OD600 reached 0.5. Cells were harvested following centrifugation at 4,000 × g at 4℃ after an additional 4 h of growth. The cell pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, and 500 mM NaCl) and disrupted using an Ultrasonic processor (Cole-Parmer, USA). Cell lysate was centrifuged at 20,000 × g for 1 h at 4℃. The cleared supernatant was purified by binding to a Ni-NTA (Ni-nitrilotriacetate) affinity column (Qiagen, Germany) and eluted with an elution buffer containing 200 mM imidazole. Furthermore, purification and buffer exchanges were achieved by size-exclusion chromatography. The HslV was prepared with a 10 mg/mL concentration in 50 mM Tris-HCl, pH 7.5, and 200 mM NaCl and loaded onto a Superdex 75 (100/300GL) column (GE Healthcare Life Science, USA) that was previously equilibrated with the same buffer. The purified HslV was concentrated at 10 mg/ml by ultrafiltration in 10,000 Da molecular mass cut-off spin columns (Millipore, USA).

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was conducted according to the Laemmli method using 15% (w/v) polyacrylamide gel (Laemmli 1970). The samples were treated with 1% (w/v) SDS and 5% (v/v) 2-mercaptoethanol before electrophoresis in a vertical Mini Gel system (Bio-Rad Laboratories, USA). The proteins were stained with Coomassie Brilliant Blue R250 (Thermo Scientific, USA).

Results

Oligomeric state of S. aureus HslV

Size-exclusion chromatography was employed to determine the oligomeric state of S. aureus HslV. A major peak appeared at a molecular mass of approximately 20 kDa, indicating that the protein is predominately monomeric. The corresponding SDS-PAGE indicates the main band at approximately 20 kDa with > 95% purity (Fig. 2). Considering the calculated mass of the S. aureus HslV (19.8 kDa), the size-exclusion chromatography result is consistent with the monomeric state. Although HslV only functions when it forms multiple complexes, monomeric S. aureus HslV shows stable properties. The S. aureus HslV monomer is stable overnight at room temperature and does not indicate any sign of degradation. This finding does not agree with the previous study, which revealed a trimeric state of S. aureus HslV in solution (Jeong et al. 2020). This discrepancy would be due to the different final buffer conditions that affect electrostatic and hydrophobic inter-residue interactions (pH, metal, and salt concentration). Additionally, the hexahistidine affinity tag was attached to the N-terminal in the previous study, but here, we used the C-terminal hexahistidine tagged construct. Since S. aureus HslV undergoes N-terminal cleavage for activation, an additional tag would alter the oligomerization state. The monomer state shows a structurally stable sandwich fold, certifying steady monomeric characterization in solution.

Figure 2. Monomeric state of S. aureus HslV. The size-exclusion chromatography of S. aureus HslV and corresponding SDS-PAGE are shown. The major peak at the molecular weight of approximately 20 kDa indicates a monomeric state.

Crystallization of S. aureus HslV

Crystals of S. aureus HslV were grown using sitting-drop vapor diffusion at 20℃. Initial crystallization conditions were established using screening kits from Hampton Research (Crystal screen I and II, Index, PEG/Ion, and MembFac) and Emerald Biosystems (Wizard I, II, III, and IV). For the optimal growth of HslV crystals, 1 μL (10 mg/mL) of the protein was mixed with 1 μL of precipitant solution (30% PEG400, 100 mM CAPS, and pH 10.5) and was equilibrated against a 1-mL reservoir of the precipitant solution. This condition yields short rod-shaped crystals that grew to dimensions 0.1 × 0.05 × 0.05 mm in 5 days (Fig. 3). Crystals were flash frozen in a stream of nitrogen at 100 K, and diffraction data were collected at the BL-5C beamline of the Pohang Accelerator Laboratory, Republic of Korea.

Figure 3. Morphology of S. aureus HslV crystals. Rod-shaped S. aureus HslV crystals are obtained in 5 days.

Data collection and processing

For data collection under cryogenic conditions, the crystals were transferred to a cryoprotectant solution with 20% (v/v) glycerol under crystallization conditions for several minutes before being flash frozen in a stream of nitrogen gas at 100 K. Data were obtained from ADSC Quantum 270 CCD in beamline 5C of Pohang Accelerator Laboratory, Republic of Korea (Fig. 4). The peaks obtained from consecutive diffraction images were indexed and integrated using the Scalepack package of HKL2000 software (Otwinowski and Minor 1997). The integrated data were processed to refine the electron density by applying a scale factor. The unit cell of S. aureus HslV crystals is a C-centered monoclinic in the Bravais lattice, and its space group was C2, with a large cell parameter: a = 221.407, b = 209,523, and c = 6309.842. This indicates that many monomers are packed in an asymmetric unit (Table 1).

Table 1 . Crystallographic data collection.

S. aureus HslV
Data collection
BeamlinePAL-5C
Wavelength (Å)0.97
Rotation range per image (°)1
Total rotation range (°)180
Exposure time per image (s)1
Space groupC2
Unit cell parameters (Å)a = 221.407
b = 209.523
c = 6309.842
Resolution (Å)40.0−3.5
Total no. of reflections31556
Rmerge0.76 (0.80)a)
CC1/20.335
I/sigma14.9 (13.0)

a)Numbers in parentheses indicate the statistics for the last resolution shell..



Figure 4. A representative diffraction image. A diffraction image from S. aureus HslV was obtained at the BL-5C beamline of the Pohang Accelerator Laboratory. The resolution circle for 3.0 Å is shown.

Discussion

The HslUV is a large complex with > 600 kDa molecular weight, 125 Å width, and 150 Å height. Two hexamers of HslV and 1-2 hexamers of HslU consist the HslUV arrangement. The HslV forms an internal cavity to cleave target proteins, and HslU activates HslV by N-terminal cleavage. S. aureus HslV utilizes Thr9 as a nucleophile after its exposure by removal of the N-terminal loop (Jeong et al. 2020). Herein, we showed that S. aureus HslV forms a stable monomer and can crystallize. To solve the structure, molecular replacement methods were applied with the program Phaser within the CCP4 suite using the available template HslV (PDB ID 6KR1) as the search model. Since the protein is expected to have an intact N-terminal structure, identifying the differences from the N-terminal-cleaved structure (PDB ID 6KR1) will improve the understanding of the HslUV activation mechanism.

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Acknowledgements

We thank the beamline staff members at Pohang Light Source (BL-5C), Republic of Korea. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) (No. 2022R1F1A1065273) and “Leaders in INdustry-university Cooperation 3.0” Project, supported by the Ministry of Education and National Research Foundation of Korea.

Fig 1.

Figure 1.E. coli HslUV complex. The HslUV complex consists of two HslU hexamers and the dodecameric HslV, “double-donut” hexameric rings (PDB ID 1G3I). The proteolytic active sites are located in the inner chamber of HslV. HslU identifies and translocates target proteins into this chamber using ATP.
Drug Targets and Therapeutics 2022; 1: 40-44https://doi.org/10.58502/DTT.22.007

Fig 2.

Figure 2.Monomeric state of S. aureus HslV. The size-exclusion chromatography of S. aureus HslV and corresponding SDS-PAGE are shown. The major peak at the molecular weight of approximately 20 kDa indicates a monomeric state.
Drug Targets and Therapeutics 2022; 1: 40-44https://doi.org/10.58502/DTT.22.007

Fig 3.

Figure 3.Morphology of S. aureus HslV crystals. Rod-shaped S. aureus HslV crystals are obtained in 5 days.
Drug Targets and Therapeutics 2022; 1: 40-44https://doi.org/10.58502/DTT.22.007

Fig 4.

Figure 4.A representative diffraction image. A diffraction image from S. aureus HslV was obtained at the BL-5C beamline of the Pohang Accelerator Laboratory. The resolution circle for 3.0 Å is shown.
Drug Targets and Therapeutics 2022; 1: 40-44https://doi.org/10.58502/DTT.22.007

Table 1 Crystallographic data collection

S. aureus HslV
Data collection
BeamlinePAL-5C
Wavelength (Å)0.97
Rotation range per image (°)1
Total rotation range (°)180
Exposure time per image (s)1
Space groupC2
Unit cell parameters (Å)a = 221.407
b = 209.523
c = 6309.842
Resolution (Å)40.0−3.5
Total no. of reflections31556
Rmerge0.76 (0.80)a)
CC1/20.335
I/sigma14.9 (13.0)

a)Numbers in parentheses indicate the statistics for the last resolution shell.


References

  1. Anderson KL, Roberts C, Disz T, Vonstein V, Hwang K, Overbeek R, Olson PD, Projan SJ, Dunman PM (2006) Characterization of the Staphylococcus aureus heat shock, cold shock, stringent, and SOS responses and their effects on log-phase mRNA turnover. J Bacteriol 188:6739-6756. doi: 10.1128/JB.00609-06.
    Pubmed KoreaMed CrossRef
  2. Choi WH, Kim S, Park S, Lee MJ (2021) Concept and application of circulating proteasomes. Exp Mol Med 53:1539-1546. doi: 10.1038/s12276-021-00692-x.
    Pubmed KoreaMed CrossRef
  3. Ciechanover A (2005) Proteolysis: from the lysosome to ubiquitin and the proteasome. Nat Rev Mol Cell Biol 6:79-87. doi: 10.1038/nrm1552.
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