Mol. Cells 2020; 43(8): 694-704
Published online July 22, 2020
https://doi.org/10.14348/molcells.2020.0074
© The Korean Society for Molecular and Cellular Biology
Correspondence to : hanc210@snu.ac.kr (NCH); arkwon@dhu.ac.kr (ARK)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
HslUV is a bacterial heat shock protein complex consisting of the AAA+ ATPase component HslU and the protease component HslV. HslV is a threonine (Thr) protease employing the N-terminal Thr residue in the mature protein as the catalytic residue. To date, HslUV from Gram-negative bacteria has been extensively studied. However, the mechanisms of action and activation of HslUV from Gram-positive bacteria, which have an additional N-terminal sequence before the catalytic Thr residue, remain to be revealed. In this study, we determined the crystal structures of HslV from the Gram-positive bacterium Staphylococcus aureus with and without HslU in the crystallization conditions. The structural comparison suggested that a structural transition to the symmetric form of HslV was triggered by ATP-bound HslU. More importantly, the additional N-terminal sequence was cleaved in the presence of HslU and ATP, exposing the Thr9 residue at the N-terminus and activating the ATP-dependent protease activity. Further biochemical studies demonstrated that the exposed N-terminal Thr residue is critical for catalysis with binding to the symmetric HslU hexamer. Since eukaryotic proteasomes have a similar additional N-terminal sequence, our results will improve our understanding of the common molecular mechanisms for the activation of proteasomes.
Keywords ATP-dependent protease, crystal structure, heat shock protein, HslU, HslV, methicillin-resistant Staphylococcus aureus, proteasome
Proteasomes in eukaryotic cells are responsible for the active degradation of intracellular proteins conjugated with ubiquitin. Proteasome-dependent protein degradation is essential in the regulation of cellular physiology as well as in amino acid recycling. The cytosolic 26S proteasome consists of a 19S core particle and a 20S regulatory particle (Rousseau and Bertolotti, 2018). The core particle has a proteolytic chamber for protein degradation composed of seven α-type and seven β-type subunits resembling a barrel-shaped cylinder. Three of the seven β-type subunits (β-1, β-2, and β-5) in the core particle belong to the threonine (Thr) protease family, because they employ the N-terminal Thr residue in the mature form as the catalytic nucleophile. The N-terminal 11-72 residues before the Thr residue are processed in their mature forms (Culp and Wright, 2017; Groll et al., 1997; Huber et al., 2016; Thomson and Rivett, 1996).
The bacterial heat shock protein complex HslUV consists of the protease component HslV and the AAA+ ATPase component HslU, which function together as a two-component protease (Chuang et al., 1993; Rohrwild et al., 1996). HslUV is considered a bacterial counterpart of eukaryotic proteasomes because HslV exhibits the highest sequence similarity to the β-type subunits of eukaryotic proteasomes. To date, HslUV has been most extensively studied in Gram-negative bacteria (Kwon et al., 2003; Sousa et al., 2000; Yoo et al., 1996). Like the eukaryotic β-type subunit of proteasomes, HslV from Gram-negative bacteria belongs to the Thr protease family (Yoo et al., 1997). The catalytic Thr residue (Thr2; numbering is based on the open reading frame sequence in this study) of
HslUV from
Among Gram-positive bacteria, CodWX (previously known as HslUV) of
The Gram-positive bacterium
The gene encoding HslV (National Center for Biotechnology Information reference sequence: BAB57415.1) was polymerase chain reaction-amplified from genomic DNA of
The
The enzymatic assays of HslUV were performed at 25°C in 100-μl reaction mixtures containing 100 μM substrate Z-GGL-AMC (Sigma-Aldrich, USA), 10 μM HslV and/or HslU in SEC buffer, and 1 mM ATP. Degradation of the substrate was measured based on the fluorescence from the liberated AMC at excitation and emission wavelengths of 380 nm and 460 nm, respectively (Rohrwild et al., 1996).
The SaHslUV complex was pre-incubated in SEC buffer in the presence or absence of 1 mM ATP for 30 min at room temperature, and then each sample was loaded onto an SEC column (Superdex 200 increase 10/300 GL; GE Healthcare). The first peak was collected, and each fraction of the first peak was re-subjected to SEC with and without 1 mM ATP in SEC buffer.
SaHslV and SaHslU were prepared with 100 µl of 2 mg/ml protein sample in SEC buffer with and without 1 mM ATP or ADP. Each sample was subjected to SEC on a Superdex 200 increase 10/300 GL column (GE Healthcare). The molecular sizes and oligomerization states of the complexes were measured by MALS (DAWN HELIOS II; Wyatt Technology, USA).
The purified protein complex from SEC in the presence of 1 mM ATP was subjected to 4%-20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, USA) and the protein bands were blotted onto a PVDF membrane (GE Healthcare) in a buffer containing 10 mM CAPS (pH 11.0) and 20% methanol. After staining with Coomassie Brilliant Blue R-250, the shorter HslV band was cut from the membrane and subjected to N-terminal amino acid sequence analysis by Proteinworks (Korea) using Edman degradation.
The atomic coordinates and structure factors (codes 6KR1, 6KUI, and 6KWW) have been deposited in the Protein Data Bank (http://wwpdb.org/).
We performed a sequence alignment with HslV protein from
We first characterized the oligomeric properties of SaHslV, which are essential for the function of the complex. The oligomeric state of SaHslV in solution was measured with the purified protein (the calculated monomer size is ~20 kDa) by SEC-MALS. The results indicated that SaHslV failed to form a stable hexameric ring arrangement since it mostly behaved as a trimer (~61 kDa) with a minor portion of a higher oligomer (Fig. 1B). This was different from the results of HslV from
To examine the structural features of SaHslV, we determined a crystal structure of the SaHslV protein at 2.0 Å resolution. The asymmetric unit contained 12 protomers displaying the typical stacked double hexameric ring arrangement observed in
The conformations of the group A and B protomers are similar to those of
This SaHslV structure also revealed a substantial conformational difference in the regions of the β-hairpin motif (residues 27-39), which may be responsible for the interaction with the main body of the adjacent protomer (Fig. 2D). The β-hairpin motifs of the group A and B protomers are similar to the corresponding region of
By contrast, the β-hairpin of group C is oriented outward from the center of the hexameric ring, which is the opposite of those of groups A and B, and
To analyze the structural changes of SaHslV upon complex formation with SaHslU, we attempted to determine the complex structure of SaHslV and SaHslU with a 1:1 molar ratio of the two proteins in the presence of ATP. However, the crystal contained the SaHslV protein alone, but not the SaHslU protein; overall, this SaHslV structure was similar to the HslV conformation that formed symmetric hexamers, as observed in the dodecameric forms of
Remarkably, the additional N-terminal sequence was not seen in the electron density maps of this symmetric hexamer structure. Thr9 was the first N-terminal residue seen in the electron density maps of all chains of the symmetric HslV structure (Fig. 3B). To identify the actual N-terminal residue of SaHslV in this symmetric HslV structure, we proceeded with the following studies.
Due to the structural differences in the N-terminal region between the twisted and symmetric hexamers, we analyzed the sizes of the proteins by SDS-PAGE. A down-shifted band (a shorter fragment) representing ~20% of the SaHslV protein was generated by incubation with SaHslU and 1 mM ATP. However, a more prolonged incubation did not increase the proportion of the shorter fragment. To identify the actual N-terminal residue of the shorter fragment, we performed N-terminal sequencing analyses of the band by Edman degradation. The shorter fragment of SaHslV started from Thr9 in the N-terminal sequencing analysis, which is in good agreement with the crystal structure of the symmetric SaHslV structure. These results indicated that SaHslV is processed into a shorter fragment with Thr9 as the first N-terminal residue.
To examine whether the ATP hydrolysis is required or just ATP binding is enough for this cleavage of SaHslV, we performed the N-terminal cleavage experiment with non-hydrolysable ATP analogue (AMP-PNP) (Supplementary Fig. S4). As a result, we found that the AMP-PNP activated the HslU-dependent processing of HslV as well as ATP. These results indicate that the ATP binding to HslU, but not ATP hydrolysis, is required for this cleavage at the N-terminal region of HslV.
We next examined the ability of SaHslV to bind SaHslU. Mixtures of the processed and unprocessed forms of SaHslV with SaHslU in the presence or absence of 1 mM ATP or ADP were subjected to SEC (Fig. 4A). The peak indicating the SaHslUV complex at a larger molecular size appeared only in the presence of ATP. SDS-PAGE analysis of the peak fractions showed that most of the SaHslV in the complex was in the processed form rather than the full-length form. However, the SaHslV in the ADP-containing buffer was not processed and did not form a complex with SaHslU (Supplementary Fig. S5).
To examine the stability of the HslUV complex, we subjected the peak fractions of the complex to SEC in the presence or absence of ATP in the buffer (Fig. 4B). The HslUV complex was intact in the ATP-containing buffer, whereas the complex was dissociated when ATP was absent from the buffer. These results indicate that ATP is required to process SaHslV and to form and maintain the complex with SaHslU (Supplementary Fig. S6).
Proteolytic assays using the synthetic peptide substrate Z-GGL-AMC revealed that only the processed SaHslV in complex with SaHslU was active (Fig. 4C). By contrast, the unprocessed SaHslV did not exhibit proteolytic activity even in the presence of SaHslU and ATP. These results indicate that processed SaHslV, SaHslU, and ATP are indispensable for substrate cleavage by the functional complex. Since the processed and functional SaHslV has a Thr residue at the N-terminus of the protein like typical Thr proteases, it is evident that SaHslV belongs to the Thr protease family, which is different from CodW from
In the case of
To examine the processing of the N-terminal regions in the mutant SaHslV proteins, we analyzed the proteins in the presence of SaHslU and ATP by SDS-PAGE. The amount of the processed form of SaHslV (SaHslV* in Fig. 5B) showed a correlation with the relative proteolytic activities of the complex shown in Fig. 5A. Thus, our results indicate that the apparent proteolytic activity represents the processing efficiency of SaHslV, and Thr9 plays a crucial role in the processing of the N-terminal region. Moreover, our results further indicate that Ser2, Thr4, and His7 in the additional N-terminal sequence partly contribute to this N-terminal processing.
To examine the structural characteristics of SaHslU, we determined the crystal structure of SaHslU without nucleotides at a resolution of 3.0 Å (Supplementary Fig. S7). The asymmetric unit contained 24 protomers, consisting of two copies of a dodecameric unit (two hexameric rings). The overall structure of the hexameric SaHslU units was similar to those of HslU from
The C-terminal region of HslU only in the ATP-bound form is known to interact with HslV in the complex directly (Bochtler et al., 2000). Thus the conformation of the C-terminal region is essential for binding to HslV. The C-terminal region of SaHslU is folded into the NBD as a short helix in our nucleotide-free structure, which is different from that of HslU from
We determined the crystal structures of SaHslV and SaHslU from the Gram-positive bacterium
Prokaryotic HslV proteases were believed to be activated by removing only the N-terminal formyl-Met residue. Given the sequence similarity to CodW from
In this study, we showed that ATP-bound SaHslU was involved in processing the N-termianal extension region of SaHslV. We suggest that the C-terminal binding motifs of SaHslU would be extended out to bind SaHslV for the processing and activation of SaHslV in the presence of ATP. However, we don’t believe that only the C-terminal peptide of HslU could promote the processing of SaHslV because two of six grooves for the binding interface for the C-terminal motif of HslU are absent in the twisted form of SaHslV. The proper hexameric arrangement and orientation of the C-terminal motif of SaHslU would be more critical for the binding to the twisted form of SaHslV. Since the processed form of SaHslV is more suitable for binding to the ATP-bound form of SaHslU, the binding between the C-terminal motif of SaHslU and SaHslV would be mutually cooperative with the processing of SaHslV. The further complex structure of SaHslU and SaHslV would be required to gain the structural explanation of the cooperative activation of SaHslV.
Although this study demonstrated the SaHslU-dependent cleavage of the additional N-terminal sequence of SaHslV is required for protease activity, only a small portion of SaHslV was cleaved under our experimental conditions. We speculate that other factors or stimuli increase or trigger the cleavage and activation of SaHslV in collaboration with SaHslU. Further study is necessary to identify these factors or stimuli, which will provide insight into the cellular roles of HslUV in the bacteria.
Eukaryotic and archaeal protease components undergo autoprocessing of the additional N-terminal sequence to activate protease activity. In this respect, the activation mechanism of SaHslV requiring SaHslU and ATP is similar to the autolysis-dependent activation of the β-5 subunit in the yeast 26S proteasome. The structure of the yeast β-5 subunit, which is catalytically active, suggests that six residues, Thr1, Asp17, Lys33, Arg19, Ser129, and Asp166 (numbering is based on the matured structure), are involved in autolysis-dependent activation (Huber et al., 2016). Of these, Thr1, Asp17, Lys33, and Ser129 of the yeast β-subunit align with Thr9, Asp25, Lys42, and Ser133 of SaHslV, respectively, in the structural superposition (Supplementary Fig. S9). The residues of the yeast β-5 subunit were better matched to the processed form of SaHslV (Supplementary Fig. S9A) than to the unprocessed structures of SaHslV (Supplementary Figs. S9B-S9D). These findings suggest that the structural change of SaHslV by SaHslU is required for the correct alignment of the residues for autolysis.
This study revealed the overall similarities of SaHslV to eukaryotic proteasomes in terms of the activation by N-terminal processing. However, the detailed activation mechanisms and triggers are still unclear in both SaHslV and eukaryotic proteasomes. Thus, identifying the stimuli that trigger cleavage of the N-terminal extension region will provide important clues about the cellular functions of SaHslUV complex-like Thr proteases. Determining the detailed activation mechanisms and functions of the SaHslUV complex will also improve our understanding of eukaryotic proteasomes.
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through Agriculture, Food and Rural Affairs Research Center Support Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant 710012-03-1-HD120 to N.C.H.), and also was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science & ICT (grant NRF-2019M3E5D6063871 to N.C.H.). S.J. was supported by the BK21 Plus Program of the Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea. This work was also supported through the National Research Foundation of Korea (NRF) funded by the Ministry of Education of the Korean government (2017R1D1A1B03033857 to A.R.K.). We used beamlines 5C and 7A at Pohang Accelerator Laboratory (Pohang, Republic of Korea) and the MALS facility at the Korea Basic Science Institute (Ochang, Republic of Korea).
The authors have no potential conflicts of interest to disclose.
X-ray diffraction data
Twisted SaHslV | Symmetric SaHslV | SaHslU | |
---|---|---|---|
Data collection | |||
Beam line | PAL7A | PAL5C | PAL5C |
Wavelength (Å) | 0.97933 | 0.97960 | 0.97960 |
Space group | |||
Cell dimensions | |||
a, b, c (Å) | 94.3, 94.3, 227.4 | 95.6, 108.7, 122.9 | 146.5, 189.5, 215.6 |
α, β, γ (°) | 90, 90, 120 | 90, 90, 90 | 90, 93, 90 |
Resolution (Å) | 50.0-2.00 (2.03-2.00) | 50.0-2.33 (2.37-2.33) | 50.0-3.00 (3.05-3.00) |
Rmerge | 0.076 (0.453) | 0.070 (0.351) | 0.052 (0.619) |
Rpim | 0.028 (0.260) | 0.021 (0.184) | 0.032 (0.255) |
I/σI | 17.65 (2.3) | 24.5 (2.5) | 19.06 (1.96) |
Completeness (%) | 98.6 (96.6) | 98.6 (90.7) | 93.1 (87.4) |
Redundancy | 6.5 (3.6) | 8.7 (3.9) | 6.2 (5.7) |
Refinement | |||
Resolution (Å) | 33.2-2.0 | 41.3-2.3 | 36.4-3.0 |
No. of reflections | 125,784 | 25,104 | 212,267 |
Rwork/Rfree | 0.215/0.264 | 0.229/0.2856 | 0.2349/0.2792 |
No. of total atoms | 16,581 | 4,060 | 57,793 |
Wilson B-factor (Å) | 31.8 | 35.15 | 50.0 |
R.M.S deviations | |||
Bond lengths (Å) | 0.002 | 0.003 | 0.002 |
Bond angles (°) | 0.397 | 0.600 | 0.51 |
Ramachandran plot | |||
Favored (%) | 96.9 | 96.67 | 96.60 |
Allowed (%) | 3.1 | 3.3 | 3.37 |
Outliers (%) | 0.00 | 0.00 | 0.03 |
PDB code | 6KR1 | 6KUI | 6KWW |
Numbers in parentheses indicate the statistics for the last resolution shell.
Mol. Cells 2020; 43(8): 694-704
Published online August 31, 2020 https://doi.org/10.14348/molcells.2020.0074
Copyright © The Korean Society for Molecular and Cellular Biology.
Soyeon Jeong1 , Jinsook Ahn1
, Ae-Ran Kwon2,*
, and Nam-Chul Ha1,*
1Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, and Research Institute for Agriculture and Life Sciences, CALS, Seoul National University, Seoul 08826, Korea, 2Department of Beauty Care, College of Medical Science, Daegu Haany University, Gyeongsan 38610, Korea
Correspondence to:hanc210@snu.ac.kr (NCH); arkwon@dhu.ac.kr (ARK)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
HslUV is a bacterial heat shock protein complex consisting of the AAA+ ATPase component HslU and the protease component HslV. HslV is a threonine (Thr) protease employing the N-terminal Thr residue in the mature protein as the catalytic residue. To date, HslUV from Gram-negative bacteria has been extensively studied. However, the mechanisms of action and activation of HslUV from Gram-positive bacteria, which have an additional N-terminal sequence before the catalytic Thr residue, remain to be revealed. In this study, we determined the crystal structures of HslV from the Gram-positive bacterium Staphylococcus aureus with and without HslU in the crystallization conditions. The structural comparison suggested that a structural transition to the symmetric form of HslV was triggered by ATP-bound HslU. More importantly, the additional N-terminal sequence was cleaved in the presence of HslU and ATP, exposing the Thr9 residue at the N-terminus and activating the ATP-dependent protease activity. Further biochemical studies demonstrated that the exposed N-terminal Thr residue is critical for catalysis with binding to the symmetric HslU hexamer. Since eukaryotic proteasomes have a similar additional N-terminal sequence, our results will improve our understanding of the common molecular mechanisms for the activation of proteasomes.
Keywords: ATP-dependent protease, crystal structure, heat shock protein, HslU, HslV, methicillin-resistant Staphylococcus aureus, proteasome
Proteasomes in eukaryotic cells are responsible for the active degradation of intracellular proteins conjugated with ubiquitin. Proteasome-dependent protein degradation is essential in the regulation of cellular physiology as well as in amino acid recycling. The cytosolic 26S proteasome consists of a 19S core particle and a 20S regulatory particle (Rousseau and Bertolotti, 2018). The core particle has a proteolytic chamber for protein degradation composed of seven α-type and seven β-type subunits resembling a barrel-shaped cylinder. Three of the seven β-type subunits (β-1, β-2, and β-5) in the core particle belong to the threonine (Thr) protease family, because they employ the N-terminal Thr residue in the mature form as the catalytic nucleophile. The N-terminal 11-72 residues before the Thr residue are processed in their mature forms (Culp and Wright, 2017; Groll et al., 1997; Huber et al., 2016; Thomson and Rivett, 1996).
The bacterial heat shock protein complex HslUV consists of the protease component HslV and the AAA+ ATPase component HslU, which function together as a two-component protease (Chuang et al., 1993; Rohrwild et al., 1996). HslUV is considered a bacterial counterpart of eukaryotic proteasomes because HslV exhibits the highest sequence similarity to the β-type subunits of eukaryotic proteasomes. To date, HslUV has been most extensively studied in Gram-negative bacteria (Kwon et al., 2003; Sousa et al., 2000; Yoo et al., 1996). Like the eukaryotic β-type subunit of proteasomes, HslV from Gram-negative bacteria belongs to the Thr protease family (Yoo et al., 1997). The catalytic Thr residue (Thr2; numbering is based on the open reading frame sequence in this study) of
HslUV from
Among Gram-positive bacteria, CodWX (previously known as HslUV) of
The Gram-positive bacterium
The gene encoding HslV (National Center for Biotechnology Information reference sequence: BAB57415.1) was polymerase chain reaction-amplified from genomic DNA of
The
The enzymatic assays of HslUV were performed at 25°C in 100-μl reaction mixtures containing 100 μM substrate Z-GGL-AMC (Sigma-Aldrich, USA), 10 μM HslV and/or HslU in SEC buffer, and 1 mM ATP. Degradation of the substrate was measured based on the fluorescence from the liberated AMC at excitation and emission wavelengths of 380 nm and 460 nm, respectively (Rohrwild et al., 1996).
The SaHslUV complex was pre-incubated in SEC buffer in the presence or absence of 1 mM ATP for 30 min at room temperature, and then each sample was loaded onto an SEC column (Superdex 200 increase 10/300 GL; GE Healthcare). The first peak was collected, and each fraction of the first peak was re-subjected to SEC with and without 1 mM ATP in SEC buffer.
SaHslV and SaHslU were prepared with 100 µl of 2 mg/ml protein sample in SEC buffer with and without 1 mM ATP or ADP. Each sample was subjected to SEC on a Superdex 200 increase 10/300 GL column (GE Healthcare). The molecular sizes and oligomerization states of the complexes were measured by MALS (DAWN HELIOS II; Wyatt Technology, USA).
The purified protein complex from SEC in the presence of 1 mM ATP was subjected to 4%-20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, USA) and the protein bands were blotted onto a PVDF membrane (GE Healthcare) in a buffer containing 10 mM CAPS (pH 11.0) and 20% methanol. After staining with Coomassie Brilliant Blue R-250, the shorter HslV band was cut from the membrane and subjected to N-terminal amino acid sequence analysis by Proteinworks (Korea) using Edman degradation.
The atomic coordinates and structure factors (codes 6KR1, 6KUI, and 6KWW) have been deposited in the Protein Data Bank (http://wwpdb.org/).
We performed a sequence alignment with HslV protein from
We first characterized the oligomeric properties of SaHslV, which are essential for the function of the complex. The oligomeric state of SaHslV in solution was measured with the purified protein (the calculated monomer size is ~20 kDa) by SEC-MALS. The results indicated that SaHslV failed to form a stable hexameric ring arrangement since it mostly behaved as a trimer (~61 kDa) with a minor portion of a higher oligomer (Fig. 1B). This was different from the results of HslV from
To examine the structural features of SaHslV, we determined a crystal structure of the SaHslV protein at 2.0 Å resolution. The asymmetric unit contained 12 protomers displaying the typical stacked double hexameric ring arrangement observed in
The conformations of the group A and B protomers are similar to those of
This SaHslV structure also revealed a substantial conformational difference in the regions of the β-hairpin motif (residues 27-39), which may be responsible for the interaction with the main body of the adjacent protomer (Fig. 2D). The β-hairpin motifs of the group A and B protomers are similar to the corresponding region of
By contrast, the β-hairpin of group C is oriented outward from the center of the hexameric ring, which is the opposite of those of groups A and B, and
To analyze the structural changes of SaHslV upon complex formation with SaHslU, we attempted to determine the complex structure of SaHslV and SaHslU with a 1:1 molar ratio of the two proteins in the presence of ATP. However, the crystal contained the SaHslV protein alone, but not the SaHslU protein; overall, this SaHslV structure was similar to the HslV conformation that formed symmetric hexamers, as observed in the dodecameric forms of
Remarkably, the additional N-terminal sequence was not seen in the electron density maps of this symmetric hexamer structure. Thr9 was the first N-terminal residue seen in the electron density maps of all chains of the symmetric HslV structure (Fig. 3B). To identify the actual N-terminal residue of SaHslV in this symmetric HslV structure, we proceeded with the following studies.
Due to the structural differences in the N-terminal region between the twisted and symmetric hexamers, we analyzed the sizes of the proteins by SDS-PAGE. A down-shifted band (a shorter fragment) representing ~20% of the SaHslV protein was generated by incubation with SaHslU and 1 mM ATP. However, a more prolonged incubation did not increase the proportion of the shorter fragment. To identify the actual N-terminal residue of the shorter fragment, we performed N-terminal sequencing analyses of the band by Edman degradation. The shorter fragment of SaHslV started from Thr9 in the N-terminal sequencing analysis, which is in good agreement with the crystal structure of the symmetric SaHslV structure. These results indicated that SaHslV is processed into a shorter fragment with Thr9 as the first N-terminal residue.
To examine whether the ATP hydrolysis is required or just ATP binding is enough for this cleavage of SaHslV, we performed the N-terminal cleavage experiment with non-hydrolysable ATP analogue (AMP-PNP) (Supplementary Fig. S4). As a result, we found that the AMP-PNP activated the HslU-dependent processing of HslV as well as ATP. These results indicate that the ATP binding to HslU, but not ATP hydrolysis, is required for this cleavage at the N-terminal region of HslV.
We next examined the ability of SaHslV to bind SaHslU. Mixtures of the processed and unprocessed forms of SaHslV with SaHslU in the presence or absence of 1 mM ATP or ADP were subjected to SEC (Fig. 4A). The peak indicating the SaHslUV complex at a larger molecular size appeared only in the presence of ATP. SDS-PAGE analysis of the peak fractions showed that most of the SaHslV in the complex was in the processed form rather than the full-length form. However, the SaHslV in the ADP-containing buffer was not processed and did not form a complex with SaHslU (Supplementary Fig. S5).
To examine the stability of the HslUV complex, we subjected the peak fractions of the complex to SEC in the presence or absence of ATP in the buffer (Fig. 4B). The HslUV complex was intact in the ATP-containing buffer, whereas the complex was dissociated when ATP was absent from the buffer. These results indicate that ATP is required to process SaHslV and to form and maintain the complex with SaHslU (Supplementary Fig. S6).
Proteolytic assays using the synthetic peptide substrate Z-GGL-AMC revealed that only the processed SaHslV in complex with SaHslU was active (Fig. 4C). By contrast, the unprocessed SaHslV did not exhibit proteolytic activity even in the presence of SaHslU and ATP. These results indicate that processed SaHslV, SaHslU, and ATP are indispensable for substrate cleavage by the functional complex. Since the processed and functional SaHslV has a Thr residue at the N-terminus of the protein like typical Thr proteases, it is evident that SaHslV belongs to the Thr protease family, which is different from CodW from
In the case of
To examine the processing of the N-terminal regions in the mutant SaHslV proteins, we analyzed the proteins in the presence of SaHslU and ATP by SDS-PAGE. The amount of the processed form of SaHslV (SaHslV* in Fig. 5B) showed a correlation with the relative proteolytic activities of the complex shown in Fig. 5A. Thus, our results indicate that the apparent proteolytic activity represents the processing efficiency of SaHslV, and Thr9 plays a crucial role in the processing of the N-terminal region. Moreover, our results further indicate that Ser2, Thr4, and His7 in the additional N-terminal sequence partly contribute to this N-terminal processing.
To examine the structural characteristics of SaHslU, we determined the crystal structure of SaHslU without nucleotides at a resolution of 3.0 Å (Supplementary Fig. S7). The asymmetric unit contained 24 protomers, consisting of two copies of a dodecameric unit (two hexameric rings). The overall structure of the hexameric SaHslU units was similar to those of HslU from
The C-terminal region of HslU only in the ATP-bound form is known to interact with HslV in the complex directly (Bochtler et al., 2000). Thus the conformation of the C-terminal region is essential for binding to HslV. The C-terminal region of SaHslU is folded into the NBD as a short helix in our nucleotide-free structure, which is different from that of HslU from
We determined the crystal structures of SaHslV and SaHslU from the Gram-positive bacterium
Prokaryotic HslV proteases were believed to be activated by removing only the N-terminal formyl-Met residue. Given the sequence similarity to CodW from
In this study, we showed that ATP-bound SaHslU was involved in processing the N-termianal extension region of SaHslV. We suggest that the C-terminal binding motifs of SaHslU would be extended out to bind SaHslV for the processing and activation of SaHslV in the presence of ATP. However, we don’t believe that only the C-terminal peptide of HslU could promote the processing of SaHslV because two of six grooves for the binding interface for the C-terminal motif of HslU are absent in the twisted form of SaHslV. The proper hexameric arrangement and orientation of the C-terminal motif of SaHslU would be more critical for the binding to the twisted form of SaHslV. Since the processed form of SaHslV is more suitable for binding to the ATP-bound form of SaHslU, the binding between the C-terminal motif of SaHslU and SaHslV would be mutually cooperative with the processing of SaHslV. The further complex structure of SaHslU and SaHslV would be required to gain the structural explanation of the cooperative activation of SaHslV.
Although this study demonstrated the SaHslU-dependent cleavage of the additional N-terminal sequence of SaHslV is required for protease activity, only a small portion of SaHslV was cleaved under our experimental conditions. We speculate that other factors or stimuli increase or trigger the cleavage and activation of SaHslV in collaboration with SaHslU. Further study is necessary to identify these factors or stimuli, which will provide insight into the cellular roles of HslUV in the bacteria.
Eukaryotic and archaeal protease components undergo autoprocessing of the additional N-terminal sequence to activate protease activity. In this respect, the activation mechanism of SaHslV requiring SaHslU and ATP is similar to the autolysis-dependent activation of the β-5 subunit in the yeast 26S proteasome. The structure of the yeast β-5 subunit, which is catalytically active, suggests that six residues, Thr1, Asp17, Lys33, Arg19, Ser129, and Asp166 (numbering is based on the matured structure), are involved in autolysis-dependent activation (Huber et al., 2016). Of these, Thr1, Asp17, Lys33, and Ser129 of the yeast β-subunit align with Thr9, Asp25, Lys42, and Ser133 of SaHslV, respectively, in the structural superposition (Supplementary Fig. S9). The residues of the yeast β-5 subunit were better matched to the processed form of SaHslV (Supplementary Fig. S9A) than to the unprocessed structures of SaHslV (Supplementary Figs. S9B-S9D). These findings suggest that the structural change of SaHslV by SaHslU is required for the correct alignment of the residues for autolysis.
This study revealed the overall similarities of SaHslV to eukaryotic proteasomes in terms of the activation by N-terminal processing. However, the detailed activation mechanisms and triggers are still unclear in both SaHslV and eukaryotic proteasomes. Thus, identifying the stimuli that trigger cleavage of the N-terminal extension region will provide important clues about the cellular functions of SaHslUV complex-like Thr proteases. Determining the detailed activation mechanisms and functions of the SaHslUV complex will also improve our understanding of eukaryotic proteasomes.
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through Agriculture, Food and Rural Affairs Research Center Support Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant 710012-03-1-HD120 to N.C.H.), and also was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science & ICT (grant NRF-2019M3E5D6063871 to N.C.H.). S.J. was supported by the BK21 Plus Program of the Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea. This work was also supported through the National Research Foundation of Korea (NRF) funded by the Ministry of Education of the Korean government (2017R1D1A1B03033857 to A.R.K.). We used beamlines 5C and 7A at Pohang Accelerator Laboratory (Pohang, Republic of Korea) and the MALS facility at the Korea Basic Science Institute (Ochang, Republic of Korea).
The authors have no potential conflicts of interest to disclose.
. X-ray diffraction data.
Twisted SaHslV | Symmetric SaHslV | SaHslU | |
---|---|---|---|
Data collection | |||
Beam line | PAL7A | PAL5C | PAL5C |
Wavelength (Å) | 0.97933 | 0.97960 | 0.97960 |
Space group | |||
Cell dimensions | |||
a, b, c (Å) | 94.3, 94.3, 227.4 | 95.6, 108.7, 122.9 | 146.5, 189.5, 215.6 |
α, β, γ (°) | 90, 90, 120 | 90, 90, 90 | 90, 93, 90 |
Resolution (Å) | 50.0-2.00 (2.03-2.00) | 50.0-2.33 (2.37-2.33) | 50.0-3.00 (3.05-3.00) |
Rmerge | 0.076 (0.453) | 0.070 (0.351) | 0.052 (0.619) |
Rpim | 0.028 (0.260) | 0.021 (0.184) | 0.032 (0.255) |
I/σI | 17.65 (2.3) | 24.5 (2.5) | 19.06 (1.96) |
Completeness (%) | 98.6 (96.6) | 98.6 (90.7) | 93.1 (87.4) |
Redundancy | 6.5 (3.6) | 8.7 (3.9) | 6.2 (5.7) |
Refinement | |||
Resolution (Å) | 33.2-2.0 | 41.3-2.3 | 36.4-3.0 |
No. of reflections | 125,784 | 25,104 | 212,267 |
Rwork/Rfree | 0.215/0.264 | 0.229/0.2856 | 0.2349/0.2792 |
No. of total atoms | 16,581 | 4,060 | 57,793 |
Wilson B-factor (Å) | 31.8 | 35.15 | 50.0 |
R.M.S deviations | |||
Bond lengths (Å) | 0.002 | 0.003 | 0.002 |
Bond angles (°) | 0.397 | 0.600 | 0.51 |
Ramachandran plot | |||
Favored (%) | 96.9 | 96.67 | 96.60 |
Allowed (%) | 3.1 | 3.3 | 3.37 |
Outliers (%) | 0.00 | 0.00 | 0.03 |
PDB code | 6KR1 | 6KUI | 6KWW |
Numbers in parentheses indicate the statistics for the last resolution shell..
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