EcoliWiki - User contributions [en]

PMID:20637416

2010-10-22T16:09:35Z

Hourylab: Table edited by Hourylab via TableEdit

{{RightTOC}}
<protect>
 '''Kimber, MS, Yu, AY, Borg, M, Leung, E, Chan, HS and Houry, WA''' (2010) Structural and theoretical studies indicate that the cylindrical protease ClpP samples extended and compact conformations.''Structure'' '''18''':798-808

{|{{Prettytable}} align = 'center' width= '70%'
|-
|{{TableHeadingColor}} |'''Abstract''' || <protect>The highly conserved ClpP protease consists of two heptameric rings that interact by the interdigitation of an alpha-helix beta strand handle domain motif to form a tetradecameric cylinder. We previously proposed that protease dynamics results in the temporary unstructuring of interacting pairs of handle domains, opening transient equatorial side pores that allow for peptide egress. Here, we report the structure of an Escherichia coli ClpP mutant in which each opposing pair of protomers is linked by a disulfide bond. This structure resembles the compact structures of Streptococcus pneumoniae, Mycobacterium tuberculosis, and Plasmodium falciparum ClpPs, rather than the active, extended structures that have previously been determined for E. coli ClpPs. The structural data, along with normal mode analysis, support a model whereby the ClpP cylinder switches dynamically between an active extended state required for substrate degradation and an inactive compact state allowing peptide product release.</protect>
|-
|{{TableHeadingColor}} nowrap |'''Links''' ||
* [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=20637416 PubMed]
Online version:[http://dx.doi.org/10.1016/j.str.2010.04.008 10.1016/j.str.2010.04.008]
|}
</protect>
==Significance {{HelpIcon|Help:Literature}} ==
{{LitSignificance}}

== Useful Materials and Methods {{HelpIcon|Help:Literature}}==
{{LitMaterials}}

==Annotations {{HelpIcon|Help:Literature}}==
<protect>

{| id="D4c98fc00b6919" class=" tableEdit GO_table_reference"
|- {{table heading style}}
!|Gene product!!Qualifier!!GO ID!!GO term name!!Evidence Code!!with/from!!Aspect!!Notes!!Status
|-
|
ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog
|

|
GO:0000166
|
nucleotide binding
|
IEA: Inferred from Electronic Annotation
|
InterPro:IPR001907
InterPro:IPR018215

|
F
|

|
complete
|-
|
ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog
|

|
GO:0008236
|
serine-type peptidase activity
|
IEA: Inferred from Electronic Annotation
|
InterPro:IPR001907
InterPro:IPR018215

|
F
|

|
complete

|- class="tableEdit_footer"
|[{{SERVER}}{{SCRIPTPATH}}?title=Special:TableEdit&id=63ce1649aab8b3180235b0af7618f667.176149.D4c98fc00b6919&page=176149&pagename={{FULLPAGENAMEE}}&type=0&template=GO_table_reference edit table] || || || || || || || ||
|}
</protect>

==EcoliWiki Links {{HelpIcon|Help:Literature}} ==
{{LitLinks}}

==See also==

==References==
{{RefHelp}}
<references/>

PMID:20637416

2010-10-15T22:36:24Z

Hourylab: Table edited by Hourylab via TableEdit

{{RightTOC}}
<protect>
 '''Kimber, MS, Yu, AY, Borg, M, Leung, E, Chan, HS and Houry, WA''' (2010) Structural and theoretical studies indicate that the cylindrical protease ClpP samples extended and compact conformations.''Structure'' '''18''':798-808

{|{{Prettytable}} align = 'center' width= '70%'
|-
|{{TableHeadingColor}} |'''Abstract''' || <protect>The highly conserved ClpP protease consists of two heptameric rings that interact by the interdigitation of an alpha-helix beta strand handle domain motif to form a tetradecameric cylinder. We previously proposed that protease dynamics results in the temporary unstructuring of interacting pairs of handle domains, opening transient equatorial side pores that allow for peptide egress. Here, we report the structure of an Escherichia coli ClpP mutant in which each opposing pair of protomers is linked by a disulfide bond. This structure resembles the compact structures of Streptococcus pneumoniae, Mycobacterium tuberculosis, and Plasmodium falciparum ClpPs, rather than the active, extended structures that have previously been determined for E. coli ClpPs. The structural data, along with normal mode analysis, support a model whereby the ClpP cylinder switches dynamically between an active extended state required for substrate degradation and an inactive compact state allowing peptide product release.</protect>
|-
|{{TableHeadingColor}} nowrap |'''Links''' ||
* [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=20637416 PubMed]
Online version:[http://dx.doi.org/10.1016/j.str.2010.04.008 10.1016/j.str.2010.04.008]
|}
</protect>
==Significance {{HelpIcon|Help:Literature}} ==
{{LitSignificance}}

== Useful Materials and Methods {{HelpIcon|Help:Literature}}==
{{LitMaterials}}

==Annotations {{HelpIcon|Help:Literature}}==
<protect>

{| id="D4c98fc00b6919" class=" tableEdit GO_table_reference"
|- {{table heading style}}
!|Gene product!!Qualifier!!GO ID!!GO term name!!Evidence Code!!with/from!!Aspect!!Notes!!Status
|-
|
ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog
|

|
GO:0000166
|
nucleotide binding
|
IEA: Inferred from Electronic Annotation
|
InterPro:IPR018215
InterPro:IPR001907

|
F
|

|
complete

|- class="tableEdit_footer"
|[{{SERVER}}{{SCRIPTPATH}}?title=Special:TableEdit&id=63ce1649aab8b3180235b0af7618f667.176149.D4c98fc00b6919&page=176149&pagename={{FULLPAGENAMEE}}&type=0&template=GO_table_reference edit table] || || || || || || || ||
|}
</protect>

==EcoliWiki Links {{HelpIcon|Help:Literature}} ==
{{LitLinks}}

==See also==

==References==
{{RefHelp}}
<references/>

clpP:Gene Product(s

2010-10-15T18:27:29Z

Hourylab:

ClpP is a highly conserved serine protease present throughout bacteria and eukaryota, ; it is absent in archaea, mollicutes, and some fungi <ref name='15037239:'/>. ClpP was first identified in Escherichia coli <ref name='3549708:'/><ref name='3049606:'/>, and more than 400 studies have been published on it since its discovery. The X-ray structure of ClpP has been solved from several different organisms. All the structures show similar features: the protease is comprised of 14 subunits arranged into two heptameric rings forming a cylindrical-like structure which encloses a large chamber containing the protease active sites. Entrance into the chamber occurs through axial pores in the cylinder. ClpP typically forms complexes with AAA+ (ATPases Associated with various cellular Activities) chaperones that denature substrates and then translocate them through the axial pores into the proteolytic chamber of the protease for degradation. ClpP degrades proteins into peptides of about 7 to 8 residues <ref name='16229481:'/> which are subsequently released from the chamber. It is one of the five major proteases in E. coli which includes ClpXP, ClpAP, HslUV, FtSH and Lon protease. Proteases play an essential role in protein quality control by removing short-lived regulatory proteins, as well as proteins that are misfolded and damaged, thus maintaining cellular homeostasis. A significant proportion of protein degradation in the cell is carried out by oligomeric, cylindrical, self-compartmentalized, energy-dependent proteases. ClpP (Caseinolytic Protease) is a representative member of these cylindrical proteases.

ClpP is found in all bacteria sequenced to date except for Mollicutes <ref name='12937164:'/>. The majority of bacteria contain only one copy of ClpP, although there are exceptions such as for actinobacteria, chlamydiae, cyanobacteria, and others which have multiple isoforms of the protease. In addition to ClpP, cyanobacteria contain a ClpP paralog that does not have all 3 residues of the Ser-His-Asp catalytic triad. This inactive version of ClpP is called ClpR <ref name='11278690:'/> and is mostly present in plants in addition to cyanobacteria. For example, cyanobacteria Synechocystis PCC6803 contains three copies of ClpP and one copy of ClpR <ref name='11278690:'/><ref name='12101312:'/>.

ClpP was first identified and mostly studied in Escherichia coli, which contains only one copy of the protease. E. coli ClpP consists of 207 amino acids including an N-terminal prosequence which acts as a regulatory peptide and undergoes autocatalytic cleavage during folding to yield a mature ClpP of 193 residues <ref name='2197276:'/>. In E. coli, ClpP forms complexes with AAA+ chaperones, ClpX and ClpA, which belong to the Clp/Hsp100 family. ClpA is an 83 kDa ATPase that contains two AAA+ domains, while ClpX contains only one AAA+ domain (46 kDa), which is homologous to the second AAA+ domain of ClpA. ClpA and ClpX are hexameric chaperones that can stack onto one or both ends of ClpP to form ClpAP or ClpXP holoenzyme complexes. Substrates are recognized, unfolded by ClpX or ClpA, and then threaded into the ClpP proteolytic chamber through the narrow axial pores for degradation. Only the unfolding and threading by the chaperones require ATP binding and hydrolysis, while proteolysis by ClpP does not require nucleotide hydrolysis. The mechanism by which ClpX or ClpA binds to and unfolds substrates is poorly understood. Other bacteria contain ClpA paralogs, such as ClpC, ClpE, and ClpL <ref name='16629660:'/>.

The most general substrates for the E. coli ClpXP or ClpAP system are those with an SsrA C-terminal tag <ref name='8584937:'/>. The SsrA tag consists of 11 hydrophobic residues that are added by a tmRNA, also called SsrA or 10Sa RNA, to the C-terminus of nascent chains whose translation is stalled on the ribosome, targeting them for degradation <ref name='9573050:'/><ref name='10485712:'/><ref name='11737633:'/>. SsrA tagging can be thought of as the prokaryotic equivalent to eukaryotic ubiquitination.

Without ATPase components, only small peptides can enter the ClpP chamber <ref name='8027082:'/><ref name='9390551:'/>; hence, ClpP on its own cannot degrade folded proteins. Recently, however, a group at Bayer described the discovery of acyldepsipeptides (ADEPs) that, when added to ClpP, allow the protease to degrade folded native proteins in the absence of its cognate chaperones <ref name='16200071:'/>. The drug was initially found to rescue mice challenged with lethal infections of Enterococcus faecalis and Staphylococcus aureus, and the target for the drug was then identified to be ClpP. ClpP is activated by ADEPs to degrade, or at least to cleave, folded proteins in the absence of the cognate Clp ATPases. The consequences of the action of the drug in vivo, is that the ADEPs cause unregulated proteolysis by ClpP, subsequently, triggering cell death in gram-positive bacteria. Gram-negative bacteria, on the other hand, were found to be able to survive drug treatment because they have efficient efflux pumps to remove the drug from the cell, but deletion of those pumps or the use of permeabilizing agents made gram-negative bacteria vulnerable to ADEP treatment as well <ref name='16200071:'/>. The mechanism of how ADEPs activate ClpP in the absence of its cognate chaperones is currently unknown. The discovery of ADEPs may lead to development of a novel class of antimicrobial agents.

Understanding how the ClpP system functions has direct implications on understanding the mechanism of function of other more complex cylindrical proteases such as the proteasome. ClpP shares many similarities with the proteasome both functionally and structurally. The arrangement of subunits in ClpP is strikingly analogous to that of eukaryotic and archaeal 20S proteasomes, which is composed of 2 heptameric β-subunit rings enclosing the active site sandwiched between 2 heptameric rings of α subunits. Another protease HslV (ClpQ), also known as the ‘bacterial proteasome’, exhibits analogous packing except that the cylinder is composed of two hexameric rings. The similarity in the cylindrical structural arrangement probably underlines the commonality in the mechanism of function of these proteases. Efforts currently underway in several laboratories should further enhance our understanding of the mechanism of function of these degradative machines. These ongoing efforts might have direct clinical consequences as ClpP has now been shown to be a legitimate antibacterial drug target.

References:
<ref name='15037239:'/> Wong, P. and Houry, W.A. (2004) Chaperone networks in bacteria: analysis of protein homeostasis in minimal cells. J. Struct. Biol. 146, 79-89.
<ref name='3549708:'/> Katayama-Fujimura, Y., Gottesman, S. and Maurizi, M.R. (1987) A multiple-component, ATP-dependent protease from Escherichia coli. J. Biol. Chem. 262, 4477-4485.
<ref name='3049606:'/> Katayama, Y., Gottesman, S., Pumphrey, J., Rudikoff, S., Clark, W.P. and Maurizi, M.R. (1988) The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component. J. Biol. Chem. 263, 15226-15236.
<ref name='16229481:'/> Choi, K.H. and Licht, S. (2005) Control of peptide product sizes by the energy-dependent protease ClpAP. Biochemistry. 44, 13921-13931.
<ref name='12937164:'/> Wojtyra, U.A., Thibault, G., Tuite, A. and Houry, W.A. (2003) The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function. J. Biol. Chem. 278, 48981-48990.
<ref name='11278690:'/> Peltier, J.B., Ytterberg, J., Liberles, D.A., Roepstorff, P. and van Wijk, K.J. (2001) Identification of a 350-kDa ClpP protease complex with 10 different Clp isoforms in chloroplasts of Arabidopsis thaliana. J. Biol. Chem. 276, 16318-16327.
<ref name='12101312:'/> Schelin, J., Lindmark, F. and Clarke, A.K. (2002) The clpP multigene family for the ATP-dependent Clp protease in the cyanobacterium Synechococcus. Microbiology 148, 2255-2265.
<ref name='2197276:'/> Maurizi, M.R., Clark, W.P., Kim, S.H. and Gottesman, S. (1990) Clp P represents a unique family of serine proteases. J. Biol. Chem. 265, 12546-12552.
<ref name='16629660:'/> Butler, S.M., Festa, R.A., Pearce, M.J. and Darwin, K.H. (2006) Self-compartmentalized bacterial proteases and pathogenesis. Mol. Microbiol. 60, 553-562.
<ref name='8584937:'/> Keiler, K.C., Waller, P.R. and Sauer, R.T. (1996) Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271, 990-993.
<ref name='9573050:'/> Gottesman, S., Roche, E., Zhou, Y. and Sauer, R.T. (1998) The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev. 12, 1338-1347.
<ref name='10485712:'/> Weber-Ban, E.U., Reid, B.G., Miranker, A.D. and Horwich, A.L. (1999) Global unfolding of a substrate protein by the Hsp100 chaperone ClpA. Nature 401, 90-93.
<ref name='11737633:'/> Gillet, R. and Felden, B. (2001) Emerging views on tmRNA-mediated protein tagging and ribosome rescue. Mol. Microbiol. 42, 879-885.
<ref name='8027082:'/> Thompson, M.W., Singh, S.K. and Maurizi, M.R. (1994) Processive degradation of proteins by the ATP-dependent Clp protease from Escherichia coli. Requirement for the multiple array of active sites in ClpP but not ATP hydrolysis. J. Biol. Chem. 269, 18209-18215.
<ref name='9390551:'/> Gottesman, S., Maurizi, M.R. and Wickner, S. (1997) Regulatory subunits of energy-dependent proteases. Cell 91, 435-438.
<ref name='16200071:'/> Brotz-Oesterhelt, H. et al. (2005) Dysregulation of bacterial proteolytic machinery by a new class of antibiotics. Nat. Med. 11, 1082-1087.

clpP:Gene Product(s

2010-10-15T15:45:48Z

Hourylab:

ClpP is a highly conserved serine protease present throughout bacteria and eukaryota, ; it is absent in archaea, mollicutes, and some fungi <ref name='15037239:'/>. ClpP was first identified in Escherichia coli <ref name='3549708:'/><ref name='3049606:'/>, and more than 400 studies have been published on it since its discovery. The X-ray structure of ClpP has been solved from several different organisms. All the structures show similar features: the protease is comprised of 14 subunits arranged into two heptameric rings forming a cylindrical-like structure which encloses a large chamber containing the protease active sites. Entrance into the chamber occurs through axial pores in the cylinder. ClpP typically forms complexes with AAA+ (ATPases Associated with various cellular Activities) chaperones that denature substrates and then translocate them through the axial pores into the proteolytic chamber of the protease for degradation. ClpP degrades proteins into peptides of about 7 to 8 residues <ref name='16229481:'/> which are subsequently released from the chamber. It is one of the five major proteases in E. coli which includes ClpXP, ClpAP, HslUV, FtSH and Lon protease. Proteases play an essential role in protein quality control by removing short-lived regulatory proteins, as well as proteins that are misfolded and damaged, thus maintaining cellular homeostasis. A significant proportion of protein degradation in the cell is carried out by oligomeric, cylindrical, self-compartmentalized, energy-dependent proteases. ClpP (Caseinolytic Protease) is a representative member of these cylindrical proteases.

ClpP is found in all bacteria sequenced to date except for Mollicutes <ref name='12937164:'/>. The majority of bacteria contain only one copy of ClpP, although there are exceptions such as for actinobacteria, chlamydiae, cyanobacteria, and others which have multiple isoforms of the protease. In addition to ClpP, cyanobacteria contain a ClpP paralog that does not have all 3 residues of the Ser-His-Asp catalytic triad. This inactive version of ClpP is called ClpR <ref name='11278690:'/> and is mostly present in plants in addition to cyanobacteria. For example, cyanobacteria Synechocystis PCC6803 contains three copies of ClpP and one copy of ClpR <ref name='11278690:'/><ref name='12101312:'/>.

ClpP was first identified and mostly studied in Escherichia coli, which contains only one copy of the protease. E. coli ClpP consists of 207 amino acids including an N-terminal prosequence which acts as a regulatory peptide and undergoes autocatalytic cleavage during folding to yield a mature ClpP of 193 residues <ref name='2197276:'/>. In E. coli, ClpP forms complexes with AAA+ chaperones, ClpX and ClpA, which belong to the Clp/Hsp100 family. ClpA is an 83 kDa ATPase that contains two AAA+ domains, while ClpX contains only one AAA+ domain (46 kDa), which is homologous to the second AAA+ domain of ClpA. ClpA and ClpX are hexameric chaperones that can stack onto one or both ends of ClpP to form ClpAP or ClpXP holoenzyme complexes. Substrates are recognized, unfolded by ClpX or ClpA, and then threaded into the ClpP proteolytic chamber through the narrow axial pores for degradation. Only the unfolding and threading by the chaperones require ATP binding and hydrolysis, while proteolysis by ClpP does not require nucleotide hydrolysis. The mechanism by which ClpX or ClpA binds to and unfolds substrates is poorly understood. Other bacteria contain ClpA paralogs, such as ClpC, ClpE, and ClpL <ref name='16629660:'/>.

The most general substrates for the E. coli ClpXP or ClpAP system are those with an SsrA C-terminal tag <ref name='8584937:'/>. The SsrA tag consists of 11 hydrophobic residues that are added by a tmRNA, also called SsrA or 10Sa RNA, to the C-terminus of nascent chains whose translation is stalled on the ribosome, targeting them for degradation <ref name='9573050:'/><ref name='10485712:'/><ref name='11737633:'/>. SsrA tagging can be thought of as the prokaryotic equivalent to eukaryotic ubiquitination.

Without ATPase components, only small peptides can enter the ClpP chamber <ref name='12101312:'/>[14,15]; hence, ClpP on its own cannot degrade folded proteins. Recently, however, a group at Bayer described the discovery of acyldepsipeptides (ADEPs) that, when added to ClpP, allow the protease to degrade folded native proteins in the absence of its cognate chaperones <ref name='12101312:'/>[16]. The drug was initially found to rescue mice challenged with lethal infections of Enterococcus faecalis and Staphylococcus aureus, and the target for the drug was then identified to be ClpP. ClpP is activated by ADEPs to degrade, or at least to cleave, folded proteins in the absence of the cognate Clp ATPases. The consequences of the action of the drug in vivo, is that the ADEPs cause unregulated proteolysis by ClpP, subsequently, triggering cell death in gram-positive bacteria. Gram-negative bacteria, on the other hand, were found to be able to survive drug treatment because they have efficient efflux pumps to remove the drug from the cell, but deletion of those pumps or the use of permeabilizing agents made gram-negative bacteria vulnerable to ADEP treatment as well <ref name='12101312:'/>[16]. The mechanism of how ADEPs activate ClpP in the absence of its cognate chaperones is currently unknown. The discovery of ADEPs may lead to development of a novel class of antimicrobial agents.

Understanding how the ClpP system functions has direct implications on understanding the mechanism of function of other more complex cylindrical proteases such as the proteasome. ClpP shares many similarities with the proteasome both functionally and structurally. The arrangement of subunits in ClpP is strikingly analogous to that of eukaryotic and archaeal 20S proteasomes, which is composed of 2 heptameric β-subunit rings enclosing the active site sandwiched between 2 heptameric rings of α subunits. Another protease HslV (ClpQ), also known as the ‘bacterial proteasome’, exhibits analogous packing except that the cylinder is composed of two hexameric rings. The similarity in the cylindrical structural arrangement probably underlines the commonality in the mechanism of function of these proteases. Efforts currently underway in several laboratories should further enhance our understanding of the mechanism of function of these degradative machines. These ongoing efforts might have direct clinical consequences as ClpP has now been shown to be a legitimate antibacterial drug target.

References:
[1] Wong, P. and Houry, W.A. (2004) Chaperone networks in bacteria: analysis of protein homeostasis in minimal cells. J. Struct. Biol. 146, 79-89.
[2] Katayama-Fujimura, Y., Gottesman, S. and Maurizi, M.R. (1987) A multiple-component, ATP-dependent protease from Escherichia coli. J. Biol. Chem. 262, 4477-4485.
[3] Katayama, Y., Gottesman, S., Pumphrey, J., Rudikoff, S., Clark, W.P. and Maurizi, M.R. (1988) The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component. J. Biol. Chem. 263, 15226-15236.
[4] Choi, K.H. and Licht, S. (2005) Control of peptide product sizes by the energy-dependent protease ClpAP. Biochemistry. 44, 13921-13931.
[5] Wojtyra, U.A., Thibault, G., Tuite, A. and Houry, W.A. (2003) The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function. J. Biol. Chem. 278, 48981-48990.
[6] Peltier, J.B., Ytterberg, J., Liberles, D.A., Roepstorff, P. and van Wijk, K.J. (2001) Identification of a 350-kDa ClpP protease complex with 10 different Clp isoforms in chloroplasts of Arabidopsis thaliana. J. Biol. Chem. 276, 16318-16327.
[7] Schelin, J., Lindmark, F. and Clarke, A.K. (2002) The clpP multigene family for the ATP-dependent Clp protease in the cyanobacterium Synechococcus. Microbiology 148, 2255-2265.
[8] Maurizi, M.R., Clark, W.P., Kim, S.H. and Gottesman, S. (1990) Clp P represents a unique family of serine proteases. J. Biol. Chem. 265, 12546-12552.
[9] Butler, S.M., Festa, R.A., Pearce, M.J. and Darwin, K.H. (2006) Self-compartmentalized bacterial proteases and pathogenesis. Mol. Microbiol. 60, 553-562.
[10] Keiler, K.C., Waller, P.R. and Sauer, R.T. (1996) Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271, 990-993.
[11] Gottesman, S., Roche, E., Zhou, Y. and Sauer, R.T. (1998) The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev. 12, 1338-1347.
[12] Weber-Ban, E.U., Reid, B.G., Miranker, A.D. and Horwich, A.L. (1999) Global unfolding of a substrate protein by the Hsp100 chaperone ClpA. Nature 401, 90-93.
[13] Gillet, R. and Felden, B. (2001) Emerging views on tmRNA-mediated protein tagging and ribosome rescue. Mol. Microbiol. 42, 879-885.
[14] Thompson, M.W., Singh, S.K. and Maurizi, M.R. (1994) Processive degradation of proteins by the ATP-dependent Clp protease from Escherichia coli. Requirement for the multiple array of active sites in ClpP but not ATP hydrolysis. J. Biol. Chem. 269, 18209-18215.
[15] Gottesman, S., Maurizi, M.R. and Wickner, S. (1997) Regulatory subunits of energy-dependent proteases. Cell 91, 435-438.
[16] Brotz-Oesterhelt, H. et al. (2005) Dysregulation of bacterial proteolytic machinery by a new class of antibiotics. Nat. Med. 11, 1082-1087.

clpP:Gene Product(s

2010-10-15T15:30:31Z

Hourylab:

ClpP is a highly conserved serine protease present throughout bacteria and eukaryota, ; it is absent in archaea, mollicutes, and some fungi <ref name='15037239:'/>[1]. ClpP was first identified in Escherichia coli [2,3], and more than 400 studies have been published on it since its discovery. The X-ray structure of ClpP has been solved from several different organisms. All the structures show similar features: the protease is comprised of 14 subunits arranged into two heptameric rings forming a cylindrical-like structure which encloses a large chamber containing the protease active sites. Entrance into the chamber occurs through axial pores in the cylinder. ClpP typically forms complexes with AAA+ (ATPases Associated with various cellular Activities) chaperones that denature substrates and then translocate them through the axial pores into the proteolytic chamber of the protease for degradation. ClpP degrades proteins into peptides of about 7 to 8 residues [4] which are subsequently released from the chamber. It is one of the five major proteases in E. coli which includes ClpXP, ClpAP, HslUV, FtSH and Lon protease. Proteases play an essential role in protein quality control by removing short-lived regulatory proteins, as well as proteins that are misfolded and damaged, thus maintaining cellular homeostasis. A significant proportion of protein degradation in the cell is carried out by oligomeric, cylindrical, self-compartmentalized, energy-dependent proteases. ClpP (Caseinolytic Protease) is a representative member of these cylindrical proteases.

ClpP is found in all bacteria sequenced to date except for Mollicutes [5]. The majority of bacteria contain only one copy of ClpP, although there are exceptions such as for actinobacteria, chlamydiae, cyanobacteria, and others which have multiple isoforms of the protease. In addition to ClpP, cyanobacteria contain a ClpP paralog that does not have all 3 residues of the Ser-His-Asp catalytic triad. This inactive version of ClpP is called ClpR [6] and is mostly present in plants in addition to cyanobacteria. For example, cyanobacteria Synechocystis PCC6803 contains three copies of ClpP and one copy of ClpR [6,7].

ClpP was first identified and mostly studied in Escherichia coli, which contains only one copy of the protease. E. coli ClpP consists of 207 amino acids including an N-terminal prosequence which acts as a regulatory peptide and undergoes autocatalytic cleavage during folding to yield a mature ClpP of 193 residues [8]. In E. coli, ClpP forms complexes with AAA+ chaperones, ClpX and ClpA, which belong to the Clp/Hsp100 family. ClpA is an 83 kDa ATPase that contains two AAA+ domains, while ClpX contains only one AAA+ domain (46 kDa), which is homologous to the second AAA+ domain of ClpA. ClpA and ClpX are hexameric chaperones that can stack onto one or both ends of ClpP to form ClpAP or ClpXP holoenzyme complexes. Substrates are recognized, unfolded by ClpX or ClpA, and then threaded into the ClpP proteolytic chamber through the narrow axial pores for degradation. Only the unfolding and threading by the chaperones require ATP binding and hydrolysis, while proteolysis by ClpP does not require nucleotide hydrolysis. The mechanism by which ClpX or ClpA binds to and unfolds substrates is poorly understood. Other bacteria contain ClpA paralogs, such as ClpC, ClpE, and ClpL [9].

The most general substrates for the E. coli ClpXP or ClpAP system are those with an SsrA C-terminal tag [10]. The SsrA tag consists of 11 hydrophobic residues that are added by a tmRNA, also called SsrA or 10Sa RNA, to the C-terminus of nascent chains whose translation is stalled on the ribosome, targeting them for degradation [11-13]. SsrA tagging can be thought of as the prokaryotic equivalent to eukaryotic ubiquitination.

Without ATPase components, only small peptides can enter the ClpP chamber [14,15]; hence, ClpP on its own cannot degrade folded proteins. Recently, however, a group at Bayer described the discovery of acyldepsipeptides (ADEPs) that, when added to ClpP, allow the protease to degrade folded native proteins in the absence of its cognate chaperones [16]. The drug was initially found to rescue mice challenged with lethal infections of Enterococcus faecalis and Staphylococcus aureus, and the target for the drug was then identified to be ClpP. ClpP is activated by ADEPs to degrade, or at least to cleave, folded proteins in the absence of the cognate Clp ATPases. The consequences of the action of the drug in vivo, is that the ADEPs cause unregulated proteolysis by ClpP, subsequently, triggering cell death in gram-positive bacteria. Gram-negative bacteria, on the other hand, were found to be able to survive drug treatment because they have efficient efflux pumps to remove the drug from the cell, but deletion of those pumps or the use of permeabilizing agents made gram-negative bacteria vulnerable to ADEP treatment as well [16]. The mechanism of how ADEPs activate ClpP in the absence of its cognate chaperones is currently unknown. The discovery of ADEPs may lead to development of a novel class of antimicrobial agents.

Understanding how the ClpP system functions has direct implications on understanding the mechanism of function of other more complex cylindrical proteases such as the proteasome. ClpP shares many similarities with the proteasome both functionally and structurally. The arrangement of subunits in ClpP is strikingly analogous to that of eukaryotic and archaeal 20S proteasomes, which is composed of 2 heptameric β-subunit rings enclosing the active site sandwiched between 2 heptameric rings of α subunits. Another protease HslV (ClpQ), also known as the ‘bacterial proteasome’, exhibits analogous packing except that the cylinder is composed of two hexameric rings. The similarity in the cylindrical structural arrangement probably underlines the commonality in the mechanism of function of these proteases. Efforts currently underway in several laboratories should further enhance our understanding of the mechanism of function of these degradative machines. These ongoing efforts might have direct clinical consequences as ClpP has now been shown to be a legitimate antibacterial drug target.

References:
[1] Wong, P. and Houry, W.A. (2004) Chaperone networks in bacteria: analysis of protein homeostasis in minimal cells. J. Struct. Biol. 146, 79-89.
[2] Katayama-Fujimura, Y., Gottesman, S. and Maurizi, M.R. (1987) A multiple-component, ATP-dependent protease from Escherichia coli. J. Biol. Chem. 262, 4477-4485.
[3] Katayama, Y., Gottesman, S., Pumphrey, J., Rudikoff, S., Clark, W.P. and Maurizi, M.R. (1988) The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component. J. Biol. Chem. 263, 15226-15236.
[4] Choi, K.H. and Licht, S. (2005) Control of peptide product sizes by the energy-dependent protease ClpAP. Biochemistry. 44, 13921-13931.
[5] Wojtyra, U.A., Thibault, G., Tuite, A. and Houry, W.A. (2003) The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function. J. Biol. Chem. 278, 48981-48990.
[6] Peltier, J.B., Ytterberg, J., Liberles, D.A., Roepstorff, P. and van Wijk, K.J. (2001) Identification of a 350-kDa ClpP protease complex with 10 different Clp isoforms in chloroplasts of Arabidopsis thaliana. J. Biol. Chem. 276, 16318-16327.
[7] Schelin, J., Lindmark, F. and Clarke, A.K. (2002) The clpP multigene family for the ATP-dependent Clp protease in the cyanobacterium Synechococcus. Microbiology 148, 2255-2265.
[8] Maurizi, M.R., Clark, W.P., Kim, S.H. and Gottesman, S. (1990) Clp P represents a unique family of serine proteases. J. Biol. Chem. 265, 12546-12552.
[9] Butler, S.M., Festa, R.A., Pearce, M.J. and Darwin, K.H. (2006) Self-compartmentalized bacterial proteases and pathogenesis. Mol. Microbiol. 60, 553-562.
[10] Keiler, K.C., Waller, P.R. and Sauer, R.T. (1996) Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271, 990-993.
[11] Gottesman, S., Roche, E., Zhou, Y. and Sauer, R.T. (1998) The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev. 12, 1338-1347.
[12] Weber-Ban, E.U., Reid, B.G., Miranker, A.D. and Horwich, A.L. (1999) Global unfolding of a substrate protein by the Hsp100 chaperone ClpA. Nature 401, 90-93.
[13] Gillet, R. and Felden, B. (2001) Emerging views on tmRNA-mediated protein tagging and ribosome rescue. Mol. Microbiol. 42, 879-885.
[14] Thompson, M.W., Singh, S.K. and Maurizi, M.R. (1994) Processive degradation of proteins by the ATP-dependent Clp protease from Escherichia coli. Requirement for the multiple array of active sites in ClpP but not ATP hydrolysis. J. Biol. Chem. 269, 18209-18215.
[15] Gottesman, S., Maurizi, M.R. and Wickner, S. (1997) Regulatory subunits of energy-dependent proteases. Cell 91, 435-438.
[16] Brotz-Oesterhelt, H. et al. (2005) Dysregulation of bacterial proteolytic machinery by a new class of antibiotics. Nat. Med. 11, 1082-1087.

clpP:Gene Product(s

2010-10-13T16:05:02Z

Hourylab:

clpP:Gene Product(s

2010-10-13T16:04:12Z

Hourylab:

ClpP is a highly conserved serine protease present throughout bacteria and eukaryota, ; it is absent in archaea, mollicutes, and some fungi [1]. ClpP was first identified in Escherichia coli [2,3], and more than 400 studies have been published on it since its discovery. The X-ray structure of ClpP has been solved from several different organisms. All the structures show similar features: the protease is comprised of 14 subunits arranged into two heptameric rings forming a cylindrical-like structure which encloses a large chamber containing the protease active sites. Entrance into the chamber occurs through axial pores in the cylinder. ClpP typically forms complexes with AAA+ (ATPases Associated with various cellular Activities) chaperones that denature substrates and then translocate them through the axial pores into the proteolytic chamber of the protease for degradation. ClpP degrades proteins into peptides of about 7 to 8 residues [4] which are subsequently released from the chamber. It is one of the five major proteases in E. coli which includes ClpXP, ClpAP, HslUV, FtSH and Lon protease. Proteases play an essential role in protein quality control by removing short-lived regulatory proteins, as well as proteins that are misfolded and damaged, thus maintaining cellular homeostasis. A significant proportion of protein degradation in the cell is carried out by oligomeric, cylindrical, self-compartmentalized, energy-dependent proteases. ClpP (Caseinolytic Protease) is a representative member of these cylindrical proteases.

ClpP is found in all bacteria sequenced to date except for Mollicutes [5]. The majority of bacteria contain only one copy of ClpP, although there are exceptions such as for actinobacteria, chlamydiae, cyanobacteria, and others which have multiple isoforms of the protease. In addition to ClpP, cyanobacteria contain a ClpP paralog that does not have all 3 residues of the Ser-His-Asp catalytic triad. This inactive version of ClpP is called ClpR [6] and is mostly present in plants in addition to cyanobacteria. For example, cyanobacteria Synechocystis PCC6803 contains three copies of ClpP and one copy of ClpR [6,7].

ClpP was first identified and mostly studied in Escherichia coli, which contains only one copy of the protease. E. coli ClpP consists of 207 amino acids including an N-terminal prosequence which acts as a regulatory peptide and undergoes autocatalytic cleavage during folding to yield a mature ClpP of 193 residues [8]. In E. coli, ClpP forms complexes with AAA+ chaperones, ClpX and ClpA, which belong to the Clp/Hsp100 family. ClpA is an 83 kDa ATPase that contains two AAA+ domains, while ClpX contains only one AAA+ domain (46 kDa), which is homologous to the second AAA+ domain of ClpA. ClpA and ClpX are hexameric chaperones that can stack onto one or both ends of ClpP to form ClpAP or ClpXP holoenzyme complexes. Substrates are recognized, unfolded by ClpX or ClpA, and then threaded into the ClpP proteolytic chamber through the narrow axial pores for degradation. Only the unfolding and threading by the chaperones require ATP binding and hydrolysis, while proteolysis by ClpP does not require nucleotide hydrolysis. The mechanism by which ClpX or ClpA binds to and unfolds substrates is poorly understood. Other bacteria contain ClpA paralogs, such as ClpC, ClpE, and ClpL [9].

The most general substrates for the E. coli ClpXP or ClpAP system are those with an SsrA C-terminal tag [10]. The SsrA tag consists of 11 hydrophobic residues that are added by a tmRNA, also called SsrA or 10Sa RNA, to the C-terminus of nascent chains whose translation is stalled on the ribosome, targeting them for degradation [11-13]. SsrA tagging can be thought of as the prokaryotic equivalent to eukaryotic ubiquitination.

Without ATPase components, only small peptides can enter the ClpP chamber [14,15]; hence, ClpP on its own cannot degrade folded proteins. Recently, however, a group at Bayer described the discovery of acyldepsipeptides (ADEPs) that, when added to ClpP, allow the protease to degrade folded native proteins in the absence of its cognate chaperones [16]. The drug was initially found to rescue mice challenged with lethal infections of Enterococcus faecalis and Staphylococcus aureus, and the target for the drug was then identified to be ClpP. ClpP is activated by ADEPs to degrade, or at least to cleave, folded proteins in the absence of the cognate Clp ATPases. The consequences of the action of the drug in vivo, is that the ADEPs cause unregulated proteolysis by ClpP, subsequently, triggering cell death in gram-positive bacteria. Gram-negative bacteria, on the other hand, were found to be able to survive drug treatment because they have efficient efflux pumps to remove the drug from the cell, but deletion of those pumps or the use of permeabilizing agents made gram-negative bacteria vulnerable to ADEP treatment as well [16]. The mechanism of how ADEPs activate ClpP in the absence of its cognate chaperones is currently unknown. The discovery of ADEPs may lead to development of a novel class of antimicrobial agents.

Understanding how the ClpP system functions has direct implications on understanding the mechanism of function of other more complex cylindrical proteases such as the proteasome. ClpP shares many similarities with the proteasome both functionally and structurally. The arrangement of subunits in ClpP is strikingly analogous to that of eukaryotic and archaeal 20S proteasomes, which is composed of 2 heptameric β-subunit rings enclosing the active site sandwiched between 2 heptameric rings of α subunits. Another protease HslV (ClpQ), also known as the ‘bacterial proteasome’, exhibits analogous packing except that the cylinder is composed of two hexameric rings. The similarity in the cylindrical structural arrangement probably underlines the commonality in the mechanism of function of these proteases. Efforts currently underway in several laboratories should further enhance our understanding of the mechanism of function of these degradative machines. These ongoing efforts might have direct clinical consequences as ClpP has now been shown to be a legitimate antibacterial drug target.

References:
[1] Wong, P. and Houry, W.A. (2004) Chaperone networks in bacteria: analysis of protein homeostasis in minimal cells. J. Struct. Biol. 146, 79-89.
[2] Katayama-Fujimura, Y., Gottesman, S. and Maurizi, M.R. (1987) A multiple-component, ATP-dependent protease from Escherichia coli. J. Biol. Chem. 262, 4477-4485.
[3] Katayama, Y., Gottesman, S., Pumphrey, J., Rudikoff, S., Clark, W.P. and Maurizi, M.R. (1988) The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component. J. Biol. Chem. 263, 15226-15236.
[4] Choi, K.H. and Licht, S. (2005) Control of peptide product sizes by the energy-dependent protease ClpAP. Biochemistry. 44, 13921-13931.
[5] Wojtyra, U.A., Thibault, G., Tuite, A. and Houry, W.A. (2003) The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function. J. Biol. Chem. 278, 48981-48990.
[6] Peltier, J.B., Ytterberg, J., Liberles, D.A., Roepstorff, P. and van Wijk, K.J. (2001) Identification of a 350-kDa ClpP protease complex with 10 different Clp isoforms in chloroplasts of Arabidopsis thaliana. J. Biol. Chem. 276, 16318-16327.
[7] Schelin, J., Lindmark, F. and Clarke, A.K. (2002) The clpP multigene family for the ATP-dependent Clp protease in the cyanobacterium Synechococcus. Microbiology 148, 2255-2265.
[8] Maurizi, M.R., Clark, W.P., Kim, S.H. and Gottesman, S. (1990) Clp P represents a unique family of serine proteases. J. Biol. Chem. 265, 12546-12552.
[9] Butler, S.M., Festa, R.A., Pearce, M.J. and Darwin, K.H. (2006) Self-compartmentalized bacterial proteases and pathogenesis. Mol. Microbiol. 60, 553-562.
[10] Keiler, K.C., Waller, P.R. and Sauer, R.T. (1996) Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271, 990-993.
[11] Gottesman, S., Roche, E., Zhou, Y. and Sauer, R.T. (1998) The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev. 12, 1338-1347.
[12] Weber-Ban, E.U., Reid, B.G., Miranker, A.D. and Horwich, A.L. (1999) Global unfolding of a substrate protein by the Hsp100 chaperone ClpA. Nature 401, 90-93.
[13] Gillet, R. and Felden, B. (2001) Emerging views on tmRNA-mediated protein tagging and ribosome rescue. Mol. Microbiol. 42, 879-885.
[14] Thompson, M.W., Singh, S.K. and Maurizi, M.R. (1994) Processive degradation of proteins by the ATP-dependent Clp protease from Escherichia coli. Requirement for the multiple array of active sites in ClpP but not ATP hydrolysis. J. Biol. Chem. 269, 18209-18215.
[15] Gottesman, S., Maurizi, M.R. and Wickner, S. (1997) Regulatory subunits of energy-dependent proteases. Cell 91, 435-438.
[16] Brotz-Oesterhelt, H. et al. (2005) Dysregulation of bacterial proteolytic machinery by a new class of antibiotics. Nat. Med. 11, 1082-1087.
[17] Corydon, T.J. et al. (2000) Human and mouse mitochondrial orthologs of bacterial ClpX. Mamm. Genome 11, 899-905.
[18] de Sagarra, M.R., Mayo, I., Marco, S., Rodriguez-Vilarino, S., Oliva, J., Carrascosa, J.L. and Casta n, J.G. (1999) Mitochondrial localization and oligomeric structure of HClpP, the human homologue of E. coli ClpP. J. Mol. Biol. 292, 819-825.
[19] von Heijne, G., Steppuhn, J. and Herrmann, R.G. (1989) Domain structure of mitochondrial and chloroplast targeting peptides. Eur. J. Biochem. 180, 535-545.
[20] Kang, S.G., Maurizi, M.R., Thompson, M., Mueser, T. and Ahvazi, B. (2004) Crystallography and mutagenesis point to an essential role for the N-terminus of human mitochondrial ClpP. J. Struct. Biol. 148, 338-352.
[21] Kang, S.G., Ortega, J., Singh, S.K., Wang, N., Huang, N.N., Steven, A.C. and Maurizi, M.R. (2002) Functional proteolytic complexes of the human mitochondrial ATP-dependent protease, hClpXP. J. Biol. Chem. 277, 21095-21102.
[22] Kang, S.G., Dimitrova, M.N., Ortega, J., Ginsburg, A. and Maurizi, M.R. (2005) Human mitochondrial ClpP is a stable heptamer that assembles into a tetradecamer in the presence of ClpX. J. Biol. Chem. 280, 35424-35432.
[23] Kirstein, J., Schlothauer, T., Dougan, D.A., Lilie, H., Tischendorf, G., Mogk, A., Bukau, B. and Turgay, K. (2006) Adaptor protein controlled oligomerization activates the AAA+ protein ClpC. EMBO J. 25, 1481-1491.
[24] Peltier, J.B. et al. (2004) Clp protease complexes from photosynthetic and non-photosynthetic plastids and mitochondria of plants, their predicted three-dimensional structures, and functional implications. J. Biol. Chem. 279, 4768-4781.
[25] Sjogren, L.L., Stanne, T.M., Zheng, B., Sutinen, S. and Clarke, A.K. (2006) Structural and functional insights into the chloroplast ATP-dependent Clp protease in Arabidopsis. Plant Cell 18, 2635-2649.
[26] Erbse, A., Schmidt, R., Bornemann, T., Schneider-Mergener, J., Mogk, A., Zahn, R., Dougan, D.A. and Bukau, B. (2006) ClpS is an essential component of the N-end rule pathway in Escherichia coli. Nature 439, 753-756.
[27] Halperin, T., Zheng, B., Itzhaki, H., Clarke, A.K. and Adam, Z. (2001) Plant mitochondria contain proteolytic and regulatory subunits of the ATP-dependent Clp protease. Plant Mol. Biol. 45, 461-468.
[28] Majeran, W., Friso, G., van Wijk, K.J. and Vallon, O. (2005) The chloroplast ClpP complex in Chlamydomonas reinhardtii contains an unusual high molecular mass subunit with a large apical domain. Febs J 272, 5558-5571.
[29] Majeran, W., Wollman, F.A. and Vallon, O. (2000) Evidence for a role of ClpP in the degradation of the chloroplast cytochrome b(6)f complex. Plant Cell 12, 137-150.
[30] Shikanai, T., Shimizu, K., Ueda, K., Nishimura, Y., Kuroiwa, T. and Hashimoto, T. (2001) The chloroplast clpP gene, encoding a proteolytic subunit of ATP-dependent protease, is indispensable for chloroplast development in tobacco. Plant Cell Physiol. 42, 264-273.
[31] Kuroda, H. and Maliga, P. (2002) Overexpression of the clpP 5'-untranslated region in a chimeric context causes a mutant phenotype, suggesting competition for a clpP-specific RNA maturation factor in tobacco chloroplasts. Plant Physiol. 129, 1600-1606.
[32] Rudella, A., Friso, G., Alonso, J.M., Ecker, J.R. and van Wijk, K.J. (2006) Downregulation of ClpR2 leads to reduced accumulation of the ClpPRS protease complex and defects in chloroplast biogenesis in Arabidopsis. Plant Cell 18, 1704-1721.
[33] Wang, J., Hartling, J.A. and Flanagan, J.M. (1997) The structure of ClpP at 2.3 A resolution suggests a model for ATP-dependent proteolysis. Cell 91, 447-456.
[34] Bewley, M.C., Graziano, V., Griffin, K. and Flanagan, J.M. (2006) The asymmetry in the mature amino-terminus of ClpP facilitates a local symmetry match in ClpAP and ClpXP complexes. J. Struct. Biol. 153, 113-128.
[35] Szyk, A. and Maurizi, M.R. (2006) Crystal structure at 1.9A of E. coli ClpP with a peptide covalently bound at the active site. J. Struct. Biol. 156, 165-174.
[36] Gribun, A., Kimber, M.S., Ching, R., Sprangers, R., Fiebig, K.M. and Houry, W.A. (2005) The ClpP double ring tetradecameric protease exhibits plastic ring-ring interactions, and the N termini of its subunits form flexible loops that are essential for ClpXP and ClpAP complex formation. J. Biol. Chem. 280, 16185-16196.
[37] Vedadi, M. et al. (2007) Genome-scale protein expression and structural biology of Plasmodium falciparum and related Apicomplexan organisms. Mol. Biochem. Parasitol. 151, 100-110.
[38] Ingvarsson, H., Mate, M.J., Hogbom, M., Portnoi, D., Benaroudj, N., Alzari, P.M., Ortiz-Lombardia, M. and Unge, T. (2007) Insights into the inter-ring plasticity of caseinolytic proteases from the X-ray structure of Mycobacterium tuberculosis ClpP1. Acta Crystallogr. D. Biol. Crystallogr. 63, 249-259.
[39] Wang, J., Hartling, J.A. and Flanagan, J.M. (1998) Crystal structure determination of Escherichia coli ClpP starting from an EM-derived mask. J. Struct. Biol. 124, 151-163.
[40] Maurizi, M.R., Singh, S.K., Thompson, M.W., Kessel, M. and Ginsburg, A. (1998) Molecular properties of ClpAP protease of Escherichia coli: ATP-dependent association of ClpA and clpP. Biochemistry. 37, 7778-7786.
[41] Sprangers, R., Gribun, A., Hwang, P.M., Houry, W.A. and Kay, L.E. (2005) Quantitative NMR spectroscopy of supramolecular complexes: dynamic side pores in ClpP are important for product release. Proc. Natl. Acad. Sci. U. S. A. 102, 16678-16683.
[42] Waller, R.F. and McFadden, G.I. (2005) The apicoplast: a review of the derived plastid of apicomplexan parasites. Curr Issues Mol Biol 7, 57-79.
[43] Kim, D.Y. and Kim, K.K. (2003) Crystal structure of ClpX molecular chaperone from Helicobacter pylori. J. Biol. Chem. 278, 50664-50670.
[44] Thompson, M.W. and Maurizi, M.R. (1994) Activity and specificity of Escherichia coli ClpAP protease in cleaving model peptide substrates. J. Biol. Chem. 269, 18201-18208.
[45] Kim, Y.I., Burton, R.E., Burton, B.M., Sauer, R.T. and Baker, T.A. (2000) Dynamics of substrate denaturation and translocation by the ClpXP degradation machine. Mol. Cell 5, 639-648.
[46] Babbitt, S.E. et al. (2005) ATP hydrolysis-dependent disassembly of the 26S proteasome is part of the catalytic cycle. Cell 121, 553-565.
[47] Henikoff, S. and Henikoff, J.G. (1993) Performance evaluation of amino acid substitution matrices. Proteins 17, 49-61.

clpP:Gene Product(s

2010-10-13T16:02:17Z

Hourylab: Created page with 'ClpP is a highly conserved serine protease present throughout bacteria and eukaryota, ; it is absent in archaea <Wong, P. and Houry, W.A. (2004) Chaperone networks in bacteria: a...'

ClpP is a highly conserved serine protease present throughout bacteria and eukaryota, ; it is absent in archaea <Wong, P. and Houry, W.A. (2004) Chaperone networks in bacteria: analysis of protein homeostasis in minimal cells. J. Struct. Biol. 146, 79-89>, mollicutes, and some fungi. ClpP was first identified in Escherichia coli [2,3], and more than 400 studies have been published on it since its discovery. The X-ray structure of ClpP has been solved from several different organisms. All the structures show similar features: the protease is comprised of 14 subunits arranged into two heptameric rings forming a cylindrical-like structure which encloses a large chamber containing the protease active sites. Entrance into the chamber occurs through axial pores in the cylinder. ClpP typically forms complexes with AAA+ (ATPases Associated with various cellular Activities) chaperones that denature substrates and then translocate them through the axial pores into the proteolytic chamber of the protease for degradation. ClpP degrades proteins into peptides of about 7 to 8 residues [4] which are subsequently released from the chamber. It is one of the five major proteases in E. coli which includes ClpXP, ClpAP, HslUV, FtSH and Lon protease. Proteases play an essential role in protein quality control by removing short-lived regulatory proteins, as well as proteins that are misfolded and damaged, thus maintaining cellular homeostasis. A significant proportion of protein degradation in the cell is carried out by oligomeric, cylindrical, self-compartmentalized, energy-dependent proteases. ClpP (Caseinolytic Protease) is a representative member of these cylindrical proteases.

ClpP is found in all bacteria sequenced to date except for Mollicutes [5]. The majority of bacteria contain only one copy of ClpP, although there are exceptions such as for actinobacteria, chlamydiae, cyanobacteria, and others which have multiple isoforms of the protease. In addition to ClpP, cyanobacteria contain a ClpP paralog that does not have all 3 residues of the Ser-His-Asp catalytic triad. This inactive version of ClpP is called ClpR [6] and is mostly present in plants in addition to cyanobacteria. For example, cyanobacteria Synechocystis PCC6803 contains three copies of ClpP and one copy of ClpR [6,7].

ClpP was first identified and mostly studied in Escherichia coli, which contains only one copy of the protease. E. coli ClpP consists of 207 amino acids including an N-terminal prosequence which acts as a regulatory peptide and undergoes autocatalytic cleavage during folding to yield a mature ClpP of 193 residues [8]. In E. coli, ClpP forms complexes with AAA+ chaperones, ClpX and ClpA, which belong to the Clp/Hsp100 family. ClpA is an 83 kDa ATPase that contains two AAA+ domains, while ClpX contains only one AAA+ domain (46 kDa), which is homologous to the second AAA+ domain of ClpA. ClpA and ClpX are hexameric chaperones that can stack onto one or both ends of ClpP to form ClpAP or ClpXP holoenzyme complexes. Substrates are recognized, unfolded by ClpX or ClpA, and then threaded into the ClpP proteolytic chamber through the narrow axial pores for degradation. Only the unfolding and threading by the chaperones require ATP binding and hydrolysis, while proteolysis by ClpP does not require nucleotide hydrolysis. The mechanism by which ClpX or ClpA binds to and unfolds substrates is poorly understood. Other bacteria contain ClpA paralogs, such as ClpC, ClpE, and ClpL [9].

The most general substrates for the E. coli ClpXP or ClpAP system are those with an SsrA C-terminal tag [10]. The SsrA tag consists of 11 hydrophobic residues that are added by a tmRNA, also called SsrA or 10Sa RNA, to the C-terminus of nascent chains whose translation is stalled on the ribosome, targeting them for degradation [11-13]. SsrA tagging can be thought of as the prokaryotic equivalent to eukaryotic ubiquitination.

Without ATPase components, only small peptides can enter the ClpP chamber [14,15]; hence, ClpP on its own cannot degrade folded proteins. Recently, however, a group at Bayer described the discovery of acyldepsipeptides (ADEPs) that, when added to ClpP, allow the protease to degrade folded native proteins in the absence of its cognate chaperones [16]. The drug was initially found to rescue mice challenged with lethal infections of Enterococcus faecalis and Staphylococcus aureus, and the target for the drug was then identified to be ClpP. ClpP is activated by ADEPs to degrade, or at least to cleave, folded proteins in the absence of the cognate Clp ATPases. The consequences of the action of the drug in vivo, is that the ADEPs cause unregulated proteolysis by ClpP, subsequently, triggering cell death in gram-positive bacteria. Gram-negative bacteria, on the other hand, were found to be able to survive drug treatment because they have efficient efflux pumps to remove the drug from the cell, but deletion of those pumps or the use of permeabilizing agents made gram-negative bacteria vulnerable to ADEP treatment as well [16]. The mechanism of how ADEPs activate ClpP in the absence of its cognate chaperones is currently unknown. The discovery of ADEPs may lead to development of a novel class of antimicrobial agents.

Understanding how the ClpP system functions has direct implications on understanding the mechanism of function of other more complex cylindrical proteases such as the proteasome. ClpP shares many similarities with the proteasome both functionally and structurally. The arrangement of subunits in ClpP is strikingly analogous to that of eukaryotic and archaeal 20S proteasomes, which is composed of 2 heptameric β-subunit rings enclosing the active site sandwiched between 2 heptameric rings of α subunits. Another protease HslV (ClpQ), also known as the ‘bacterial proteasome’, exhibits analogous packing except that the cylinder is composed of two hexameric rings. The similarity in the cylindrical structural arrangement probably underlines the commonality in the mechanism of function of these proteases. Efforts currently underway in several laboratories should further enhance our understanding of the mechanism of function of these degradative machines. These ongoing efforts might have direct clinical consequences as ClpP has now been shown to be a legitimate antibacterial drug target.