Proteolysis in prokaryotes: protein quality control and regulatory principles

A conference entitled ‘Proteolysis in Prokaryotes: Protein Quality Control and Regulatory Principles’ was organized by Regine Hengge and Bernd Bukau from 25–27 October, 2002 in Schwetzingen, a small southern German town with a reputation for its castle and asparagus. It was the first conference sponsored by the Deutsche Forschungsgemeinschaft (DFG) as part of a 6 year priority programme to support research in this field. The scientific programme covered the molecular mechanisms of proteolytic systems, including protease and chaperone structures as well as molecular recognition, unfolding and processing of substrates. Complementary to this biochemical view of proteolysis, the physiology and biodiversity of proteolytic processes in prokaryotes, mitochondria and chloroplasts were covered with a particular focus on the recently emerging roles of proteolysis in the complex regulation of stress responses, cell cycle and development. The cellular proteases with a major function in protein quality control and regulation have a number of features in common: (i) they recognize many different substrate proteins that range from denatured polypeptides to key regulatory proteins in a fully native state, i.e. substrate recognition is at the same time highly flexible and highly specific; (ii) they form multisubunit barrel-like complexes, with the active sites sequestered inside with entry tightly controlled by chaperone subunits; (iii) for the cytosolic proteases, unfolding and translocation into the proteolytic chamber requires ATP hydrolysis; and (iv) substrate degradation is processive, i.e. complete (for recent reviews, see Dougan et al., 2002a; Jenal and Hengge-Aronis, 2003). Recent research has focussed on three model systems, i.e. the cytosolic Clp proteases, the membrane-bound FtsH (HflB) protease and the periplasmic DegP (HtrA) protease. The ClpXP and ClpAP proteases each consist of two seven-subunit ClpP rings that form the proteolytic chamber and six-subunit ClpX or ClpA rings attached at both ends that bind and unfold substrates (Guo et al., 2002a; Wang et al., 1997). Electron microscopy has been used to locate protein substrate binding sites at the outer ring surface in ClpXP and ClpAP complexes (Mike Maurizi, NIH, Bethesda, USA). Direct binding measurements with several substrate proteins (λO protein, RepA, GFP-SsrA) indicate that only one protein molecule binds per ClpX or ClpA ring at a time. Surprisingly, small peptides containing ClpA or ClpX recognition tags also bind with a stoichiometry of one peptide per ClpA6 or ClpX6. Two models can explain these data: all six subunits contribute to a single binding site centrally located on a hexamer, or binding sites are located on each subunit but display high negative cooperativity. Cross-linking with peptide substrates is underway to distinguish between these models and to identify the regions of ClpA and ClpX that make up the binding sites. To date, only a handful of cellular substrates of ClpXP and ClpAP have been identified. Using a tagged and inactive variant of ClpP (ClpPtrap), substrates for ClpXP and ClpAP were trapped in vivo, purified and identified by mass spectrometry (reported by Briana Burton, from Tania Baker's laboratory, MIT, Cambridge, USA; Flynn et al., 2003). The trapped proteins include transcription factors, metabolic enzymes and proteins involved in the starvation and oxidative stress responses. Analysis of the newly identified ClpXP substrates provided insight into the mechanisms of substrate recognition. Sequence analysis, binding studies, fusion proteins and mutagenesis revealed the presence of two related C-terminal motifs and three related N-terminal motifs that may be recognized by ClpX. Nearly all of the trapped substrates have one or more of the recognition signals (Flynn et al., 2003). Use of the ClpPtrap also revealed that some substrate proteins (e.g. the SOS-regulator LexA) are recognized only after they are initially cleaved by another protease. RecA-induced autocleavage uncovers recognition signals in the two separated protein domains that render the LexA protein fragments susceptible to ClpXP-dependent proteolysis in vivo and in vitro (Neher et al., 2003). Additional recognition or targeting factors, whose expression and/or activity can be regulated by intra- or extracellular signals, help to ensure that degradation of specific substrates occurs at the right time and place (Dougan et al., 2002a). Proteolysis of the general stress sigma factor σS or RpoS (which occurs in rapidly growing cells but is inhibited by various stress conditions) requires initial direct binding by the phosphorylated response regulator RssB (Becker et al., 1999; Klauck et al., 2001; Zhou et al., 2001). Recognition of σS by the RssB/ClpXP proteolytic machinery is a complex process involving at least two distinct regions in σS (Fig. 1; R Hengge, Freie Universität, Berlin, Germany). Region 2.5 of σS, which forms a long α helix, is sufficient for binding of phosphorylated RssB. In addition, a region close to the N-terminus acts as a binding site for ClpX6. Native σS, however, is not bound by ClpX6. Interaction of each factor with its respective binding site alone is not sufficient to trigger degradation of σS or σS-derived reporter proteins. These data indicate the following sequence of events: RssB initially interacts with σS and triggers a change of conformation that exposes the ClpX6-binding site close to the N-terminus. Once σS is bound to ClpX6, RssB plays a second role (either in unfolding or during the beginning transfer of unfolded σS into the ClpXP protease), before it is released from the complex and σS is completely degraded. Experiments reported by Susan Gottesmann (NIH) suggest that a region near or at the C-terminus of the output domain of RssB might play a role in this process that goes beyond σS binding. She reported that deletions that remove 25–100 amino acids from the C-terminus apparently sequester σSin vivo, with the consequence that σS becomes stable but inactive. Surprisingly, this inhibition of activity requires both ClpX and ClpP, suggesting that a stable σS–RssB–ClpXP protease complex forms but is unable to release σS for degradation by ClpXP. Consistent with a critical role for phosphorylation in the interaction of RssB and σS and with a physiologically relevant interaction of RssB and σS in the C-terminally deleted mutants, a C-terminally deleted derivative of RssB that also lacks the phosphorylation site (D58P) no longer sequestered σS. Interaction of σS(RpoS) and RpoS::LacZ hybrid proteins with the recognition factor RssB and the hexameric ClpX chaperone (provided by R. Hengge, Freie Universität, Berlin). Numbers refer to the common nomenclature for functional regions in sigma factors. NTE: N-terminal element for ClpX6 recognition. For other details, see text. The SspB protein also serves as a specificity factor for the ClpXP protease by specifically stimulating proteolysis of proteins bearing an SsrA tag. The SsrA tag is an 11 residue peptide that is added co-translationally to the C-terminus of polypeptides whose biosynthesis has been stalled (Karzai et al., 2000). SspB enhances binding of tagged substrates to ClpX and stimulates their unfolding by ClpX and their degradation by ClpXP (Levchenko et al., 2000). SspB protein is a stable homodimer with two independent binding sites for SsrA-tagged substrates. SspB itself binds to ClpX and stimulates its ATPase activity. The mechanism of substrate delivery involves the formation of a tertiary complex consisting of a ClpX hexamer, a SspB dimer and two SsrA-tagged substrate proteins (Wah et al., 2002). As reported by Igor Levchenko (MIT, Cambridge, USA), the crystal structure of Haemophilus influenzae SspB protein at 1.8 Å resolution (Fig. 2) reveals that the SspB dimer is 65 Å wide in its maximal dimension. The dimerization interface of the protein is formed by hydrophobic contacts between two amino-terminal α-helices. The core domain of each monomer of SspB is formed by two four-stranded β-sheets. At the extreme ends of each dimer there is a well-defined hydrophobic ‘cleft’ formed by one of the β-strands and two loop structures. This cleft is involved in crystallographic contact with the C-terminus of the neighbouring SspB dimer, where the backbone atoms of Asn53 in the cleft interact with the side-chain of Gln120. This cleft–peptide interaction has been observed in two independent crystal forms, and probably reflects an interaction important in forming the SspB–SsrA complex. Structure of the SspB dimer with the SsrA tag peptide modelled into hydrophobic clefts at opposite ends of the dimer (provided by I. Levchenko, R. Grant and T. Baker; MIT). ClpA, ClpB and ClpC are representatives of the typical HSP100 protein family, which are chaperones with two ATPase domains. These chaperones can cooperate with specific adaptor proteins or specificity factors. ClpS is such a factor for the ClpA chaperone that improves recognition of denatured proteins by ClpA (Dougan et al., 2002b). A 2.5 Å resolution crystal structure of ClpS in a complex with the N-terminal domain of ClpA (Fig. 3A) was recently determined (Guo et al., 2002b; Zeth et al., 2002). Using mutagenesis, it was demonstrated that two contact residues (Glu 79 and Lys 84) are essential not only for ClpAS complex formation but also for ClpAPS-mediated substrate degradation. The corresponding residues are absent from ClpB, providing a structural rationale for the unique specificity exhibited by ClpS despite the high overall similarity between ClpA and ClpB. In order to elucidate the location of ClpS within the ClpA hexamer, the N-terminal domain of ClpA was modelled onto a structurally defined, homologous, AAA+ protein (Fig. 3B). From this a molecular mechanism to explain the in ClpA substrate specificity has been in with Dougan and Bernd Germany). Structure of a complex between ClpS and the N-terminal domain of ClpA as well as a model of hexameric ClpA with ClpS subunits attached as and (provided by The ClpA6 model was by onto the related hexameric et al., and Bernd Bukau reported on the roles of chaperones in the of proteins in the A consisting of ClpB and the the and of proteins. The of small proteins into protein the process in vitro and the to small protein in the of ClpB. Consistent with these exhibited protein and and function essential at were and Bukau reported on the of a ClpB This has in both ATPase activity the overall structure et al., 2003). The ClpB protein in a stable with substrates in an and inhibited the of ClpC is an HSP100 protein involved in general stress of A complex of ClpC with the protease ClpP and the adaptor protein by regulated proteolysis of the regulator et al., et al., the major chaperone of ClpC were to In ClpC to and protein et al., 2003). the degradation of unfolded or proteins by native protein was not degraded. This that adaptor proteins such as with their are not to specific regulatory Germany). of substrate complex proteases have to three major substrate translocation and degradation. These were in model protein substrates and transfer To the unfolding a of the N-terminal domain was with the at opposite ends of the but close in the native To the degradation the were close on the but that occurs in the two with forming a the unfolding and degradation be a in unfolding and a to release of substrate from sites be in the of active ClpA and inactive ClpP, inside the ClpP The degradation with a longer with the unfolding is unfolded and to near and bound to ClpA in an unfolded conformation of time. not related at the amino sequence Clp proteases and the and have structure and The of the six ATPase To they unfold and proteins into the for the ATPase complex from and the substrate were from laboratory, USA). Using small or tags and attached near its or was to the ATPase in a to translocation not but which occurs on the surface of the ATPase ring and 2001). various proteins at the complex and also the were to proteins (e.g. only from the and other proteins (e.g. in both Surprisingly, the domain was or of a the of the fusion was not and was released in a the of translocation and degradation with different substrates and also the of the ATPase to unfold a domain to be determined by the of the regions or by the location of a specific degradation The crystal structure of the ATPase domain of FtsH (HflB) was determined and a form was providing into of binding and (Fig. et al., 2002). In the hexameric there is a in which the The function of was by FtsH in which is by each of the other common amino that residues at this are essential for of suggesting an of in unfolded polypeptides the and of a modelled of the ATPase domain of is bound are in (provided by The of the FtsH protease as well as its role in protein quality control were by (Fig. The by FtsH can be from the or from the C-terminal of a protein that into the stimulates the degradation which can be in and 2003). In FtsH as an complex with FtsH with the as by the of the of FtsH and The is the control of the stress response which to proteins. between two proteases, and has also been observed for the degradation of the factor which in the of the stress response et al., et al., 2002). in the (provided by for details, see FtsH substrates are the sigma factor and the which plays a key role in the of proteins can be from that the of are not essential for degradation by Analysis of hybrid proteins between and a stable variant from revealed that an region in the N-terminal part of was to be for provided that the C-terminal of acts as a and sufficient for of this tag or that two residues at the C-terminus of the tag not the structure or regulatory activity of but the degradation by protein, from degradation in in vitro for the protein demonstrated that proteolysis by FtsH to from degradation. also the proteolysis of a number of other FtsH substrates. The region for the C-terminal of with a that a small of of the of the in with the in the These the first of a that a region that both at the and protein et al., 2002). The DegP (HtrA) of proteins of but not of the are proteins. are in such as the of the of and the chloroplasts of are involved in and as well as in The of the is the of a domain with one or more C-terminal domains et al., 2002). The of substrates that DegP is a key factor protein in the cell DegP of has both chaperone and protease These are in a the protease activity at et al., The crystal structure of DegP was et al., 2002). of rings forms the DegP (Fig. The proteolytic sites are located in a that is only The are formed by which the and of the and probably the initial binding of hydrophobic that as sites for unfolded polypeptides the In the chaperone the protease domain of DegP in an inactive state, in which substrate binding as well as are and Germany). In addition, the complex between DegP and the protease be structural data suggest that as in the the protease The protease form be to structural for substrate specificity and for the in activity. reported that the of this can also be observed in the and forms of DegP in the chaperone conformation (provided by T. The N-terminal domains are in the protease domains in and the and domains in and A major function for proteolysis is the of proteins. These can be denatured and where the cell has to make the between i.e. and or degradation of the This is to by between chaperones (e.g. or see and (e.g. are polypeptides from or In such the and are released by the SsrA which co-translationally a tag to polypeptides during their The fusion proteins are to degradation by the protease (Karzai et al., 2000). It recently that SsrA can also on proteins et al., and 2002). A change from a to a sequence in the for to The of was with a the of the sequence and the presence of in its the of of the and to It is that an function of is to at the formation of proteins from laboratory, Proteolysis can be important to the activity of an essential and the to the overall The first in biosynthesis in has a and is et al., 2002). It is a model to proteolysis, as well as of proteins by chaperones at The amino part of the molecule is a degradation for proteases and the becomes stable only in The complex and from requires and ClpB. it was that occurs in mutants, a to other proteins also on In the the which the is a ClpXP In reported by of and the and biochemical of two ClpX both at the and was In vivo the protein no activity in the as a cells are not In activity in vivo, i.e. cells are In to their in vivo ClpX variant binds or the ClpXP-dependent degradation of or in to the protein stable binding to These that both degradation and its binding to the ClpX chaperone can be important in the of the of active The regulation of the sigma factor and of the general stress response sigma factor σS are the for a key role of proteolysis in the control of stress responses. and σS are but are they are by FtsH and RssB/ClpXP and can be in response to various This an of these sigma factors. a single i.e. an in denatured proteins and of the chaperone is the key in et al., signals or have to be in the control of σS This occurs (i) by the phosphorylation of the recognition factor (ii) by of RssB by of σS as well as (iii) by the of σS and for core as σS within the is proteolysis and Hengge-Aronis, 2002). Control of σS proteolysis a complex The σS to is by a mechanism RssB that up to a where RssB are not which in RssB and a into the of stable from R. laboratory, Freie Berlin, initial by from Germany). a protein that to in cells to starvation or oxidative is a ClpXP substrate (Flynn et al., 2003). degradation is controlled by stress σS, is by starvation in to σS proteolysis is inhibited by oxidative stress not by and is not on RssB from R. laboratory, Freie Berlin, Germany). biosynthesis is a highly regulated which in signals that The regulator of the regulatory is the which has to be a ClpXP substrate et al., 2002). the complex as well as alone are by alone is a of or of a proteolytic in only bound to The regulatory role of of is not of display a that to and in and and substrates for the FtsH protease were identified by the of and cells Germany). At least identified and proteins were in in the of the protease. of is a of the and FtsH was to control its transcription that of several other of this is an sigma factor that involved in with cell biosynthesis et al., 2002). the FtsH protease acts as a of the of suggesting that might be a FtsH substrate and that the expression of the be to proteolytic control et al., 2003). In was observed to be Germany). This control involves a regulatory protein, (for which is for of and As the highly is both in the and a it is that and expression in is controlled degradation of by the Clp protease. of denatured i.e. may in of This mechanism to be as proteins and in of are in all is used as a model to regulatory of the cell cycle and cell of protein and that up to of the proteins are during one cell cycle and that the of the proteins are also in a cell et al., 2001). This that protein is a critical regulatory element of cell and cell cycle This was by the that the Clp and FtsH proteases are critical for these processes in and et al., 2002). The ClpA ATPase subunit is in vivo for the cell degradation of the protein As reported by Jenal a of hydrophobic amino acids at the C-terminus was identified as a up to hydrophobic residues were to for and cell proteolysis, longer hydrophobic the protein to an as protease and degradation cells ClpP or ClpX a cell cycle In order to the role of the ClpXP protease and to identify which have to be during the cell protein in a was by At least proteins were at the that they might on ClpXP for degradation. Protein and a molecular of their roles in cell cycle and are in this only the structural but also some regulatory of the in are controlled by These include the which the to the cell This degradation requires ClpXP and residues at the of the proteins. from the for the proteins the to be suggesting that the complex be to degradation. The on the degradation of the is probably to as the response regulator which is phosphorylated by is in the presence of of the the degradation of the et al., and of the protein requires ClpX for its which that the ClpXP protease is one of the major of the in Clp proteins in and and functional and 2002). The Clp protein has been in the model a has distinct chaperone and proteolytic as well as the adaptor protein Clp proteins in are essential for or are for and to different stress for is for of to high and It was recently that degradation of the complex of i.e. the that to the of to in order to to the process and and cell i.e. the degradation of has been in in response to the model et al., 2001). is by the of metabolic activity and by a degradation of cellular proteins Germany). In the state, the cells of and protein which is a model for a programme that is by proteolytic proteases involved in this their and control are to protease are with to protein and in vivo proteolytic regulation of on the function of various proteases in Using various with or deletions in from the the role of all has been and Germany). revealed two that control the of cells to As the functional of the a major for the to be the proteins that bind the and the of during to The a different as the i.e. is probably involved in other of the was in The functional specificity of the in and is et al., 2002). proteolysis of proteins is recognized as the of many A number of proteases are of or completely proteins and 2002). The proteolytic of the of mitochondria of several highly by Germany). These include the and proteases with sites at opposite (Fig. These form a quality control and regulatory during cells both proteases are of one of these proteases in In addition, the complex chaperone activity and the degradation of proteins by the protease. cellular in and which might of protein on the peptides by the protease into the their release from a of have with the protease. in mitochondria (provided by T. for details, see and which are of the ClpB and ClpX play a role in protein degradation within the et al., 2002). Proteolysis of substrate which were in vitro into was on the of ATP in the and was by the protease were to be independent of the of the reporter substrates. from cells exhibited a in the degradation at a mitochondria activity. is a of the proteolysis that is for the degradation of substrate proteins in the Germany). many other in proteolysis to be a of and have been in of the of the also revealed a for a protein with high sequence similarity to In to the which is a into the domain Germany). of that is in both in cells and as a protein in was by and purified as a complex that as a on In purified ATPase activity and and degradation of substrate proteins. In the protease might the functional of the membrane-bound FtsH which has not been identified in It is that proteolytic are not only molecular but also complex processing As a molecular recognition as well as processing and by proteolytic systems, which include specific recognition as well as chaperone and protease have major of is to the that these molecular processes in a i.e. within a complex protein quality control that all cellular and within the cellular regulatory proteins that are regulated by proteolysis may be a in the they to be key of stress responses, the cell cycle and that proteolysis has a major on overall cellular regulation and Hengge-Aronis, 2003). by the of the the of the have that this conference on Proteolysis to a The place in the of