Characterization of Gain-of-Function Mutant Provides New Insights

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Characterization of Gain-of-function Mutant Provides New Insights into ClpP Structure Tengfeng Ni, Fei Ye, Xing Liu, Jie Zhang, Hongchuan Liu Liu, Jiahui Li, Zhang Yingyi, Yingqiang Sun, Meining Wang, Cheng Luo, Hualiang Jiang, Lefu Lan, Jianhua Gan, Ao Zhang, Hu Zhou, and Cai-Guang Yang ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00390 • Publication Date (Web): 12 May 2016 Downloaded from http://pubs.acs.org on May 14, 2016

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ACS Chemical Biology

Characterization of Gain-of-function Mutant Provides New Insights into ClpP Structure Tengfeng Ni§, †, ⊥, Fei Ye‡, ≠, ⊥, Xing LiuƩ, ⊥, Jie Zhang§, ⊥, Hongchuan Liu§, Jiahui Li§, †, Yingyi Zhang¶, Yingqiang SunΔ, Meining WangƩ, Cheng Luo≠, Hualiang Jiang≠, †, Lefu Lan§, †, Jianhua Ganǁ, Ao ZhangƩ, †, Hu ZhouƩ, †, Cai-Guang Yang§, †, * §

Laboratory of Chemical Biology, State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; †

University of Chinese Academy of Sciences, Beijing 100049, China;

‡

College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China;

≠

Drug Design and Discovery Center, State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; Ʃ

CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, Shanghai 201203, China; ¶

National Center for Protein Science Shanghai, Institute of Biochemistry and Cell

Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 201210, China; Δ

Experiment center for science and Technology, Shanghai University of Traditional

Chinese Medicine, Shanghai 201203, China ǁ

School of Life Sciences, Fudan University, Shanghai 200433, China.

Keywords: Clp protease; dysfunctional activation; gain-of-function mutant; mechanism of domino effect

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ABSTRACT: ATP-dependent Clp protease (ClpP), a highly conserved serine protease in vast bacteria, could be converted into a non-controllable enzyme capable of degrading mature proteins in the presence of acyldepsipeptides (ADEPs). Here, we design such a gain-of-function mutant of Staphylococcus aureus ClpP (SaClpP) capable of triggering the same level of dysfunctional activity that occurs upon ADEPs treatment. The SaClpPY63A mutant degrades FtsZ in vivo, and inhibits staphylococcal growth. The crystal structure of SaClpPY63A indicates that Asn42 would be an important domino to fall for further activation of ClpP. Indeed, the SaClpPN42AY63A mutant demonstrates promoted self-activated proteolysis, which is a result of an enlarged entrance-pore as observed in cryo-electron microscopy images. In addition, the expression of the engineered clpP allele phenocopies treatment with ADEPs; inhibition of cell division occurs as does showing sterilizing with rifampicin antibiotics. Collectively, we show that gain-of-function SaClpPN42AY63A mutant becomes a fairly nonspecific protease and kills persisters by degrading over 500 proteins, thus providing new insights into the structure of the ClpP protease.

The ClpP protease system plays a critical role in protein quality control and cellular homeostasis

1, 2

. In an unperturbed cell, ClpP needs to be associated with

AAA+ (ATPases associated with diverse cellular activities) chaperones in order to degrade a variety of misfolded or damaged proteins as well as native regulatory proteins, including those involved in regulating stress responses and virulence-factor

2


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production

3, 4

. ClpX and ClpA are hexameric chaperones, which recognize target

proteins and use ATP hydrolysis to unfold them, and then translocate them into the tetradecameric ClpP proteolytic chamber through axial pores lined by axial loops for 5-10

degradation

. Taken together the structural evidences

11-17

combined with

biochemical analysis 18-22 reveal that major contacts occur between the IGF/L loops of ATPases and the hydrophobic pockets of ClpP proteases. These hydrophobic pockets are located between adjacent monomers, surrounding the axial entrance-pore at N-terminus. Cryo-electron microscopy (EM) has revealed that binding of ATPases causes entrance-pores to open up 23. Because of the lack of high-resolution structure of ClpP/ATPase complex, however, the activation mechanism that depends on the protein-protein interaction of the two partners is currently not known. ClpP is able to degrade small peptides without the involvement of ATPase chaperones

24

. Recently, acyldepsipeptides (ADEPs) have been identified as a novel

class of antibiotics that reprogram ClpP and convert it to an unregulated protease capable

of

degrading

misfolded

and

native

cellular

proteins

in

an

ATPase-independent manner 25-27. AEDPs-activated ClpP dysfunctionally degrade cell division protein FtsZ, resulting in inhibition of cell division 26, 28. In addition, ADEPs kill bacterial persister cells of pathogenic S. aureus in the presence of rifampicin, thus suggesting a potential avenue for developing therapies in order to treat chronic infections

29

. These discoveries have paved a new avenue for understanding the

mechanism for ClpP protease activation. Crystal structures of Bacillus subtilis ClpP (BsClpP), Escherichia coli ClpP (EcClpP), and Mycobacterium tuberculosis ClpP

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(MtClpP1P2) bound to ADEPs antibiotics have revealed that the activators occupy the hydrophobic pockets of ClpP proteases, thus mimicking the interaction between AAA+ chaperones and ClpP proteases 30-32. ADEPs-binding triggers a reorientation of the ClpP subunits and enlargement of the axial entrance-pore, thereby converting ClpP protease to an “open-gate” activated form, thus facilitating the passage of substrate proteins into the degradation chamber

30-34

. The “open-gate” form is

different from the other three states observed in crystal structures of apo-form ClpP, the extended, compact, and compressed states 35-38. How the structural dynamics of activation ClpP are regulated remains unclear, however. Here we succeed in designing an engineered gain-of-function mutant that converts SaClpP protease to an uncontrollable enzyme. The conformational twist of the side chain of Tyr63, a key component in the hydrophobic pocket that interacts with ATPases and ADEPs, initiates the dysfunctional activation of ClpP toward the proteolysis of mature proteins. In order to avoid steric clash, the side chain of Met31 falls down as the first domino, and subsequently pushes the side chain of Asn42 to swing away, which facilitates the whole activation of SaClpP. Taken together, these results advance our understanding of ClpP activation mechanism that the allosteric domino effect controls the dynamic changes of the protease and triggers its activation. RESULTS AND DISCUSSION Mutation of Tyr63 leads to gain-of-function of SaClpP. Both ATPases and ADEP antibiotics allosterically regulate the ClpP barrel conformation and activate

4


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proteolysis (Figure 1A). The crystal structure of BsClpP/ADEP1 has showed that Tyr63 forms two hydrogen bonds between the hydroxyl group and the carbonyl backbone of ADEP1 (Figure S1A). Other major interactions include prominent hydrophobic interactions and van der Waals contacts 30. Of note, the structural superimpositions of BsClpP (PDB code 3PTG) and ADEP1-bound BsClpP (PDB code 3PTI) clearly reveals that the aromatic side chain of Tyr63 is rotated 90° upon ADEP1 binding (Figure 1B). This phenomenon is also observed in the structural complexes of EcClpP/ADEP1 and MtClpP1P2/ADEP/agonist (Figure S1B-C)

31, 32

. As a result, the benzyl ring plane

overlaps with the Cα-Cβ-Cγ plane, an “eclipsed” conformation in our terms (Figure S1D). While in the extended, compact, and compressed state of the SaClpP structures, the side chains of Tyr63 have been consistently observed to take the staggered conformation (Figure S1D). For tyrosine in the staggered conformation, the energy barriers for rotation of the ring dihedral angle have been calculated, revealing the location of the barrier maximum near 90° rotation and its symmetric form

39

.

Consequently, apo-ClpP could not achieve the “open-gate” activated form perhaps due to the energy barrier; rotation of the side chain of Try63 upon ADEPs-binding provides the dynamic energy to overcome the transformation barrier, rendering the activation of ClpP peptidase. Since an energy barrier was observed during rotation of the side chain of Tyr63, we wondered if certain mutations would convert ClpP into a non-controllable protease capable of degrading mature proteins, as occurs in the presence of ADEPs. In order to test this hypothesis, we investigated the proteolysis of Tyr63 mutations in

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SaClpP in the absence of ATPase chaperones or ADEP activators. Interestingly, several mutants, including SaClpPY63A, SaClpPY63I, and SaClpPY63F could efficiently degrade the poorly folded protein β-casein in vitro (Figure 1C and S2A). Furthermore, SaClpPY63A and SaClpPY63I are capable of degrading cell division protein FtsZ, while ClpPY63F is inactive (Figure 1D, Figure S2B-S2G). Presumably, the bulk of the tyrosine residue locks ClpP into a conformation that closes the axial pore due to the energy barrier. Removal of this sterically encumbering residue likely releases the restriction, enabling the ClpP peptidase to dynamically occupy conformations in which the pore is open, thus leading to self-activated ClpP. Indeed, replacement of Tyr63 with a bigger tryptophan residue abolishes the activity to degrade the unfolded protein β-casein or FtsZ (Figure S2A and S2G). In order to quantitatively compare the differential proteolysis of these ClpP mutants, we measure the initial rates of the hydrolysis of FITC-casein protein. SaClpPY63A mutant presents the highest self-activation activity (Figure 1E). Next, we performed a fluorescence-based assay in order to measure the thermal stability of the SaClpP variants (Figure 1F). The melting temperature (Tm) of SaClpP at 2.0 uM is measured to be 52.1 °C, while those of the SaClpP variants are all observably increased. However, no obvious linear correlation could be seen between Tm of certain SaClpP mutant and its dysfunctional activity. It is important to note no difference in the quantity of enzymes were used in these reactions, nor did the solubility of the ClpP variants differ, which rules out the possibility the apparent differences are the consequence of having different amounts of enzyme. Taken together, we designed a dysfunctional SaClpP protease by release

6


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of the restriction of Tyr63 side chain. It has been established that the IGF tripeptide motif of ClpX is important for ClpX binding to ClpP and for the initiation of protein degradation of SsrA 18. However, it remains unclear if Tyr63 is involved in the ClpX-dependent degradation of folded proteins. To this end, we tested if ATPase chaperone SaClpX was able to activate the SaClpPY63A mutant for protein degradation of SsrA. The wild-type S. aurues ClpX/ClpP complex efficiently degrades the folded GFP-SsrA protein, while the SaClpPY63A mutant displays significantly retarded activity (Figure 1G). We perform pull-down experiments in order to evaluate whether or not SaClpX binds to SaClpPY63A mutant. As expected, the His-tagged SaClpX was observed binding to SaClpP specifically, while this binding could be completely inhibited upon the treatment of ADEP8 that occupies the hydrophobic pockets, therefore preventing the binding of IGF tripeptide motif of ClpX (Figure 1H). The SaClpPY63A mutant still binds to SaClpX, to some extent, but weakly, which might result in the dysregulated activity of SaClpPY63 for SsrA degradation in the manner of ClpX-dependence. Crystal structure of SaClpPY63A. ADEPs-binding to ClpP protease triggers the enlargement of the N-terminal entrance-pore, resulting in an “open-gate” activated state

30-32

. In order to investigate if the self-activated SaClpPY63A protease also

demonstrates a similar phenomenon, we solved the crystal structure of SaClpPY63A at a resolution of 1.75 Å by molecular replacement using the extended SaClpP monomer (PDB code 3STA) as a search template. The supplementary Table S1 summarizes our data collection and structural refinement statistics. The structural

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alignment of the extended SaClpP to the SaClpPY63A mutant yields very small Cα RMSD values, 0.205 Å2 for the monomer, and 0.58 Å2 for the tetradecamer, respectively, which indicates that the SaClpPY63A barrel possesses an extended state under the crystallization conditions (Figure S3A). The SaClpPY63A mutant likely switches between the extended and the "open-gate" conformation as observed in the activators-bound state. The “closed-gate” conformation is more stable than the one in which the axial pore is open; therefore, it is unsurprising that the SaClpPY63A mutant does not display the “open-gate” activated conformation under crystallization conditions (Figure 2A). Different from wild-type SaClpP in the extended state, however, alignment of all 14 monomers in SaClpPY63A tetradecamer clearly shows that the conformations of the side chain of Asn42 are largely variable (Figures 2B and S3B). These conformations can be classified in three distinct states. First, similar to the ADEP1-bound BsClpP protease, the side chain of Asn42 bends downward; this phenomenon is observed in five monomers in the SaClpPY63A tetradecamer. Second, the orientation of Asn42 side chain is similar to that of the wild-type extended SaClpP (Figure S3C), which is found in only one SaClpPY63A monomer. Therein the side chain of Asn42 forms hydrogen-bonding with Tyr21 from the neighboring monomer. The side chain orientation of the left eight Asn42 resides in the third conformation, a dual conformation with both upward and downward sections. With these structural observations, we are able to describe the mechanism for ClpP activation that is stimulated by the binding of ADEPs (Figure S3D). In the ADEP1-bound BsClpP, the aromatic side chain of Tyr63 is

8


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twisted 90° compared to that in the extended state, which results in the downward movement of the side chain of Met31 in order to avoid steric clashes. In order to accommodate the fall of the first domino, the side chain of Asn42 bends downward, breaking the hydrogen-bonding between Asn42 and Tyr21 as a result, and a new interaction network is formed between Asn42 and Gly33, Asn65 from the neighboring monomer. In summary, distinct conformations of the side chain of Asn42 in SaClpPY63A support the substitution of Tyr63 with the smaller alanine residue that could result in a more dynamic ClpP protease that adopts more conformations, including the “closed-gate” and "open-gate" conformations. SaClpPN42AY63A mutant displays enhanced protease activity comparable to activator-bound ClpP. The side chain of Asn42 displayed dual conformations in the SaClpPY63A structure, which suggests that the allosterically conformational changes of Asn42 would contribute proteolysis activity to SaClpPY63A protease. Indeed, the SaClpPN42AY63A mutant exhibits elevated activity for FtsZ degradation compared to SaClpY63A in vitro (Figure 2C, S2D, and S2I). However, SaClpPN42A is unable to degrade FtsZ under the same experimental conditions (Figure 2C and S2H). Next, we tested if the mutation of Tyr63 and Asn42 alters the oligomeric state or thermal stability of SaClpP protein. As shown in Figure 2D, the predominant oligomeric states observed in the size exclusion chromatography assay are in equilibrium of tetradecamer for both SaClpPY63A and SaClpPN42AY63A, suggesting that there were no gross structural perturbations of the assemblies of these two SaClpP mutants. In addition, the thermal stability is measured and the Tm values of SaClpP

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mutants are higher than that of the wild-type enzyme, which are 54 °C for SaClpPY63A, 58.0 °C for SaClpPN42A, and 57.3 °C for SaClpPN42AY63A (Figure 1F), respectively, indicating that these mutations stabilize the folding of the tetradecameric protease. Electron microscopy shows the “open-gate” activated state of SaClpPN42AY63A mutant. ADEPs-binding triggers the enlargement of the axial entrance-pore in ClpP, thus converting the protease into an “open-gate” activated form

30-33

. Since the

N42AY63A mutation causes SaClpP to malfunction and thus degrades proteins in an uncontrolled manner, we wondered if SaClpPN42AY63A mutant might undergo similar conformational changes, as happens in wild-type ClpP in the presence of ADEPs. In order to assess the changes of the axial entrance-pore in SaClpPN42AY63A, we observed a particle of free SaClpP, ADEP8-activated SaClpP, and SaClpPN42AY63A mutant, respectively, by performance of cryo-electron microscopy (Figure 2E). As expected, microscopy images clearly imply that the entrance-pore of SaClpP is enlarged in the presence of ADEP8. Similarly, the SaClpPN42AY63A mutant also displays additional flexibility in the entrance-pore region. This result reveals the mechanism of the gain-of-function of the engineered SaClpPN42AY63A protease, which is similar to that of the activator-bound ClpP proteases. The expression of the allele encoding SaClpPN42AY63A compromises the growth of S. aureus and destabilizes FtsZ. ADEP-activated ClpP eradicates the pathogenic bacteria SaClpP

would

26

, so we explore whether the gain-of-function mutation of

influence

the

growth

of

10


S.

aureus.

The

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complemented ΔclpP/pYJ335::clpP strain enters into a logarithmic growth phase at almost

the

same

time

as

wild-type

strain,

while

the

growth

of

ΔclpP/pYJ335::clpPY63A mutant strain is slightly inhibited (Figure 3A). Interestingly, the presence of TET at 0.025 µg/ml minimally impaired the growth of the wild-type staphylococcal, but clearly inhibited the growth of the ΔclpP/pYJ335::clpPY63A mutant strain with an inducible promoter. A bare amount of growth was observed in the staphylococcal allele encoding the ClpPN42AY63A mutant (Figure 3A). In addition, we performed a colony-forming units (CFU) assay in order to quantitatively determine the growth of the clpP alleles (Figure 3B, top panel). Consistently, cell viability of the ΔclpP/pYJ335::clpPY63A or ΔclpP/pYJ335::clpPY63A mutant strain is much lower than that of the wide-type wt/pYJ335 or the ΔclpP/pYJ335::clpP complementary strain. Western blot was used to detect the inducible expression of SaClpP proteases (Figure 3B, bottom panel). Abnormal degradation of FtsZ could lead to bacterial growth restriction and even cell death, due to failure of cell division 40. Since the SaClpP mutants degrade FtsZ in vitro (Figure 1D), we wondered if these mutants could lead to the destabilization of the FtsZ protein in the staphylococcal. The cellular abundance of FtsZ in the complemented

ΔclpP/pYJ335::clpP,

ΔclpP/pYJ335::clpPY63A,

and

ΔclpP/pYJ335::clpPN42AY63A mutant strain was monitored, respectively, by Western blot at the time point of 3 hr during S. aureus growth. As shown in Figure 3C, the major portion of the hybridized bands positioned at molecular weight 25 kD in the mutant strains, which are likely composed of protein fragments gained from the

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degradation of FtsZ, revealing that the self-activated SaClpPN42AY63A protease is in an uncontrolled dysfunctional state in vivo. The expression of the engineered clpP allele phenocopies treatment with ADEP. We have described genetic experiments in which the expression of the allele-encoding SaClpPN42AY63A severely compromises the growth of S. aureus and destabilizes FtsZ. These observations mirror the treatment of S. aureus with an ADEP that dysregulates the activity of ClpP. An alternative explanation of these observations is that cells expressing the engineered clpP allele could be sick and exhibiting poor growth because the protein is misfolded and therfore toxic. We measured the size of staphylococcal cells using light microscopy, which could reflect the cell division activities of different strains (Figure 3D and S4). The average diameter of 1,000 bacterial cells was measured at 0.867 µm, 0.876 µm, and 0.885 µm for the wild-type S. aureus, the wild-type S. aureus with pYJ335 vector, and the ΔclpP/pYJ335::clpP

strain,

respectively.

The

measured

diameter

of

the

ΔclpP/pYJ335::clpPY63A mutant strain is slightly larger than those of the three control strains. Of note, we have observed that the ΔclpP/pYJ335::clpPN42AY63A mutant strain has a diameter comparable to that of the wild-type strain upon ADEP8 treatment shows. This observed phenotype of the inhibition of cell division could be a result of the destabilization of FtsZ protein by dysregulated activity of SaClpPN42AY63A mutant. ADEPs-activated ClpP becomes a fairly non-specific protease and kills persister cells; as a result, bacteria displays increased susceptibility to rifampicin only that kills

12


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them

29

. As expected, we have observed that addition of rifampicin to surviving

persisters has no effect on the viability of wide-type S. aureus Newman strain, while the combination of ADEP8 with rifampicin eradicates a stationary population of bacteria to the limit of detection (Figure 3E). Since the expression of the allele-encoding SaClpPN42AY63A severely compromises the growth of S. aureus and destabilizes

FtsZ,

we

expect

that

the

dormant

cells

of

the

ΔclpP/pYJ335::clpPN42AY63A mutant S. aureus strain could be effectively killed in the presence of the antibiotic rifampicin. It is quite interesting that the addition of rifampicin to the exponentially growing ΔclpP/pYJ335::clpPN42AY63A mutant cultures leads to the eradication of persisters below the limit of detection after 72 hrs (Figure 3E). However, persisters could be still observed after treatment of the S. aureus strain expressing the engineered clpP alleles with other antibiotics such as vancomycin, linezolid, and ciprofloxacin (Figure S5). It is not clear why this sterilizing effect is seen upon treatment of the S. aureus strain expressing the engineered clpP alleles with rifampicin rather than with other antibiotics. Taken together, these observations support the expression of the engineered clpP alleles that phenocopies treatment with ADEP. Proteomic analysis of protein degradation in the engineered clpP allele. A previous study showed that ADEP4-activated ClpP kills persisters due to the degradation of over 400 proteins in S. aureus 29. Here, in order to analyze the protein degradation in the clpP allele encoding SaClpPN42AY63A, we compared the proteome of the clpP allele with that of the wild-type S. aureus strain. As shown in

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Figure S6A, the LFQ intensity of each protein is almost at the same level across samples, precluding biases toward different samples. The correlation co-efficient of the LFQ intensities between two LC-MS/MS runs was higher than 0.86 (Figure S6B), which indicates that the experimental setup of the relative label-free quantitation profiling was highly reproducible between the two LC-MS/MS runs, intra each cohort, or inter two cohorts. Therefore, we performed an accurate comparison of the expression of 1,646 proteins (63% of the predicted open reading frames) between the clpP allele encoding SaClpPN42AY63A and wild-type S. aureus strain (see Supporting Information for Full list of all of the peptides). A heat-map of the intensities of 1,646 proteins was displayed using hierarchical clustering analysis (HCA). The 5 samples of the clpP allele encoding ClpPN42AY63A group and those in the control group were intriguingly classified into two respective clusters, demonstrating a profound proteomic regulation between the two groups (Figure 4A). Proteomic analysis of the clpP allele Newman strain encoding SaClpPN42AY63A resulted in 552 proteins (P≤0.05) showing decreased abundance; what’s more, 221 proteins show significant changes in abundance (2-fold decrease, P≤0.05). We mapped the 221 proteins to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Twelve pathways with at least five proteins involved were enriched, including

biosynthesis

of

antibiotics,

purine

metabolism,

and

glycolysis/gluconeogenesis, for example (Figure 4B). Purine metabolism and glycolysis/gluconeogenesis were included in the top three enriched pathways. Gene

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Ontology (GO) was also carried out in order to annotate the molecular functions of these 221 diminished proteins (Figure S6C). The most common molecular functions involve ion binding, oxidoreductase activity, and transmembrane transporter activity, indicating these degraded proteins belong to various functional pathways. Of note, 117 degraded proteins were overlapped with the proteins diminished in S. aureus upon ADEP4 treatment, including proteins RpIL (50S ribosomal protein L7/L12), RpsU (30S ribosomal protein S21), Tuf (Elongation factor Tu), FtsZ, Efp, and RpsA (Figure 4C and S7). Gain-of-function ClpP mutant in different bacteria. ClpP proteases display a high degree of sequence similarity in various organisms. In particular Tyr63 and Asn42 are well conserved in different bacterial ClpP proteases (Figure 5A). In order to test the generality of the self-activation property induced by mutations of Tyr63 and Asn42, we introduced alanine mutations to the corresponding residue and constructed EcClpPY76A and BsClpPY63A mutants, respectively. As expected, both EcClpPY76A and BsClpPY63A were observably gain-of-function for the efficient degradation of β-casein protein without ATPases involvement (Figure 5B). Moreover, the EcClpPN55AY76A mutant becomes a nonspecific protease capable of the efficient degradation of S. aureus FtsZ protein in vitro (Figure 5C), thus validating the generality of this gain-of-function phenomenon among different ClpP proteases. CONCLUSION The ClpP protease demonstrates its pivotal importance to both the survival and the virulence of pathogenic bacteria during host infection

15


41-44

. The inhibitors have


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

been developed in order to attenuate the production of extracellular virulence factors of S. aureus through covalent modifications on active site serine in SaClpP 45-47

or disruption of oligomerization

48, 49

. Intriguingly, ADEPs antibiotics can drive

ClpP into a dysfunctional state capable of killing persisters with rifampicin treatment 29

. In addition, ADEP derivatives have been identified as ClpP activators as well as

non-ADEP compounds

50-52

. The hydrophobic pockets near the N-terminal

entrance-pore on the protease surface act as major regulatory sites in order to activate ClpP. ADEPs cooperatively bind to these pockets, thus triggering the transition of ClpP 53. How the conformational change influences ClpP on its activation remains exclusive, however. Besides ATPases- and activators-triggered activation of ClpP, herein we have designed and characterized gain-of-function mutation of SaClpP, which converted it into a dysregulated enzyme independent of ATPases- or activators-promotion. The SaClpPY63A mutant leads to self-activation and promotes protein degradation of FtsZ (Figure 1 and 2). The SaClpPN42AY63A mutant shows enhanced proteolytic activity, leads to inhibition of cell division, and retarded proliferation of S. aureus (Figure 3). These data imply that removal of these sterically encumbering residues likely enable the ClpP peptidase to dynamically occupy conformations in which the pore is open. Similar to ADEPs-promoted killing dormant cells, persisters could not be observed after treatment of the S. aureus strain expressing the engineered clpP alleles with rifampicin due to the degradation of over 500 proteins (Figure 3 and 4). Given the highly conserved sequences of ClpP proteins among different species, these findings

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potentially reflect a mechanism for an activation process that might be shared by the whole ClpP family in general and thus aid us in better understanding the principles of ClpP protease function and structure (Figure 5). In summary, we have described the engineering and characterization of an allele of the clpP gene in S. aureus that encodes a peptidase having dysregulated catalytic activity. The primary engineered allele has a mutation that substitutes a tyrosine residue in the hydrophobic pockets with an alanine, which results in a “domino effect” that reorients several adjacent residues such that an “open-gate” conformation can be achieved. In addition, Asn42 acts like a swing that further facilitates the activation of ClpP. Therefore, the allele encoding SaClpPN42AY63A displays a phenotype that is reminiscent of observations made upon the treatment of wild-type S. aureus strain with ADEPs antibiotics that dysregulate the catalytic activity of ClpP. Taken together, characterization of the gain-of-function mutant provides new insights into the structure of ClpP and further advances our understandings of mechanism for ClpP activation. METHODS A description of the methods used in this work is located in the Supporting Information. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Methods, Figures S1-S7, and Table S1 (PDF) 17



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Full list of all of the peptides in our study on proteomic analysis (XLSX) Accession Code The crystallographic coordinates for SaClpPY63A protein are deposited in the RCSB Protein Data Bank under accession ID code 5C90. AUTHOR INFORMATION Corresponding Author [email protected] Author Contributions ⊥

These authors contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank all beamline staff for data collection support at the BL17U of the Shanghai Synchrotron Radiation Facility, and S.F. Reichard, MA for editing the manuscript. We thank H. Brötz-Oesterhelt (University of Düsseldorf) for providing us compound ADEP1 when we initiated ClpP project several years ago. This work was supported by the National Natural Science Foundation of China (21172234 to C.Y.), the Self-deployment

Project

of

Shanghai

Institute

of

Materia

Medica

(CASIMM0120154016 to C.Y.), and the Public Projects of Zhejiang Province (2015C33159 to F.Y.).

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ClpP protease by ADEP antibiotics: insights from hydrogen exchange mass spectrometry, J. Mol. Biol. 425, 4508-4519.

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FIGURE LEGEND Figure 1. In vitro activity of Y63 mutants of SaClpP. (A) Cartoon representative pathways of ClpP activation. Both APTase-chaperones and ADEP antibiotics activate ClpP protease via conformational control. (B) Structural superimposition of the residue Tyr63 of BsClpP in the extended (PDB ID: 3PTG) state and ADEP1-bound state (PDB ID: 3PTI). The ADEP1 is colored in orange, Tyr63 in ADEP1-bound state in magenta and in the extended state in cyan, respectively. The black dashed lines indicate hydrogen bonding. The dihedral angle between the two side chains of Tyr63 is about 90º. (C) Hydrolysis of β-casein protein by Y63A and Y63I mutants of SaClpP. (D) Degradation of FtsZ protein by SaClpP enzymes. (E) Kinetics study of hydrolysis of FITC-casein protein by Tyr63 variants. The initial rate is calculated. (F) Tm of SaClpP variants. This was measured by DSF assay and performed in triplicate. (G) The ClpX-dependent degradation of GFP-SsrA protein is inhibited by the Y63A mutation in SaClpP. (H) Pull-down assay was performed in order to investigate the binding between SaClpX and SaClpPY63A. T is total sample, W is washing, and E is eluent with imidazole, respectively.

Figure 2. The crystal structure of SaClpPY63A indicates a domino effect mechanism for ClpP activation. (A) Crystal structure of SaClpPY63A. The representative surface is colored in gray, and the N-terminal residues that form the entrance-pore are colored in blue. The N-terminal pore is estimated to be 10 Å in diameter. The seven activators-binding sites are indicated. (B) Close view of the activators-binding site in

23



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SaClpPY63A. The side chains of Met31 and Asn42 adopt dual conformations. The residue is colored in cyan from one monomer, and the Asn42 from the neighboring monomer is colored in magenta. The black dashed lines indicate hydrogen bonding. (C) SaClpPN42AY63A mutant exhibits enhanced activity for FtsZ degradation in vitro. SaClpPN42A is unable to degrade FtsZ. (D) SaClpPN42AY63A mutant mainly assembles as a tetradecamer as SaClpPY63A or the wild-type SaClpP. The assembly is determined on size exclusive gel-filtration. (E) Cryo-electron microscopy images to show entrance-pore enlargements. Class averages of top views from free SaClpP, ADEP8-bound SaClpP, and SaClpPN42AY63A, respectively. The inner diameter of the ClpP protease is shown in average.

Figure 3. The expression of engineered SaClpPN42AY63A allele triggers similar responses as ADEP treatment. (A) Bacterial growth of S. aureus strains with or without TET (0.025 μg/ml) induction. (B) CFU assay shows the cell viability of the engineered clpP allele was severely compromised upon TET induction (top panel). The expressions of the engineered SaClpP variants are quantified using Western blot (bottom panel). (C) The expression of the allele encoding SaClpPN42AY63A severely destabilizes FtsZ inside cells. (D) SEM analysis of the diameters of the variants of S. aureus strains. 1,000 cells of each S. aureus strain were manually measured. The average diameter is shown. The scale bar represents 1 μm. (E) The combination of ADEP8 activator and rifampicin antibiotic completely kills dormant S. aureus cells

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within 72 hr. Similarly, rifampicin leads to the eradication of persisters of SaClpPN42AY63A mutant strain below the limit of detection.

Figure 4. Label-free quantitative proteomic analysis. (A) The hierarchical clustering analysis (HCA) of 1,646 proteins. W1-W5 are the wild-type S. aureus Newman strains, and M1-M5 are the clpP alleles encoding SaClpPN42AY63A. (B) Function-enrichment analysis of significantly degraded proteins in the SaClpPN42AY63A mutants cells. The altered proteins were mapped to the KEGG pathway database (http://www.genome.jp/kegg/pathway.html) using the blast2go program. (C) Box plots for the LFQ intensity from the significantly degraded proteins, including FtsZ and FbaA, which are overlapped to that in S. aureus upon ADEP4 treatment.

Figure 5. The self-activation of S. aureus ClpP mediated by Tyr63 and Asn42 is general in bacterium ClpP. (A) Sequence alignment of ClpP from different species reveals the conservation of asparagine and tyrosine. (B) Both EcClpPY76A and BsClpPY63A mutants are gain-of-function for β-casein degradation in vitro. (C) EcClpPN55AY76A becomes an uncontrolled protease that actively hydrolyzes SaFtsZ protein in vitro.

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Characterization of Gain-of-function Mutant Provides New Insights into ClpP Structure Tengfeng Ni•, †, , Fei Ye‡, ≠, , Xing LiuƩ, , Jie Zhang§, , Hongchuan Liu§, Jiahui Li•, †, Yingyi Zhang¶ , Yingqiang SunΔ, Meining WangƩ, Cheng Luo≠, Hualiang Jiang≠, †, Lefu Lan•, †, Jianhua Ganǁ, Ao ZhangƩ, †, Hu ZhouƩ, †, Cai-Guang Yang•, †, * §

Laboratory of Chemical Biology, State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; †

University of Chinese Academy of Sciences, Beijing 100049, China;

‡

College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China;

≠

Drug Design and Discovery Center, State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; Ʃ

CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, Shanghai 201203, China; ¶

National Center for Protein Science Shanghai, Institute of Biochemistry and Cell

Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 201210, China; Δ

Experiment center for science and Technology, Shanghai University of Traditional

Chinese Medicine, Shanghai 201203, China ǁ

School of Life Sciences, Fudan University, Shanghai 200433, China.



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Characterization of Gain-of-Function Mutant Provides New Insights

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