Author response: Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level Article Swipe

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Daniel Coutandin , Christian Osterburg , Ratnesh Kumar Srivastav , Manuela Sumyk , Sebastian Kehrloesser , Jakob Gebel , Marcel Tuppi , Jens Hannewald , Birgit Schäfer , E. Salah , Sebastian Mathea , Uta Müller-Kuller , James Doutch , Manuel Grez , Stefan Knapp , Volker Dötsch · YOU? · · 2016 · Open Access · · DOI: https://doi.org/10.7554/elife.13909.033

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction. https://doi.org/10.7554/eLife.13909.001 eLife digest The irradiation and chemotherapy drugs that are used to destroy cancer cells also damage healthy cells. Germ cells – from which egg cells and sperm cells develop – are particularly vulnerable as they contain sensitive quality control mechanisms that kill any cell that contain damaged DNA. Consequently, after surviving cancer many patients are confronted with infertility. A protein called p63, which is closely related to another protein that suppresses the formation of tumors, plays an essential role in detecting and responding to DNA damage. In immature egg cells (also known as oocytes), p63 mostly exists in an inactive form. The protein then switches to an active form when DNA damage is detected to trigger the process of cell self-destruction. Now, Coutandin, Osterburg et al. have performed a range of biochemical, biophysical and cell culture experiments to study how p63 is kept in its inactive form in the oocytes of mice. The experiments showed that in the inactive form, the two ends of the protein form a sheet that closes a key site on the protein and prevents it from changing into its active form. However, this closed form can be thought of as being like a spring-loaded trap – it doesn't take much energy to spring the trap and open the protein into its active form. Once this change has occurred, it is irreversible. Coutandin, Osterburg et al. also found that the oocytes of mice already contain all the proteins necessary to activate p63. This means that once the switch to the active form is triggered there is no delay waiting for other proteins to be made, which makes oocytes extremely sensitive to DNA damage. Further work is now needed to investigate the exact molecular mechanisms behind the activation of p63. https://doi.org/10.7554/eLife.13909.002 Introduction The p53 protein family with its three members p53, p63 and p73 plays very important roles in the surveillance of genetic and cellular stability (Levine et al., 2011). Probably the most ancient function of this family is the maintenance of genetic quality in germ cells since even short lived eukaryotic animals express a p63-like protein in their germ cells (Ollmann et al., 2000; Derry et al., 2001; Brodsky et al., 2000; Suh et al., 2006; Ou et al., 2007). In mammals, up to 10 diverse p63 isoforms exist with the longest one, TAp63α, being highly expressed in primary oocytes that are arrested in prophase of meiosis I. After homologous recombination, oocytes are kept in this dictyate arrest phase until they are recruited for ovulation, a period that can take decades in humans. Once oocytes reenter the cell cycle, expression of TAp63α is lost (Suh et al., 2006). Since p63 can initiate apoptosis the high expression level of TAp63α in oocytes requires that its activity is tightly regulated. Recently we could show that TAp63α assembles into a closed and only dimeric conformation in which the protein is inactive (Deutsch et al., 2011). Detection of DNA damage leads to activation of p63 triggered by phosphorylation (Suh et al., 2006; Bolcun-Filas et al., 2014) that results in the formation of open tetramers with a twentyfold higher DNA binding affinity and the induction of apoptosis. This p63-based quality control is unique to oocytes, making them very sensitive to DNA damage. Irradiation with 0.45 Gy is sufficient to eliminate all p63-expressing oocytes in mice while all surrounding cells of the ovaries survive. To understand the mechanism of inhibition and activation we have started to characterize the structural requirements for the formation of the closed and dimeric state of TAp63α. In previous experiments we have shown that the very C-terminus contains a transactivation inhibitory domain (TID) that is of central importance for creating the closed dimeric state (Serber et al., 2002; Straub et al., 2010). We have suggested a model in which both the C-terminal TID and the N-terminal transactivation domain (TAD) interact with the central tetramerization domain (TD) thereby preventing the formation of tetramers. This central TD is a dimer of dimers suggesting that blocking the interface by which two dimers form a tetramer is the most likely mechanism of inhibition. In the past we have identified mutations in all three domains – TAD, TD and TID – that break the inhibitory mechanism, establishing that at least these three domains are involved in this process. In the absence of a high resolution structure we have now used systematic alanine scanning and charge swap mutagenesis in combination with SAXS (small angle X-ray scattering) experiments to build a model of the closed and dimeric complex. In addition, we show that the inhibited conformation is a kinetically trapped state and that the oocyte contains all factors necessary to activate p63 without requirement of further protein expression. Together our data show that activation of TAp63α follows a spring-loaded mechanism and explains why oocytes are far more sensitive to DNA damage than the surrounding follicular cells. Results Defining the minimal sequence required for formation of the closed dimeric conformation TAp63α contains three folded domains, the DNA binding domain (DBD), the tetramerization domain (TD) and the SAM domain that are linked by unstructured regions. NMR experiments with a tetrameric construct containing all three folded domains showed that these domains behave independently as pearls on a string (Figure 1—figure supplement 1). All sequences outside of these folded domains are not structured in isolation but may be folded when interacting with other segments of the protein as part of the inhibitory mechanism. To identify the exact sequence elements required to form the closed state, we systematically deleted sequences in these linker regions. Deletion of sequences crucial for the formation of the closed state results in the formation of an open conformation. Previously we have shown that the open state can be detected by a conformation sensitive pull-down experiment: tetrameric mutants with an intact TAD can be pulled down with a GST-TID construct (569–616) (Straub et al., 2010). Thus, mutants that cannot be pulled down are assumed to adopt the closed dimeric state. After several rounds of deletion mutagenesis, a minimal dimeric construct was obtained. Size exclusion chromatography combined with multi angle light scattering (SEC-MALS) confirmed that this minimal construct (TAp63αmin) comprising deletions Δ(1–9; 64–119; 417–453; 460–505; 571–593; 615–641) is a stable dimer in solution (Figure 1A and Figure 1—figure supplement 2B). In addition, deletion of amino acids 322–342 between DBD and TD does not disrupt the dimeric state (Figure 1—figure supplement 3), but results in quite low expression levels in E. coli. For the experiments described below we have, therefore, used either TAp63αmin, wild type TAp63α or a slightly shortened version TAp63α(10–614) lacking unstructured sequences in the N- and C-terminus (Figure 1—figure supplement 2). Figure 1 with 10 supplements see all Download asset Open asset Mapping of structurally important regions within dimeric TAp63α. (A) Domain organization of TAp63α: transactivation domain (TAD), DNA binding domain (DBD), tetramerization domain (TD), sterile alpha motif (SAM) domain, transactivation inhibitory domain (TID). The minimal construct of TAp63α (TAp63αmin) lacks the first 9 and the last 27 amino acids as well as linker regions between TAD and DBD (64–119), TD and SAM (417–453; 460–505) and SAM and TID (571–593). Residues 454–459 were used as a linker between TD and SAM. (B) WB and corresponding bar diagram of pull-down experiments with constructs lacking either the DBD or the SAM domain using immobilized TID. Ratio of pull-down (P) and input (I) is shown relative to TAp63α(10–614) (set to 1). Pull-downs were performed in technical triplicates and error bars denote standard deviation. (C,D,F,H) TAp63α(10–614) constructs were expressed in rabbit reticulocyte lysate (RRL) and subjected to size exclusion chromatography (SEC). SEC profiles were obtained by WB (using an anti-myc antibody). (C,D) SEC profiles of TAp63α (10–614) ΔSAM (C; pink) and TAp63α (10–614) R(DBD; sfGFP) (D; green) compared with wild type (TAp63α(10–614), grey). R(DBD; sfGFP) indicates the replacement of the DBD by sfGFP. (E) Secondary structure prediction and mapping of structural motifs that stabilize the dimeric TAp63α. Cylinders and arrows represent α-helices and β-strands, respectively. Mutations (color-coded and indicated by filled circles) were introduced into TAp63α(10–614) on different faces of predicted secondary structure elements. The TAD is subdivided into TA1 (residues 10–26), TA2A (33–41) and TA2B (46–61). The TA1 forms an α-helix and the F16/W20/L23 motif constitutes the single interaction motif of the TA1. See Figure 1—figure supplement 5 for a thorough mapping of the TA1. (F) The two faces of the β-stranded TA2B were mutated (residues i, i+2, i+4 to alanine). SEC profiles of I50A I52A M54A (orange) and K49A E51A S53A (blue). See Figure 1—figure supplement 6 for a thorough mapping of the TA2. SEC of I50A I52A M54A was performed in technical triplicates and error bars denote standard deviation. (G) Transcriptional activities of TAp63α TD mutants on the p21 promoter in SAOS2 cells. Triple and double alanine mutations were introduced on the central hydrophobic interface of the TD. Bar diagrams show n-fold induction relative to the activity of the empty vector. Experiments were performed in biological triplicates and error bars denote standard deviation. (H) Mutations were introduced on the two faces of the TID β-strand. SEC profile of R598A I600A (red), E597A V599A D601A (blue), V603A F605 L607A (green) and R604A R608A (purple), Q609A I611A F613A (green) and R604A R608A (purple). See Figure 1—figure supplement 7 for SEC profiles of other mutants. (I) Central hydrophobic interface of the dimeric TD, showing the important I378 L382 M385 motif. (J) Transactivation assay of TAp63α(10–614) mutants that appeared tetrameric in previous experiments (see F, H and Figure 1—figure supplement 7). Transcriptional activities on the p21 promoter in SAOS2 cells were normalized to the protein level (determined by WB and referenced on GAPDH level). Experiments were performed in biological triplicates and error bars denote standard deviation. https://doi.org/10.7554/eLife.13909.003 The SAM domain and the DBD are not essential to retain the dimeric state In contrast to the TD, an involvement of the SAM domain and DBD in the formation of the closed dimeric state is not immediately obvious. To investigate whether these domains participate in the stabilization of the closed conformation we deleted each domain separately in TAp63α(10–614) and performed pull-down experiments with GST-TID. Interestingly, deletion of the SAM domain did not show any significant pull-down and size exclusion chromatography confirmed the formation of closed dimers (Figure 1B and C). On the contrary, deletion of the DBD resulted in a strong pull-down signal suggesting an open state (Figure 1B). Initially we expected the DBD to participate in essential domain-domain contacts that stabilize the closed conformation and therefore conducted an extensive mutagenesis screen of surface residues of the DBD (Supplementary file 1). However, none of the mutants formed tetramers making this hypothesis unlikely. Alternatively, the DBD may be important for geometric reasons, acting as a spacer between TAD and TD. To test this hypothesis, we replaced the DBD by superfolder GFP (sfGFP) which is very stable and of similar size as the DBD. SEC analysis of this chimeric protein expressed in rabbit reticulocyte lysate (RRL) suggested that it adopts a closed dimeric conformation (Figure 1D). Moreover, mutations F16A W20A L23A within the TAD and F605A T606A L608A within the TID resulted in the formation of a tetrameric state similar to experiments with wild type TAp63α (Straub et al., 2010) (Figure 1—figure supplement 4C and E). Similarly, replacement of the DBD by MBP enables the formation of a closed dimeric state (Figure 1—figure supplement 4F). These results suggest that the DBD does not participate in essential domain-domain interactions necessary to form the dimeric state and that the closed dimeric state of TAp63α is formed by interaction of the N-terminal TAD, the central TD and the C-terminal TID. Nonetheless, constructs that only contain these three domains did not form dimers but aggregated, suggesting that the DBD or a domain of similar size is necessary for structural reasons or for the folding process. Mapping of the TAD-TD-TID interaction To build a first model of the closed state we used secondary structure prediction programs to identify potential secondary structure elements within the TAD and TID and alanine scanning in combination with SEC analysis to experimentally verify these predictions. The theoretical analysis predicted the existence of an α-helix in the TA1 region, two β-strands in the TA2A and TA2B regions of the TAD and a β-strand in the TID (Figure 1E). Alanine scanning of the TA1 confirmed that only mutations of F16, W20 and L23 that have previously been identified as crucial for binding of the TA1 to the TD (Deutsch et al., 2011), disrupted the closed conformation while mutations on the three remaining faces of the hypothetical helix had no effect (Figure 1—figure supplement 5). To test the existence of the various β-sheets we mutated all amino acids on one side of each predicted β-strand to alanine (i, i+2, i+4). While mutations on both faces of the presumed first beta-strand (TA2A) did not affect the oligomeric state (Figure 1—figure supplement 6B), the mutations I50A I52A M54A located on one face of the predicted TA2B β-strand disrupted the dimeric state (Figure 1F). Alanine scanning of the TID showed that mutations on both sides of the presumed β-strand disrupt the dimeric state (Figure 1H and Figure 1—figure supplement 7B). Stabilizing the dimeric state is most likely achieved by blocking the tetramerization interface of the TD and we also used alanine scanning of the TD to identify essential residues (Figure 1—figure supplement 8). Since mutations in the tetramerization interface that destabilize the dimeric state most likely also inhibit the formation of the tetramer, we did not use SEC analysis. Previously, we have shown that an open dimeric state is transcriptionally more active than the closed dimeric state (Deutsch et al., 2011). Mutating the hydrophobic amino acids I378, L382 and M385 alongside the second half of the α-helix of the TD led to high transcriptional activity as expected for an open conformation (Figure 1G and I, Figure 1—figure supplement 8). We also used the measurement of the transcriptional activity as well as pull-down experiments with GST-TID to validate the results of our SEC analysis with the different alanine mutants (Figure 1J and Figure 1—figure supplement 9). As expected, all mutants that behaved like open and tetrameric conformations showed high transcriptional activity. The only exception was the F16A W20A L23A mutant since these mutations compromise the function of the TAD (Figure 1—figure supplement 9). TA2B and TID form a β-sheet The experiments described above support the prediction that TA2B and TID form regular secondary structure elements, most likely β-strands. In the closed dimer, two TID and two TA2B sequences must be involved in the stabilization of the closed state. For symmetry reasons, the β-strands probably adopt an antiparallel orientation. Based on the results of the alanine scanning experiments we speculated that the two TID strands form the inner pair since mutations on both faces of the predicted β-sheet show strong effects. Further, we propose that the two TA2B strands form the two outer strands of a four stranded anti-parallel β-sheet which might be further extended by β-strands contributed by the TA2A segment. Such an arrangement would create one hydrophobic surface formed by I50/I52/M54 of TA2B and V603/F605/L607 of TID and a hydrophilic surface with residues E51/D55 of TA2B and R604/R608 of TID. The arrangement shown in Figure 2B brings charged amino acids on neighboring strands in close proximity, making it possible to test this hypothetical model by charge change and charge swap mutagenesis. Exchanging R604 and R608 in the TID to glutamic acids disrupted the dimeric state (Figure 2C). In our model these mutants created in combination with the negative charges on the TA2B strands a cluster of negatively charged amino acids that destabilized the dimer. Additional charge reversal of E51R and D55R in TA2B resulted in the formation of a stable dimer. Similarly, the R595E and R598E mutants are open tetramers and the additional charge reversal of D61R, D63R in TA2B rescued the dimer (Figure 2D). To refine our model and to identify the register of the proposed β-strands we used further pairwise charge swap mutations. The results of these experiments that all support our structural model are summarized in Figure 2—figure supplement 1. Since the predicted β-sheet has one hydrophobic face and the interface used by the TD to form tetramers is also hydrophobic, we propose that the β-sheet covers the tetramerization interface of the TD, thus inhibiting the formation of tetramers (Figure 3B and C). In addition, the TA1 helix binds to the TD as well, further stabilizing the closed and compact conformation. Figure 2 with 1 supplement see all Download asset Open asset TA2B and TID form an anti-parallel β-sheet with a polar and a hydrophobic face. (A) Domain organization of TAp63α and secondary structure elements of TAD and TID. (B) Proposed interaction of TA2 and TID through β-sheet formation. This interaction is thought to be stabilized by hydrophobic amino acids clustered on one face of the β-sheet (bottom) and electrostatic interactions between charged amino acids on the other face (top). Extensive charge swap experiments (see Figure 2—figure supplement 1) revealed interactions between TA2B and TID. Interactions are depicted in green. (C, D) Introduction of negative charges in the TID and charge swaps between TID and TA2B show interaction via β-sheet formation. (C) SEC profiles of TAp63α R604E R608E (orange) and the charge swap mutant TAp63α E51R D55R R604E R608E (blue). (D) SEC profiles of TAp63α R595E R598E (orange) and the charge swap mutant TAp63α D61R D63R R595E R598E (blue). https://doi.org/10.7554/eLife.13909.014 Figure 3 Download asset Open asset Model of the closed dimeric conformation of TAp63α . (A) Domain organization of TAp63α. All domains and structural elements are color coded. (B) The TD of p63 forms a dimer of dimers (colored in dark and light grey). Its two tetrameric interfaces (in light blue and rose) must be blocked in the inactive dimer to inhibit tetramerization. The TA1 was shown to bind to the upper interface (in rose) (Deutsch et al., 2011). The I378 L382 M385 motif in the central interface (in light blue) must be covered by hydrophobic amino acids. The hydrophobic interface of the proposed 6-stranded β-sheet is expected to cover this central tetrameric interface of the TD. (C) Model of the intramolecular interactions between TAD, TD and TID. The angles between structural elements are speculative. The TD was placed on top of the TA2/TID β-sheet so that the hydrophobic amino acids mask each other. The second helix of the TD is not modelled. (D) Pair distribution function P(r) from inline SEC-SAXS (small-angle X-ray scattering) data of TAp63αmin. Derived function transformed smoothly and to central part with short (E) SAXS of without and with using and The similar the of symmetry in TAp63αmin. were and using and were obtained from inline (F) model from to wild type and using of segments when the (G) of the to data wild type in and in (H) WB and corresponding bar diagram of the pull-down experiments with TAp63α and from using either immobilized or WB signal for input and pull-down are The pull-down of was to Pull-downs were performed in technical triplicates and error bars the standard deviation. (I) of the p63 DBD to DNA and a model of the p63 DBD to on the structure between the p53 DBD and (J) TAD, TD and TID are placed the SAXS The are likely at the outside of the the to be by TAD, TD and TID. The SAM domain is not modelled. angle X-ray scattering a dimeric structure of TAp63α with the at the outside The analysis described above predicted the formation of a compact structure with To verify this we performed SAXS with TAp63αmin. To identify the of the we also SAXS data on a construct containing mutated at its resolution from of the SAXS data showed a symmetry (Figure with the located in the of the (Figure Based on these results and the of the domains we propose that the are at the outside while the formed by the TAD, TD and TID the of the (Figure In this model the SAM domain is also located in the the showed the To additional information on the of the DBD we performed binding with the and domain of the protein This protein is known to bind to the DNA binding interface of the DBD (Figure In pull-down experiments we were not to interaction of TAp63α with while the open and tetrameric showed strong interaction (Figure This that the DNA binding interface of the DBD is not but the of the The dimeric conformation of TAp63α constitutes a kinetically trapped state Activation of TAp63α of the interactions described above to the tetramerization interface to the formation of active tetramers. In oocytes this is triggered by In phosphorylation could a interface interactions that stabilize the tetrameric state, making it more stable while the dimeric state would be in the absence of However, the that of the open tetrameric state using does not in TAp63α to a dimer this model (Deutsch et al., 2011). would be that the tetrameric state is the most stable one and the dimeric state is a kinetically trapped conformation. would then function as a trigger to a and p63 into the Such spring-loaded mechanisms have been for in the activation of et al., and for this type of activation mechanism is that the kinetically trapped conformation by of in or an in the to the more stable conformation even without the Since the stability of the three folded domains of TAp63α is quite high et al., 2001; et al., (Figure supplement 1) we that using low to of might disrupt the inhibitory thus the formation of the tetramer without the folding of the the SAM or the TD. To investigate activation of TAp63α follows a spring-loaded mechanism we a SEC with different of in containing the concentration and the of dimer and Figure that a concentration of leads to an of dimer and tetramer and at above 3 no dimer was resulted in further on the SEC probably conformations (Figure supplement 2). To validate the data we performed at of 2 and (Figure and The first SEC had a of and the second a of 2 with the first one a tetrameric and the second one a dimeric conformation. Figure with 2 supplements see all Download asset Open asset The closed dimeric conformation of TAp63α constitutes a kinetically trapped state. (A) were for 1 at different and subjected to size exclusion chromatography at corresponding (B) were in and into a 6 with at different (C) SEC profiles of after for in of tetrameric and dimeric protein are in and respectively. SEC profiles of tetrameric (E) and dimeric (D) from SEC shown in after to for of at different to the tetrameric of the in and denote tetramer and dimer respectively. used to the molecular and standard deviation. (F) of in 2 in 2 for at (G) of in in for at (H) WB and corresponding bar diagram of pull-down experiments with TAp63α R604E R608E and TAp63α either during or after expression in at for with p73 TD or a mutant that is not to form is achieved by of p73 TD with of pull-down (P) and input (I) is shown relative to TAp63α after expression with p73 TD (set to 1). were performed in technical triplicates and error bars denote standard deviation. the of the spring-loaded activation is of would not the formation of a p63 dimer. To test this hypothesis, we the dimer and the tetramer at a concentration of on the SEC (Figure and both without of these by SEC revealed that the dimeric dimeric (Figure and the tetrameric tetrameric with a to (Figure These experiments suggest that the dimeric state of TAp63α is a kinetically trapped conformation that is by a spring-loaded mechanism. of the TAp63α dimer can only be A spring-loaded activation requires that the protein is trapped in a high energy state during protein p53 it is known that this protein for

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Author response: Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level

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Daniel Coutandin, Christian Osterburg, Ratnesh Kumar Srivastav, Manuela Sumyk, Sebastian Kehrloesser, Jakob Gebel, Marcel Tuppi, Jens Hannewald, Birgit Schäfer, E. Salah, Sebastian Mathea, Uta Müller-Kuller, James Doutch, Manuel Grez, Stefan Knapp, Volker Dötsch

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Mechanism (biology), Spring (device), Cell biology, Control (management), Quality (philosophy), Chemistry, Computational biology, Biophysics, Biology, Computer science, Physics, Engineering, Artificial intelligence, Mechanical engineering, Quantum mechanics

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