Many eukaryotic protein kinases are activated by the intramolecular autophosphorylation of activation loop residues. Smk1 is a meiosis-specific mitogen-activated protein kinase (MAPK) in yeast that autophosphorylates its activation loop tyrosine and thereby upregulates catalytic output. This reaction is controlled by an inhibitor, Isc10, that binds the MAPK during meiosis I and an activator, Ssp2, that binds Smk1/Isc10 during meiosis II. Upon completion of the meiotic divisions, Isc10 is degraded, and Smk1 undergoes autophosphorylation to generate the high activity form of the MAPK that controls spore formation. How Isc10 inhibits Smk1 is not clear. Here, we use a bacterial coexpression/reconstitution system to define a domain in the carboxy-terminal half of Isc10 that specifically inhibits Smk1 autophosphorylation. Nevertheless, Smk1 bound by this domain is able to phosphorylate other substrates, and it phosphorylates the amino-terminal half of Isc10 on serine 97. In turn, the phosphorylated motif in Isc10 inhibits the Smk1 active site. These data show that Isc10 inhibits autophosphorylation and the phosphorylation of substrates by separate mechanisms. Furthermore, we demonstrate Isc10 can inhibit the autophosphorylation of the mammalian intestinal cell kinase ICK1 , suggesting a conserved mechanism of action. These findings define a novel class of developmentally regulated molecules that prevent the self-activation of MAPKs and MAPK-like enzymes. Autophosphorylation of the activation loop is a common mechanism that upregulates eukaryotic protein kinases . In somecis) mechanism. Although mature CMGC group kinases typically phosphorylate downstream substrates on serine/threonine (S/T) residues, the most commonly autophosphorylated activation loop residue is tyrosine (Y). It has been proposed that these reactions involve a transitional “Prone-to-Autophosphorylate” (PA) conformation . In most cases, these reactions occur by an intramolecular  complexes (Smk1/Ssp2) that undergo autophosphorylation. The doubly phosphorylated Smk1 bound to Ssp2 then triggers key steps in spore morphogenesis. The “inhibited/poised/active (IPA)” model for Smk1 activation thereby provides an explanation for how key steps in spore differentiation take place only after MII has been completed  upon completion of the meiotic divisions  upon com, 11. Preompleted .in vitro biochemical reactions, and Saccharomyces cerevisiae undergoing meiotic development to show that Isc10 inhibits Smk1 through two mechanisms. First, a C-terminal segment in Isc10 specifically inhibits autophosphorylation of Smk1’s activation loop on Y209. This segment is referred to as the Y Autophosphorylation Inhibitor motif, or YAI hereafter. Although the YAI reduces the catalytic activity of Smk1 by preventing activation loop autophosphorylation, Smk1 that has not undergone autophosphorylation has a modest level of catalytic activity and Smk1 bound to the YAI can phosphorylate substrates. We find that Smk1 bound to Isc10 and Ssp2 phosphorylates the Isc10 protein on serine 97. The phosphorylated S97 motif in turn prevents Smk1 from phosphorylating Isc10 on other residues. We also find that an Smk1-Y209F mutant is not inhibited by the pS97 motif, suggesting that the Y209 hydroxyl plays an essential role in pS97-mediated inhibition. These findings suggest that once Smk1 undergoes autophosphorylation, it can no longer be inhibited by Isc10, implying a switch-like role for Isc10 in triggering activation of the MAPK. Our findings indicate that this switch operates during yeast meiotic development since expression of the YAI during meiosis in yeast downregulates Smk1 autophosphorylation. Interestingly, we find that Isc10 also inhibits the autophosphorylation of the activation loop Y of the mammalian intestinal cell kinase (ICK1).This study was designed to investigate how Isc10 inhibits Smk1. We used a heterologous bacterial expression system, 137-371 fused to glutathione-S-transferase (Ssp2KAD-GST). Cak1, which phosphorylates Smk1’s activation loop threonine (T207), modestly increases the Ssp2-dependent autophosphorylation of Smk1 on Y209. The expression of Isc10 eliminates almost all Y209 autophosphorylation in the presence or absence of Cak1 . These findings reflect the roles of Ssp2, Cak1, and Isc10 in controlling the activation of Smk1 in meiotic yeast cells  had little effect on Isc10’s ability to inhibit Smk1 autophosphorylation . Removal of an additional 35 residues (Isc10146-267) reduced the ability of Isc10 to inhibit Smk1 autophosphorylation (pY209 becomes detectable). Deletion of 21 residues from the C terminus of Isc10 (Isc101-246) did not affect Isc10’s ability to inhibit Smk1 autophosphorylation, while deletion of an additional 44 residues (Isc101-203) reduced Isc10’s inhibitory activity . These data show that the Y209 Autophosphorylation Inhibitory (YAI) segment resides between amino acids 110 and 246 of Isc10. Notably, this segment of Isc10 contains a region with a relatively high degree of evolutionary conservation across the Saccharomycetes family while the rest of Isc10 is poorly conserved . Moreover, the AlphaFold program predicts that the conserved region of Isc10 contains a high confidence secondary structure while it does not predict significant secondary structural features in the rest of the protein . The major secondary structural features within this segment are four predicted α-helices .To define the region of Isc10 that inhibits Smk1 autophosphorylation, we assayed deletions in the MBP-Isc10 construct. Removal of residues 1 to 110 from the N terminus of Isc10  was almost as effective as the full-length Isc10 protein in inhibiting the autophosphorylation of Smk1 on Y209 . Moreover, further deletion of residues 111 to 145 to generate a fragment of Isc10 that contains only pαh’s 1 to 3 (Isc10146-203) retained partial YAI activity. pαh4’s importance for YAI activity when the first 110 residues of Isc10 are present but not when they are absent might suggest that this segment of Isc10 can modify the activity of the YAI through pαh4. Irrespective, these findings show that pαh’s 1 to 3 in the Isc10146-203 are sufficient for core YAI functionality.While pαh’s 1 to 3 are highly conserved across A). All Isc10 fragments that inhibited Smk1 autophosphorylation formed complexes with Smk1 and Ssp2 that were stable to purification. However, some of the Isc10 mutants that did not fully inhibit Smk1 autophosphorylation also formed complexes with Smk1/Ssp2. Specifically, while the N-terminal truncated Isc10181-267 protein did not prevent autophosphorylation, it still formed complexes with Smk1/Ssp2. This suggests that while the most N-terminal helix predicted by AlphaFold (pαh1 [residues 155–168]) is required to inhibit Smk1 autophosphorylation, it is not essential for binding to Smk1/Ssp2 since pαh1 is not present in the binding competent but functionally defective deletant. Similarly, while Isc10’s inhibitory activity was significantly reduced when 64 amino acids were removed from the C terminus , this mutant and even a mutant lacking 97 C-terminal residues (Isc101-170) still bound Smk1/Ssp2. These findings suggest that pαhs 2, 3, and 4, are not required for Isc10 to bind Smk1/Ssp2 since they are not present in the binding competent but functionally defective Isc101-170 protein. Indeed, binding was not eliminated from the C-terminally deleted proteins until an additional 30 residues, which contains the entirety of pαh1, were deleted from the Isc101-170 protein (generating Isc101-140). Collectively, these findings suggest that Isc10 binds Smk1/Ssp2 through two separate binding domains  and that both BDs are required to inhibit Smk1 autophosphorylation. The data indicate that BD1 is within pαh1 and that BD2 is within the pαh2/3/4 segment (B and C). Because pαh4 is not required for YAI functionality, we speculate that BD2 is within pαh2/3.To assay the collection of MBP-Isc10 truncated mutants for binding to Ssp2 and Smk1, we purified the MBP-Isc10 proteins using amylose beads. Smk1 and Ssp2 that copurified with MBP-Isc10 in these samples were measured by immunoblotting . These assays show that BD1 and BD2 can each form complexes with Smk1 or Ssp2 in the absence of the other protein. Whether these interactions reflect mutually exclusive  or simultaneous interactions in the poised Isc10/Smk1/Ssp2 complex remain to be determined.To determine whether BD1 and BD2 can individually bind Smk1 and Ssp2, MBP fused to Isc10 residues 1 to 170 that contains BD1 or to Isc10 residues 181 to 267 that contains BD2, were produced in bacteria that coexpressed Smk1 or Ssp2. The MBP-Isc10 deletants were purified from bacterial extracts, and the presence of Smk1 or Ssp2 were tested D. These ISC10 gene in yeast. Smk1 autophosphorylation was decreased by more than 80% in post-meiotic isc10-Δ110 cells compared to wild-type (WT) cells  were present and 80% fewer spores when one copy of SMK1-HH was present (B). These findings genetically connect the meiotic function of isc10-Δ110 to SMK1. The APC/CAma1 E3 ubiquitin ligase targets full-length Isc10 for destruction as cells exit MII. One explanation for the persistent YAI activity in the isc10-Δ110 strain is that the 1 to 110 interval of Isc10 is required for its regulated degradation. To address this possibility, the WT and Δ110 forms of Isc10 were compared at different times during meiotic development (C and D). Both the full-length and deleted forms of Isc10 start to accumulate at 5 h postinduction, when cells are entering MI. As previously reported, the level of full-length Isc10 declines starting around 8 h postinduction, as cells are completing MII, and is undetectable at 24 h. In contrast, the Isc10-Δ110 protein persists after MII has been completed, and it accumulates to higher levels. It has previously been reported that the levels of full-length Isc10 are similarly increased in postmeiotic cells lacking AMA1 or the core APC/C subunit gene, SWM1 (Ama1-dependent destruction of Isc10 as cells complete MII.We next deleted the first 110 codons from the T) cells . These rWT cells . Consist present B. These elopment , C and Dne, SWM1 . Taken tA). There is an exception however—a protein that comigrates with MBP-Isc10 (indicated by the arrow) that is immunoreactive with the pT antibody. We also analyzed these samples by tandem mass spectrometry (MS). Full-length Isc10 decreased the phosphorylation of bacterial proteins by 75%. Isc10 was phosphorylated by Smk1 on numerous S and T residues with S97 being the major site of phosphorylation , bacterial substrate proteins are still phosphorylated on T and Y residues . The low Smk1 activity toward trans-substrates when full-length Isc10 is expressed and high Smk1 activity toward trans-substrates when only the YAI is expressed, suggest that a separate motif in Isc10 plays a role in inhibiting Smk1. The finding that the YAI inhibits autophosphorylation of Y209, but not the phosphorylation of Isc10 or bacterial substrates, suggests that the YAI does not prevent Smk1 from adopting an active kinase conformation. Instead, the YAI specifically inhibits the autophosphorylation of Smk1 (see Discussion).We next tested the phosphorylation of bacterial proteins by Smk1/Ssp2 in cells that coexpressed the Isc10rylation A, bactersmk1-as1 hereafter). In control experiments, the smk1-as1 yeast strain formed spores that appeared indistinguishable from those formed by the WT strain. In the presence of bulky purine (ATP) analogs, the smk1-as1 strain showed a smk1Δ-like phenotype . We coexpressed Smk1-as1 with Ssp2KAD-GST and Cak1 in bacteria, purified the Smk1-as1/Ssp2KAD-GST complex by GSH affinity chromatography, and incubated the purified Smk1-as1/Ssp2KAD-GST with and without MBP-Isc10 in a kinase assay buffer containing the as-specific ATP analog N6-benzyl-ATPγS (6-Bn-ATPγS) . We previously showed that Smk1/Ssp2 specifically phosphorylates S or T residues in an extended phosphoconsensus motif, Y-X-P-X-S/T-P . Mutation of S97 to alanine eliminated Isc10 thiophosphorylation . These data demonstrate that Smk1-as1/Ssp2KAD-GST phosphorylates Isc10 exclusively on S97 in the assay conditions tested. While these data indicate that Smk1/Ssp2 can specifically phosphorylate Isc10 on S97, these experiments do not address whether the thiophosphorylation that was observed in these experiments occurred when Isc10 was part of a ternary complex or when Isc10 was unbound (see later).To study the phosphorylation of Isc10 by Smk1 in a more defined system, we used a mutant form of Smk1 in which the “gatekeeper” residue (Q121), that is predicted to limit access to the ATP-binding pocket of the enzyme, was changed to alanine (referred to as n-ATPγS) . Thiopho-X-S/T-P . S97 in 199-P). To test whether T199 in Ssp2 is phosphorylated, Smk1-as1/Ssp2KAD-T199A-GST was purified from bacteria that also expressed Cak1, then analyzed with thiophosphorylation assays. The T199A substitution eliminated detectable thiophosphorylation of Ssp2 without affecting the phosphorylation of MBP-Isc10 . Ssp2KAD-GST is present at higher concentrations than Smk1 in the enzymatic preparations used in these experiments. Therefore, similar to the situation with Isc10, we do not know if the Ssp2 that is phosphorylated is free or stably complexed with Smk1 in the holoenzyme. We also do not know whether Ssp2KAD-GST that has been phosphorylated in bacteria (and is therefore resistant to the addition of thiophosphate in the in vitro reactions) is enriched in the Smk1/Ssp2 complex. Nevertheless, these experiments demonstrate that phosphorylated T199 is not required for Smk1 activity since Smk1/Ssp2-T199A thiophosphorylates MBP-Isc10 similar to Smk1/Ssp2.During these studies, we noticed that Ssp2-GST was also thiophosphorylated. Ssp2 has a motif that conforms to the Smk1 phosphoconsensus except at the -2 position . These findings indicate that the YAI increases the rate of S97 phosphorylation, likely by tethering Isc10 to the Smk1/Ssp2 complex via BD1 and/or 2 and thereby increasing the local concentration of the S97 phosphoconsensus motif. In summary, the data presented in this section demonstrate that Smk1 not only phosphorylates itself (on Y209) but also its activator, Ssp2 (on T199), and its inhibitor, Isc10 (on S97).We next tested whether deletion of the YAI motif influenced the ability of Smk1-as1/Ssp2 to thiophosphorylate Isc10 on S97 using the phosphorylation of Ssp2 on T199 as a control. For these experiments, identical concentrations of MBP fused to residues 1 to 110 or full-length Isc10 were incubated with Smk1-as1/Ssp2KAD-GST, and Cak1 in bacteria. The S97A or D substitutions did not influence the ability of Isc10 to inhibit Smk1 autophosphorylation or the ability of Isc10 to form stable complexes with Smk1/Ssp2 (A). These data are consistent with the ability of Isc10 deletion mutants lacking S97 (such as Isc10-Δ110) to bind Smk1/Ssp2 and inhibit autophosphorylation (A). Since the S97A substitution eliminated detectable phosphorylation of Isc10 by Ssp2/Smk1 in vitro (C), we expected the S97A substitution to ablate the phosphorylation of Isc10 in the bacterial reconstitution system. However, S97A increased the pT immunoreactivity of Isc10 (compare lanes 2 and 3). This finding suggests that when Isc10 is bound to monophosphorylated Smk1/Ssp2, that the S97A substitution increases the phosphorylation of Isc10 on nonconsensus residues. Consistent with this line of reasoning, the S97A form of Isc10 was also more immunoreactive with a pY antibody than WT Isc10. In addition, the S97A substitution increased the pT and pY immunoreactivity of bacterial proteins. The S97D phosphomimetic mutant also increased the pT and pY immunoreactivity of MBP-Isc10 and bacterial proteins, but these increases were modest compared to those seen in the S97A mutant (compare lanes 3 and 4). Taken together, these findings suggest that phosphorylation of Isc10 on S97 prevents monophosphorylated Smk1 bound to Ssp2 and Isc10 (P-complexes) from phosphorylating other residues in the Isc10 protein and bacterial proteins in trans.To gain insight into the role of S97 phosphorylation in regulating Smk1, MBP-Isc10 carrying nonphosphorylatable (A) or phosphomimetic (D) substitutions of S97 were produced with Smk1, Ssp2mk1/Ssp2 A. These rylation A. Since in vitro C, we expKAD-GST, and Cak1, and phosphorylation was assayed using phosphospecific antibodies . As expected, the S97A, YP, and YP+S97A substitutions did not substantially affect the ability of Isc10 to inhibit Smk1 autophosphorylation . Like the S97A mutant, the YP substitutions increased pT and pY immunoreactivity of MBP-Isc10 (compare lanes 3–6). The YP + S97A double mutant was indistinguishable from the YP and the S97A single mutants. Furthermore, the YP and S97A mutations reduced the electrophoretic migration of MBP-Isc10 . These findings suggest that when the S97-phosphoconsensus motif has been mutated, Smk1 in P-complexes hyperphosphorylates Isc10 on nonconsensus residues. In addition to the effects on Isc10 phosphorylation, the pS97 motif mutations also increased the phosphorylation of bacterial proteins. This is most easily observed by focusing on the pT and pY immunoreactivity of the bacterial protein that migrates slightly slower than MBP-Isc10 in Fig. 4B , and the YP mutations were combined with the S97A mutation. The WT or mutant forms of MBP-Isc10 were coexpressed with WT Smk1, Ssp2B [Y209F]). Interestingly, although Smk1-Y209F/Ssp2 is only 30% as active as WT Smk1/Ssp2 in transferring phosphate to substrates . These experiments showed that the kinase domain of ICK autophosphorylates its activation loop Y when expressed in bacteria and that MBP-Isc10 inhibits this reaction. We also found that ICK and MBP-Isc10 form a stable complex when coexpressed in bacterial cells . In addition, purified MBP-Isc10111-267 that contains the YAI motif can be used to affinity purify ICK from complex extracts . Together, these results demonstrate that the YAI motif of Isc10 binds to ICK1 and inhibits the intrinsic ability of ICK1 to autophosphorylate its activation loop.Intestinal cell kinase (ICK), also referred to as ciliogenesis associated kinase 1 (CILK1), is a member of the ICK/MAK/MOK family of MAPK-related kinases that are phosphorylated by the Cak1 homolog from humans (CCRK) and autophosphorylate their activation loop Y-residues, similar to Smk1 , 26. We Fig. 5SMK1, ISC10, and SSP2 genes are activated by the middle meiosis-specific transcription factor, Ndt80, as cells enter MI. Smk1 is phosphorylated on its activation loop T by Cak1 during MI and the monophosphorylated MAPK interacts with Isc10 to form heterodimeric I-complexes that disperse throughout the cell   . Both moMultiple CMGC kinases have been shown to undergo autophosphorylation of activation loop Ys , 34, 35.et al. have shown that hydroxylation of a proline near the CMGC/MAPK insert in the DYRK1 and GSK3ß kinases can allosterically activate the autophosphorylation of activation loop Ys , it downregulates signaling in a dominant manner in vivo. The data presented here suggest that a monophosphorylated MAPK can phosphorylate inhibitory motifs that in turn extinguish catalytic activity. Related interactions may play a role in downregulating other MAPKs and thereby connect monophosphorylated MAPKs to specific signaling outcomes in the cell.The well-studied Erk1 and 2 MAPKs from mammals are so much more active orylated , 40. Whiorylated . An inteorylated . Althougvia the YAI increases the local concentration of the motif and that binding of Ssp2 then triggers the phosphorylation of Isc10 on S97 (+2) to trigger activation of the kinase. Our data do not address the stability of the S97p motif/Smk1 active site complex, yet in the context of the Smk1/Ssp2/Isc10 holoenzyme, where the local concentration of the S97 motif is high, it can inhibit the enzyme.Isc10 is one of 42 proteins in the yeast proteome that contains a perfect Smk1 phosphoconsensus motif Y-X-P-X-S/T-P. Our mutational data show that S97, as well as the -2P and -4Y phosphoconsensus residues, are important for active site inhibition. We propose that Smk1 bound to Isc10 0 on S97 . We furttrans-phosphorylation involves high concentrations of nonphysiological substrates whose availability and local concentrations are difficult to assess. It is therefore unclear whether the pS97-dependent suppression of substrate phosphorylation observed in bacteria is relevant to meiotic yeast cells where the concentration of the enzyme is lower. Nevertheless, the concentration of Isc10 relative to Smk1 in the P-complexes produced in bacteria and meiotic yeast cells are identical, and these caveats do not influence the interpretation of the Isc10 phosphorylation data. Thus, while our findings demonstrate that S97p can suppress the phosphorylation of other, presumably nonspecific residues in Isc10, further studies are required to determine whether pS97 plays a physiological role in suppressing the phosphorylation of other downstream targets in meiotic yeast cells. The N-terminal 110 residues of Isc10 appear to play a regulatory role that controls the stability of the protein (C and D). One possibility is that the pS97 motif prevents Smk1 from phosphorylating other residues in the N-terminal segment of Isc10 that would otherwise interfere with its ability to be recognized by the APC/CAma1.A caveat of the bacterial coexpression experiments is that Smk1 is produced at supraphysiological concentrations. The readout for  protein , C and DOur data indicate that while Smk1 in P-complexes is inhibited by the S97p motif, the Smk1-Y209F mutant is resistant to the S97p inhibitory mechanism. This indicates that the same -OH that is autophosphorylated by Smk1 is essential for the S97p motif inhibitory mechanism. We speculate that once Y209 has undergone autophosphorylation, the doubly phosphorylated enzyme becomes resistant to inhibition by the S97p motif. If so, the S97p motif mechanism may reinforce the feed-forward switch-like properties that activate the kinase upon completion of the meiotic divisions.Escherichia coli cells using the plasmids listed in KAD-GST, and MBP-Isc10 have previously been described (CILK1) constructs, a fragment encoding the kinase domain (codons 1–300) was generated by PCR and cloned into the pACYC-Duet vector using Nde1/KpnI cloning sites to produce pAR130. MBP alone (from pZK11) or MBP-Isc10 (from pZK6) were cloned into pAR130 using the NcoI/NotI cloning sites to generate pAR142 and pAR134.Bacterial experiments were carried out with BL-21 DE3 escribed , 16. To escribed  using th7 cells/ml. Cells were pelleted by centrifugation, washed, and resuspended in sporulation medium  at 1.5 × 107 cells/ml and incubated in a roller drum at 30 °C.All yeast strains used in this study were in the SK1 background . Cells wisc10-Δ110-13myc strain, the protein coding segment of isc10-Δ110 was fused to a protein coding segment of ISC10 tagged at its carboxy terminus with the MYC epitope followed by the hygromycin B marker and the 3′ UTR region of ISC10  and resuspended in denaturing buffer . Subsequently, Smk1-HH was bound to nickel beads, eluted in sample buffer containing 200 mM imidazole, and analyzed by gel electrophoresis.Smk1-HH proteins were purified under denaturing conditions as previously described . In brieFor immunoblot analyses of meiotic yeast, equivalent numbers of sporulating cells at different time points were lysed with NaOH, and proteins were precipitated with TCA and solubilized with 8M urea, as described . This pr600 units/μl in loading buffer containing SDS and subjected to four cycles of 30 s boiling/10 s vortexing. Extracts were analyzed by polyacrylamide gel electrophoresis and Coomassie blue staining to assure that equivalent levels of total protein were present in each case. Subsequently, the extracts were analyzed by immunoblot analyses with the antibodies described later.For immunoblot analyses of bacterial samples, cells were collected by centrifugation and resuspended to 0.005 A202 (Thermo Fisher Scientific). Western blot images were captured at various exposures, and images within the linear range were quantified using ImageJ version 1.53i (https://imagej.nih.gov/ij/).After electrophoresis, proteins were transferred to Immobilon-P membranes and probed with antibodies. For detection of proteins expressed in the heterologous bacterial expression system, rabbit Smk1 (diluted 1:2000) and pY209 (1:2000) antisera, mouse GST , MBP , pT , and pY  mAbs were used. All of these antibodies have been validated for this application in previous studies , 16. For2, 300 mM NaCl, 1 mM DTT, 0.5% NP-40, and 1 mM PMSF]. Extracts were clarified by centrifugation at 15,000×g for 10 min at 4 °C. Soluble fractions were incubated at 4 °C with 80 μl of pre-equilibrated amylose beads (NEB). For immunoblot analyses, the beads were washed with 10 volumes of lysis buffer and eluted in 1× SDS buffer containing 10 mM maltose. For preparing the MBP-Isc10 proteins that were used in kinase assays, beads were washed with 10 volumes of lysis buffer, 10 volumes of kinase buffer , eluted in kinase buffer supplemented with 10 mM maltose, and stored in kinase buffer containing 15% (v/v) glycerol at -80 °C.For purification of MBP-Isc10 proteins, equivalent numbers of BL-21 DE3 cells were harvested and sonicated four times for 15 s each time in lysis buffer  with six rounds of 15 s pulses. The extracts were clarified by centrifugation at 30,000×g for 30 min at 4 °C. Soluble fractions were incubated overnight at 4 °C with 500 μl glutathione agarose beads (GenScript) that had been preequilibrated in lysis buffer. The beads were washed with 20 volumes of lysis buffer and 10 volumes of kinase buffer  and eluted in kinase buffer supplemented with 20 mM reduced glutathione (Acros Organics), pH 8.0. Protein concentration was quantitated by electrophoresis and Coomassie blue staining using bovine serum albumin (BSA) as a standard. The kinase complexes were frozen in kinase buffer containing 15% v/v glycerol at -80oC.Ssp2escribed . In briesmk1-as1). smk1-as1 yeast formed zymolyase-resistant spores that appeared indistinguishable from WT spores in sporulation medium lacked purine analogs. In contrast, the sporulation phenotype of the smk1-as1 strain was indistinguishable from the smk1-Δ sporulation phenotype based on microscopic appearance and zymolyase sensitivity when sporulation media contained 20 μM 1NM-PP1 or 10 μM PPI. The ability of WT cells to form spores was unaffected by both analogs at all concentrations tested. The detailed phenotypic analyses of the smk1-as1 mutant will be presented elsewhere. To produce the analog-sensitive form of active Smk1 for enzymatic assays, the Q121A mutation was introduced into Smk1 in the ampicillin-selectable Smk1/Ssp2KAD-GST coexpression plasmid pJT115 to generate pJT126. pJT126 was cotransformed into BL21-DE3 cells with the chloramphenicol-selectable Cak1 expression plasmid pJT122. Cultures at 18 °C were induced by the addition of 100 μM IPTG, and the Cak1, Smk1-as1, and Ssp2KAD-GST coexpressing cells were harvested 12 to 18 h later and Smk1-as1/Ssp2KAD-GST was purified using GSH-affinity beads as described previously . The mixtures were brought to room temperature (RT) for 5 min and reactions were initiated by the addition of 6-Bn-ATPγS and ATP . Reactions were incubated for 30 min at RT and terminated by the addition of EDTA to a final concentration of 20 mM. Thiophosphates were next alkylated with 2.5 mM p-nitrobenzyl mesylate for 2 h at RT. Samples were resolved by electrophoresis, transferred to membranes, and probed with a rabbit antithiophosphate ester mAb SD2020 .To generate the analog-sensitive form of Smk1, glutamine (Q) 121 was changed to alanine (A) . Cells were sonicated for 20 s for two cycles with incubation on ice between the sonication steps. The concentration of the samples was measured by Bradford assay, and proteins were analyzed by MS at the Wistar Proteomics Facility. Five micrograms of each sample was reduced with DTT, alkylated with iodoacetamide, and digested in-solution with trypsin (1:50 enzyme:protein in 2M urea). Starting materials and digests were run on SDS gels and stained with Coomassie blue to confirm trypsin digestion. Digested peptides were purified using Waters SepPak C18 columns. Digested peptides (1.25 mg) were subjected to two sequential TiO2 (GL Sciences) purifications to enrich for phosphorylated peptides. Enriched peptides were analyzed by LC-MS/MS on a Q Exactive HF mass spectrometer using an extended 2 h LC gradient. MS data were searched with full tryptic specificity against the UniProt E. coli (BL21-DE3) database (7/3/2021) plus the sequences of Smk1, Ssp2, Cak1, and Isc10, and a common contaminants database using MaxQuant 1.6.17.0. The false discovery rates for protein, peptide, and sites identifications were set at 1%. Phosphorylation (+79.96633 Da) on S, T, and Y were searched on the datasets. For quantitation of total bacterial protein phosphorylation, the iBAQ intensities of yeast proteins were excluded from the analysis.Approximately 100 AB were quantitated by staining cells at 72 h postinduction with 4′,6-diamidino-2-phenylindole. Cells were photographed under wet mount using a Nikon Optiphot equipped for epifluorescence as previously described  sequence for evolutionary relationship within the Saccharomycetes family guided by the phylogeny maps . For the immunoblot analyses in A and B, and The statistical analyses were performed in GraphPad Version 9.4.0 453) (GraphPad Software Inc). For analysis of pY209 levels relative to Smk1 in Figs. 2 (GraphPaAll data are contained within the article and This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Rigid occipitocervical (O-C) instrumentation can reduce the anterior pathology and has a high fusion rate in children with craniovertebral instability. Typically, axis (C2) screw fixation utilizes C1–C2 transarticular screws or C2 pars screws. However, anatomic variation may preclude these screw types due to the size of fixation elements or by placing the vertebral artery at risk for injury. Pediatric C2 translaminar screw fixation has low risk of vertebral artery injury and may be used when the anatomy is otherwise unsuitable for C1–C2 transarticular screws or C2 pars screws.We retrospectively reviewed a neurosurgical database at UCSF Benioff Children’s Hospital Oakland for patients who had undergone a cervical spinal fusion that utilized translaminar screws for occipitocervical instrumentation between 2002 and 2020. We then reviewed the operative records to determine the parameters of C2 screw fixations performed. Demographic and all other relevant clinical data were then recorded.Twenty-five patients ranging from 2 to 18 years of age underwent O-C fusion, with a total of 43 translaminar screws at C2 placed. Twenty-three patients were fused (92%) after initial surgery with a mean follow-up of 43 months. Two patients, both with Down syndrome, had a nonunion. Another 2 patients had a superficial wound dehiscence that required wound revision. One patient died of unknown cause 7 months after surgery. One patient developed an adjacent-level kyphosis.When performing occipitocervical instrumentation in the pediatric population, C2 translaminar screw fixation is an effective option to other methods of C2 screw fixation dependent on anatomic feasibility. Occipitocervical (O-C) fusion is required for a variety of childhood craniocervical abnormalities that create overt instability or significant anterior compressive pathology. Rigid O-C constructs with screw fixation at C2 have superior biomechanical profiles and are highly likely to fuse. Additionally, they are able to reduce anterior pathology –7. ScrewC2 fixation with translaminar screws was first described by Wright in both adult and pediatric patients –13. ConsConsecutive patients ages 18 years or younger who underwent O-C fusion incorporating C2 translaminar screw fixation at UCSF Benioff Children’s Hospital Oakland between 2002 and 2020 were identified. Patients who underwent O-C fusion with alternative C2 fixation techniques were not included in the study. Demographics and all other relevant patient data were then recorded.All patients underwent preoperative thin-cut CT with coronal and sagittal reconstructions and MRIs. All patients had plate-rod or rod constructs with rigid occipital screw fixation and 3.5 mm translaminar polyaxial screw fixation Fig. . The minPatients were placed prone in either cervical collar or Halo immobilization. Patients with severe instability and cord signal change or severe spinal cord compression were flipped prone in a Halo ring and vest in order to maintain the head and cervical spine in a stable neutral position. The cervical collar or posterior vest was then removed, and the patient was attached to the head holder with Mayfield pins or Halo adapter. Appropriate alignment was verified with lateral fluoroscopy. Pre- and post-flip motor-evoked and somatosensory-evoked potentials were obtained and monitored throughout the duration of the operation. The occiput, craniocervical junction, and posterior upper cervical spine was exposed in standard fashion.For C2 translaminar screw fixation in older children with adequately sized C2, the surgical technique for entry and trajectory was performed as described by Wright , 13. ForThe available surfaces of the occiput, C1 lateral facet, C2 lateral facet, and superior spinous process were then decorticated and structural iliac or rib bone graft obtained and wedged between the occiput and C2 Fig. a. The bot test and Pearson’s chi-squared test (X2) were used to compare differences in screw lengths relative to age. Statistical significance was assessed at a type I error rate of 0.05. All statistical analyses were performed using the R version 4.0.2 (http://cran.r-project.org/).Data are presented as means and standard deviations (SD) or proportions for continuous and categorical variables, respectively. Student Twenty-five consecutive pediatric patients underwent O-C fusion incorporating C2 translaminar screw fixation Table . The avep = 0.007). Four patients had hybrid constructs where one patient had an ipsilateral transarticular screw and 3 patients had contralateral pars screws. Two patients had Klippel–Feil deformity of C2 and C3, and the construct was extended to C3 using lateral mass screw. Another patient had a congenital C2–C3 kyphosis, and the construct was extended to C3 using a right C3 translaminar screw. Two patients (8%) had only unilateral translaminar screw with no contralateral screw fixation placed due to unsuitable anatomy for any screw fixation (Table Of the 25 patients, there were a total of 43 C2 translaminar screws placed, 21 of which were left-sided screws and 22 of which were right-sided screws (Table All patients had either rib and/or iliac crest bone grafts placed between the occiput and C2 which was overlaid with DBM and autologous bone chips Table . Twelve There were no postoperative neurological complications or injuries Table . One patC2 fixation represents a critical anchor point for craniocervical instrumented fusion. Transarticular C1–C2 screws, C2 pars screws, and C2 translaminar screws have been used for C2 screw fixation . TransarFor pediatric application, in addition to the two cases in Wright’s series , 13, addIn this series, largest to date in pediatric patients, we demonstrate that C2 translaminar screw is a safe and efficacious strategy when utilized in O-C fusion in the pediatric population. None of the patients in our series required C1 rigid screw fixation, as suggested by Hankinson et al. , while 4The use of adjuvant Halo is high is our series and stems from the patient population and our practice of flipping high-risk patients in a halo vest. Many of the patients in this series were highly unstable or had noted cord compression and myelopathy and were placed in a Halo preoperatively for stability or intraoperatively for maximum safety or reduction during the flip to the prone position Fig. . Once thIn this series, the minimum dimension of C2 lamina thickness was chosen as 4 mm for 3.5 mm diameter translaminar screws. In Chern’s study, 88.4% of children between 1.5 and 16 have a laminal thickness greater or equal to 4 mm, so most children can accommodate at least one translaminar screw. The technique of bilateral screw insertion in smaller C2 spinous processes that cannot accommodate crossing screws is achieved by partially resecting the spinous process and placing translaminar screws on each side at the dorsal aspect of the lamina, and the sensible limit of bilateral C2 translaminar screw placement is exceeded when there is sufficient hardware crowding of the screw heads at the dorsal aspects of the C2 to displace the placement of second translaminar screws to a more ventral direction resulting in ventral fracture or ventral laminar or foraminal breech Fig. a. In ourTranslaminar screw fixation of C2 is an effective option in pediatric O-C fusion. The ability to place translaminar screw fixation in children is limited by the size and growth considerations of pediatric C2. The choice amongst surgical techniques for O-C fusion in children depends on the particular anatomy and knowledge of the limitations of each fixation technique in order to achieve the highest rate of fusion with the lowest risk. When the anatomy is suitable, C2 TLS may be chosen as an option in pediatric patients requiring O-C fusion.


The outcomes presented couldserve to better understand the chemical basis of already characterizedpotent inhibitory tripeptides or as a blueprint to design novel andpotent inhibitory peptides and peptide-like molecules.Food proteins arean important source of bioactive peptides, andthe angiotensin I-converting enzyme (ACE) inhibitors are worthy ofattention for their possible beneficial effects in subjects with mildhypertension. However, the chemical basis underpinning their activityis not well-understood, hampering the discovery of novel inhibitorysequences from the plethora of peptides encrypted in food proteins.This work combined computational and  Nonetheless,the current state-of-the-art has showed that short peptides (dipeptidesand tripeptides) are prevalent among the most potent ACE inhibitorypeptides of food origin described so far.9 Of note, the capability of bioactive sequences to resist hydrolysisduring gastrointestinal digestion, absorption, and distribution throughthe body is crucial to ensure effects on living organisms. In thisrespect, tripeptides deserve a particular attention in the light oftheir relatively high bioavailability, potency, gastric stability,and absorption by enterocytes.10 Tripeptideshave been widely investigated in the past, providing useful—althoughpartial—insights into the structural requirements underpinningtheir capacity to inhibit ACE. Specifically, a C-terminal proline(third position) generally enhances the inhibitory activity whilepositively charged and hydrophobic amino acids are preferred at thesecond and first  position, respectively. In line withthis evidence, the LXP series includes potent in vitro ACE inhibitory peptides .11 As an example, LKP and LMP are amongthe most potent in vitro ACE inhibitory tripeptidesidentified so far, with in vivo effects being reportedfor the former and expected for the latter.12 Nevertheless,the mechanistic basis of such a structure–activity relationship,including but not limited to the clear description of how inhibitorytripeptides chemically interact with ACE and why the enzyme eitherrefuses or prefers to being inhibited by certain sequences, stillneeds clarifications. This missing piece of information ultimatelyhampers an informed and precise identification/design of potent ACEinhibitory tripeptides.Proteinhydrolysates that contain bioactive peptides could be usedto develop functional foods and bioactive peptides in silico analysisand in vitro trials presenting a rational, useful,and straightforward strategy either to better understand the 3D structure–activityrelationship of inhibitory tripeptides or to identify new, potent,and previously uncharacterized ACE inhibitory sequences. Briefly,members of the LXP and XPR series never characterized before werestudied in silico and in vitro toidentify strong candidates for further and more detailed investigations.Among the active sequences, LCP has been identified for the firsttime to the best of our knowledge as a novel and potent invitro ACE inhibitory peptide. In addition, the relevanceof our outcomes to design or identify new ACE inhibitory moleculesand broaden the horizon of knowledge of ACE inhibitory tripeptideswas discussed.In this context, this work combined https://www.certara.com) through an in-house Sybyl programminglanguage script as described previously.12 In more detail, peptides were designed using the “Build Protein”tool setting the N- and C-terminus as protonated and deprotonated,respectively. Prior to subsequent analysis, each peptide was energeticallyminimized using the Powell algorithm with a coverage gradient of ≤0.05kcal mol–1 Å–1 and a maximumof 500 cycles, in agreement with a previous study.14The 3D structuresof tripeptides analyzed in this study were automatically generatedin the Trypos “.mol2” format using the Biopolymer toolimplemented in Sybyl, version 8.1  and internal (peptide only) van derWaals energy, protein–peptide hydrogen bond energy, and ligandtorsional strain energy, and only the best-scored pose for each peptidehas been considered for the analysis . Furthermore, a semi-flexibledocking approach was applied while allowing ACE polar hydrogens torotate freely and considering peptides fully flexible. The modelsfor both C- and N-domains of ACE were derived from the Protein DataBank (http://www.rcsb.org) structureshaving PDB codes 4APH and 4BZS,respectively,17 as previously described.15 In addition, for each peptide, simulations wererun in triplicate (scores are expressed as means ± standard deviations)as GOLD adopts a genetic algorithm that may cause score fluctuations.The scores were found to be satisfactorily stable with a maximum coefficientof variation of 7% for YPR and less than 5% for all the other sequences,in agreement with previous studies.18The capabilityto inhibit ACE wasestimated for a selection of sequences through docking simulationsusing GOLD software  in agreement with previous studies.19 whilethe respective pharmacophoric fingerprints were derived using theIsoMIF algorithm,20 as previously described.12The anatomy of ACE’spockets was defined with GetCleft,21 usingGROMACS (version 2019.4),22 solvating inputstructures with SPC/E waters under a dodecahedron periodic boundarycondition and neutralizing the system with counterions (Na+ or Cl–). In brief, each system underwent an energyminimization to avoid steric clashes and to correct improper geometriesusing the steepest descent algorithm with a maximum of 5,000 steps.Then, all the systems underwent isothermal  and isobaric  100 psec simulationsbefore running 50 nsec simulations (300 K with a coupling time of0.1 psec and 1 bar with a coupling time of 2.0 psec).Moleculardynamics simulationswere used to estimate the stability of ACE in complex with LCP, inagreement with a previous study,The dockingsimulations were run in triplicates, and the statistical analysisof docking results was performed using IBM SPSS Statistics for Linux,version 25 .l-glutamine, foetalbovine serum (FBS), phosphate-buffered saline, penicillin/streptomycin,and 96-well plates were purchased from Euroclone . TheACE1 activity assay kit was from Biovision . Thepeptides LKP and LCP were synthetized by GenScript  at >95% purity.All chemicals (reagents andsolvents) were from Sigma-Aldrich . Caco-2 cellswere obtained from INSERM Paris, France; Dulbecco’s modifiedEagle’s medium (DMEM), stable 23 evaluating hippuric acid (HA) formation fromhippuryl–histidyl–leucine(HHL), as a mimic substrate for angiotensin I.Peptides were tested as already described2 atmosphere in DMEM containing 25 mM glucose, 3.7 g/L NaHCO3, 4 mM stable l-glutamine, 1% non-essential aminoacids, 100 U/L penicillin, and 100 μg/L streptomycin (completemedium), supplemented with 10% heat-inactivated FBS .Caco-2 cells were routinely sub-culturedat 50% density and maintained at 37 °C in a 90% air/10% CO4 Caco-2 cells/wellwere seeded in 96-well plates and treated with 0.1–100 μMLCP or vehicle (H2O) in complete growth media for 48 hat 37 °C under a 5% CO2 atmosphere. Subsequently,the treatment solvent was aspirated, and 100 μL/well of 3--2,5-diphenyltetrazoliumbromide (MTT) filtered solution was added. After 2 h of incubationat 37 °C under a 5% CO2 atmosphere, 0.5 mg/mL solutionwas aspirated and 100 μL/well of the lysis buffer (8 mM HCl+ 0.5% NP-40 in DMSO) was added. After 10 min of slow shaking, theabsorbance at 575 nm was read on a Synergy H1 fluorescence plate reader.A total of 3 × 104 cells/well) were seeded in 96-well plates. After 24h, cells were treated with 100 μL of LCP peptides (from 1.0to 50.0 μM) or vehicle in growth medium for 24 h at 37 °C.The next day, cells were scraped in 30 μL of ice-cold ACE1 lysisbuffer and centrifuged at 13,300 g for 15 min at 4 °C. Totalproteins were quantified from the supernatant by the Bradford method,and 1.5 μg of total proteins was added to 18 μL of ACE1 lysis buffer in each well in a black96-well plate with clear bottoms. For the background control, 20 μLof ACE1 lysis buffer was added to 20 μL of ACE1 assay buffer.Then, 20 μL of 4% ACE1 substrate (in assay buffer) was addedin each well except the background one, and the fluorescence (Ex/Em330/430 nm) was measured in a kinetic mode for 10 min at 37 °C.Caco-2 cells (5 ×10Invitro (cell-free) and cellular ACE activity for eachpeptide was expressed as a mean value of four independent experimentsin triplicate ± standard deviation. All the data sets were checkedfor normal distribution by D’Agostino and Pearson test. Sincethey were all normally disturbed with p-values <0.05, the existence of significant differences in terms of activityfor AKP, LCP, and LKP in cell-free or cell-based tests was calculatedby Student’s t-test using Graph-pad Prism 9 .24 Particularly, the 3D molecular modeling pipeline used here, whichconsisted in docking studies, pharmacophoric analysis, and moleculardynamic simulations, already proved to be a reliable means to predictthe activity of ACE inhibitory peptides.21 Therefore, it was used to assess the inhibitorypotential of uncharacterized members of the LXP series prior to experimentaltrials. In this respect, the LXP series includes some of the mostpotent ACE-inhibitory peptides identified so far, like LKP, LMP, andLGP to cite but a few ,11 with an in vitro reported activity (IC50) ranging from 0.32 μM (LKP)to 57 μM (LLP) . However, the gold benchmark database for bioactive peptidesBIOPEP-UWM reported no data for 9 out of 20 series members , which were analyzed here usinga knowledge-based approach as follows. Specifically, the current understandingof the structure–activity relationship of ACE-inhibiting tripeptidesclearly suggests an acid residue at the second position is likelyto reduce the activity and led to exclude LEP and LDP from furtheranalysis.25 LIP and LTPwere also excluded since they were analyzed in a previous work withthe same pipeline described here, showing a theoretical inhibitoryactivity worse than LKP.12 In addition,LIP sequence was highly similar to LLP, the least potent member ofthe LXP series with an IC50 of 57 μM (Table S1), and it was not expected to have astrong inhibitory activity accordingly. The remaining uncharacterizedsequences LCP, LHP, LFP, LWP, and LVP were selected for the analysisin order to identify promising candidates for further experimentalinvestigations. AKP, which is a close analogue of LKP, was also analyzedto further test the hypothesized importance of bulky hydrophobic aminoacid first position (see above).Computational methodologies already proved to be effective meansto study bioactive peptides of food origin.15 All the sequences analyzed showeda comparable architecture of binding with both the C- and N-terminalengaged in polar contacts with the enzyme, in agreement with previousstudies.12 The close inspection of thepharmacophoric fingerprint of the ACE substrate-binding site revealeda well-defined distribution of areas able to receive hydrophobic groupsor polar groups like hydrogen bond donors and acceptors. In more detail,a pharmacophoric map with six different districts was defined, andsuch a map could explain the activity of peptides belonging to theLXP series characterized so far (Table S1) in the light of the pharmacophoric map describedhere suggested that side chains with polar (hydrogen bond donors)groups are strongly preferred at the second position when they satisfythe hydrophobic requirements of the pocket and, at the same time,can engage Glu162 with polar contacts (as per LKP and reasonably forLRP). Such an interaction can contribute to the ACE–peptidebinding with hydrophobic–hydrophobic favorable contributionsarranging the hydrophobic neck of the side chain within H2 and withpolar interactions due to the positively charged terminus of the sidechain being arranged within D2. Conversely, the presence of uniquelyhydrophobic side chains seemed less preferred at this position asthey could satisfy only the H2 area, reducing thereby the overallnumber of favorable contributions to the binding event and resultingin a lower inhibitory capacity. Also, the activity seemed inverselylinked to the dimension of the hydrophobic side chain. Indeed, LAPand LGP had relatively low IC50 while peptides with a bulkierhydrophobic side chain like LLP and LMP recorded a slightly bad activitycompared to them . On this basis and in the light of the docking posescalculated for the set of peptides analyzed here, LCP was identifiedas the most promising peptide to test with experimental trials amongthe LXP members considered here. Indeed, LHP, LFP, LWP, and LVP werenot able to engage Glu162, and they were found arranging the sidechain at the second position within the hydrophobic H2 area . On this basis,they were considered similar to LLP or LMP, and an activity lowerthan LKP was expected for them accordingly although they could notbe ranked quantitatively based on docking scores (which were relativelyhigh) and the similar fitting they showed within the pharmacophoricspace of the pocket . Conversely, LCP showed Cys at the second position thathad a small and hydrophobic side chain26 and could allow a relatively potent inhibition, possibly lower thanthat of LKP and similar to that of LAP and LGP . The capability of LCP tointeract with ACE was also evaluated with molecular dynamics simulationsto assess the geometrical stability of such interaction, in agreementwith previous studies.21 As shown in Figure S2; Supporting Information) compared to the Leu’s sidechain of LKP. A lower interaction and a worse activity compared toLKP was hypothesized for AKP accordingly.These sequences underwent dockingstudies first, and the respectivedocking poses were analyzed in the light of the current understandingof the structure–activity relationship and the pharmacophoricfingerprint of ACE pockets. As shown in d so far 1. Three in vitro analysisbased on computational outcomes to characterize experimentally a strongACE inhibitory candidate peptide and to further test the role of hydrophobicside chains at the first position of LXP series, respectively. The in vitro (cell-free) ACE inhibitory potential was evaluatedusing the porcine kidney recombinant enzyme taking the results ofLKP previously described12 as reference.LCP and AKP efficiently inhibited the ACE activity by 98.38 ±0.31% and 86.14 ± 0.35% at 0.3 and 3.2 mM, respectively. Thehigh inhibitory activity found under cell-free conditions supportedtheir assessment in cell-based tests. LCP displays an IC50 value equal to 8.25 ± 0.71 μM, whereas AKP displays anIC50 value equal to 719.90 ± 16.04 μM (p < 0.05). These results agreed with the in silico outcomes and confirmed the importance of a bulky hydrophobic residueat the first position  and small hydrophobic side chainsat the second position. Furthermore, LCP was selected for furthercharacterization in cell-based assay due to the high activity showedunder the cell-free conditions. In this respect, the existence ofcytotoxic effects in the test system used, which was based in thehuman intestinal Caco-2 cells, was excluded via MTTexperiments, observing no effects on the cellular vitality in therange of concentration 0.1–100 μM . On this basis, Caco-2 cells were treated withLCP and LKP, taken as reference compounds, in the range of concentration0.1–100 μM for 24 h to evaluate the effects on the ACEactivity expressed at the human cellular level. Cells then underwentlysis, and the ACE activity was measured in the presence of a fluorescentsubstrate . The presence of Pro, which is a small and hydrophobic residue,at the second position agreed with the pocket requirements describedabove. Tyr at the first position  was not only likelyto fit the pharmacophoric space of the pocket in the H3 area but alsosupposed to cause a certain degree of hydrophobic/polar interferencesdue to the possible arrangement of its phenolic portion within thehydrophobic area H3. To verify this hypothesis, YPR underwent dockinganalysis, which revealed the same binding architecture of LXP series,the extension of the Arg side chain into the D1 area (engaging Asp453with polar contacts), and the actual arrangement of the Tyr phenolicgroup into the hydrophobic area H3, possibly causing hydrophobic/polarinterferences  taking IPR and NPR as representativesfor bulky hydrophobic and polar side chains, respectively, and GPRto observe the effects when the side chain is missing. As shown in in vitro (cell-free) ACE inhibition of nearly85% and IC50 of 460.06 ± 2.42 μM. None of thesequences belonging to the XPR series other than IPR were consideredfor the further experiments in cells due to the low activity observedin cell-free conditions  seemed not effectiveto substantially enhance the inhibitory potential of tripeptides.Taken together, theseresults highlighted the structural basisunderpinning the importance of bulky hydrophobic residues at the firstposition  of ACE inhibitory tripeptides. We also detailedwhy the second position should have either small hydrophobic aminoacids or bulky side chains with a hydrophobic neck and a polar headfor an optimal engagement of the surrounding environment of the pocket.We also showed that further polar contacts in vitro, and LCP was described as a potent ACE inhibitorypeptide for the first time to the best of our knowledge. The sequenceand mechanistic analogies with LKP, a sequence previously proved active in vivo, ultimately pointed out the relevance of furtherdedicated investigations. Generally speaking, the methodology presentedhere showed to be an effective and reliable method to investigatethe ACE inhibitory activity of peptides and provided a characterizationof ACE inhibitory activity for a substantial set of sequences. Moreover,the structural outcome presented here could serve either as a footholdto better understand the chemical basis underpinning the activityof already characterized inhibitory tripeptides or as a blueprintto design novel and potent inhibitory peptides and peptidomimeticmolecules. In addition, the geometries of binding presented may allowto design site-specific mutagenesis and crystallographic studies toexperimentally confirm the key contacts between tripeptides and ACEas a key piece of information to design novel peptido-mimetic inhibitors.In this respect, all the 3D-coordinate files of peptides and peptide–proteincomplexes are available upon request.In conclusion, this study applied a 3D molecular modeling approachto understand the structural requirements of a potent ACE inhibitorytripeptide from a mechanistic point of view, providing a detailedpharmacophoric map able to explain their activity with precision.Of note, understanding the chemical rationale of peptides bioactivitymay either lead to make predictions on previously untested sequences,as per LCP, or to extend the mechanistic understanding of peptidesthat previously tested active. Computational predictions were confirmed


Hevea brasiliensis (Hev b 8). The crystallographic models revealed that the IgE’s six complementarity-determining regions (CDRs) interact with the allergen, comprising a rigid paratope-epitope surface of 926 Å2, which includes an extensive network of interactions. Interestingly, we also observed previously unreported flexibility at Fab/IgE’s elbow angle, which did not influence the shape of the paratope. The Fab/IgE exhibits a high affinity for Hev b 8, even when using 1 M NaCl in BLI experiments. Finally, based on the encouraging cross-reactivity assays using two mutants of the maize profilin (Zea m 12), this antibody could be a promising tool in IgE engineering for diagnosis and research applications.Allergies have become a rising health problem, where plentiful substances can trigger IgE-mediated allergies in humans. While profilins are considered minor allergens, these ubiquitous proteins are primary molecules involved in cross-reactivity and pollen-food allergy syndrome. Here we report the first crystal structures of murine Fab/IgE, with its chains naturally paired, in complex with the allergen profilin from  The crystal structures of the murine IgE antibody in complex with highly cross-reactive profilins are reported, and the data of cross-reactivity among allergen mutants with antibodies may prove helpful for research applications in allergy therapeutics. Allergens are not substances produced by organisms to cause allergies; they are molecules with specific biological functions4. Different organisms carry out the same biological processes, and many of them are executed by proteins with highly conserved amino acid sequences and 3D structures. These similarities could produce cross-reactivity towards allergens from different sources in patients4, i.e., IgE can recognize a protein similar to the one that originated the sensitization, giving rise to an allergic reaction.Numerous substances can trigger IgE-mediated allergies in humans, acting as haptens or antigens5. They also share high sequence identity and thus are considered panallergens6. Profilins are involved in multiple cellular functions, such as motility, through regulation of the polymerization of actin microfilaments, binding proteins with proline-rich domains, and binding phosphatidylinositol, among others. Profilins are 12–17 kDa proteins, with 100–153 amino acids7 that vary depending on the organism of origin and the isoform8. In general, these proteins maintain a high 3D structure and sequence identity, which is approximately 70% among plant profilins9. Therefore, in patients suffering respiratory allergies, cross-reactivity between profilins from foods and aeroallergens could induce oral-allergy syndrome or even anaphylaxis.Profilins are excellent examples of allergen cross-reactivity. They are ubiquitous proteins expressed in every eukaryotic cell and some archaea10. The dynamics, allosteric properties, and flexibility studies rely on theoretical models of IgE bound to its high-affinity receptor. While there is vast information on the interactions between IgE-Fc and the FcεRI and CD23 receptors15, there are just a few examples of how recombinant Fab/IgE recognizes allergens18 and only one report on the structure of a native Fab/IgE; however, this antibody binds a hapten19. Concerning these few examples, the crystal structures of the allergens Bos d 5 (beta-lactoglobulin)16 and Phl p 217 in complex with recombinant Fabs/IgEs show planar surfaces of interaction , and both IgEs were obtained in mammalian expression systems.On the other hand, IgE is the primary antibody associated with allergies. Although, to date, there are no published crystallographic structures of intact IgE, free or in complex with an allergen, negatively stained microscopy, and small-angle X-ray scattering (SAXS) studies have exhibited significant rigidity of the IgE molecule20. This profilin is involved in the latex-fruit-pollen syndrome and oral-allergy syndrome and is highly cross-reactive21. Glycosylated Fab/IgE 2F5 exhibits an affinity for rHev b 8 in the medium-nanomolar range. Furthermore, IgE 2F5 can bind the allergen even after two annealing cycles. The structures of the free Fab/IgE 2F5 and its complex with rHev b 8 in two different conformations revealed the residues involved in the paratope-epitope interaction and provided insight into how an IgE recognizes an allergen and the structural basis of cross-reactivity.Here, we report the first crystal structures of the complex between a murine Fab/IgE  that recognizes the allergen profilin from natural rubber latex (Hev b 8)To analyze the paratope-epitope interactions of the Fab/IgE 2F5-rHev b 8 complex, we determined two crystal structures of the complex and one of the Fab/IgE free, and we performed different characterization experiments to confirm the allergenic determinants.20. We confirmed through a Western blot experiment  spectrum of rHev b 8 in the Far-UV region at 25 °C shows two negative bands centered at 218 and 208 nm and a positive band at 199 nm, indicating a significant amount of beta structures and alpha-helices. This result agrees with its 3D structure (PDB 5FDS). After two annealing procedures of increasing the temperature from 25 to 90 °C and cooling down slowly to 25 °C, the protein recovered most of its secondary structure. The band centered at 218 nm was modified after the thermal procedures; however, the alpha-helix content was recovered, although not entirely Fig. . Remarkaent Fig.  that IgE22, as explained in the methodology. The difference of 474 Da corresponds to the last four residues of the enterokinase cleavage site . Therefore, we named this construct as DDDK-rHev b 8. Better diffracting crystals of the Fab/IgE 2F5-profilin complex were obtained with the DDDK-rHev b 8 construct. To understand the strong interaction between Fab/IgE 2F5 and this profilin, we determined two X-ray crystal structures of the complex, at 3.04 and 3.34 Å resolution, from crystals that grew in different conditions  with a mass of 14,629 Da Fig. 22, as exons Fig. , c. The 23. In general, experimentally observed elbow angles for IgG Fabs vary by more than 15°24. The elbow angle for the structure at 3.04 Å is 131°, while the one at 3.34 Å is 113°. This difference was 19.2° in our models that crystallized under different conditions; this flexibility at the elbow region has not been reported for a Fab/IgE. We also found an N-linked glycosylation site in N189 of the Fab’s heavy chain constant region  of the heavy (VH) and light (VL) chains participate in the interaction with the allergen Fig. . A strucion Fig. .The overall structure of the free Fab/IgE 2F5 at 3.75 Å shows three Fabs molecules per asymmetric unit Fig. . We coul25 as controls. Then, the same gel was stained using Coomassie Blue G250, and as shown in Fig. 25. The crystal structure of Fab/IgE 2F5 showed N-linked main glycosylation at the N189/F190/T191 sequon of the Fab’s heavy chain constant region  was 57.4 kDa, while the value calculated from the sequence was 46.8 kDa .SDS-PAGE staining using the Pierce Glycoprotein Staining Kit showed that complete IgE 2F5 and its Fab were glycosylated Fig. . We deteion Fig. . Positivion Fig. , using tkDa Fig. . This re20. Profilin has a conformational epitope with a buried surface area of 926 Å2, as we determined using PDBsum28. This epitope involves conserved residues at the N- and C-terminal regions that correspond to the polyproline binding site in profilins6, and one residue in a loop that connects to β-strands 6 and 7.The DDDK-rHev b 8 model in the complex Fab/IgE 2F5-rHev b 8 exhibits three α-helixes and a β-sheet of seven β antiparallel strands connected by loops of different lengths, as has been reported previously for rHev b 8The rHev b 8 conformational epitope comprises 18 polar, acid, and basic residues. As shown in Fig. The IgE 2F5 paratope involves the heavy chain residues T31, Y32, and A33 (CDR-H1); R50, R52, T55, and N56 (CDR-H2); and H101 and V102 (CDR-H3); and the light chain residues N28, H30, N31, and Y32 (CDR-L1); N50 (CDR-L2); and F91, W92, and Y96 (CDR-L3) Fig. .30. According to the sequence alignment of different plant profilins, several could cross-react with Hev b 8  Fig. . The intThe Fab/IgE 2F5 paratope residues conform to a groove in which the hydrophobic and basic residues are surrounded by polar residues exposed to the solvent. Figure 31 and PDBsum28 interaction analysis, summarized in Fig. According to the PIC 33, obtaining a KD value of 370 ± 6.8 nM , then the koff (from the dissociation step), separately, obtaining a KD = 369 ± 3.8 nM (koff/kon) using Prism 8 20.To compare the results of the interaction Fab/IgE 2F5-rHev b 8 with the previously reported results for IgE 2F5-rHev b 8, we used NaCl 1 M in the biolayer interferometry (BLI) experiments. In the absence of NaCl, no dissociation could be obtained; therefore, the salt concentration was increased up to 1 M to measure the dissociation and to prevent reassociation effects. The interaction between rHev b 8 and the Fab/IgE 2F5 analyzed using the (BLI) OCTECT software gave a dreadful adjustment with a 1:1 global fitting  and rZea m 12 (PDB 5FEF) show the regions of high conservation involving the alpha-helices in the amino and carboxyl-terminal regions. We then identified four different residues in the epitope recognized by the Fab/IgE. rHev b 8 residues E14, N98, I118, and D128, which correspond to D14, G98, V118, and E128 in Zea m 12 Fig. .Fig. 7GeAnalysis of the structure of rHev b 8 bound to the Fab/IgE 2F5 supported that D128 on rHev b 8 is immersed in a complementary antibody cavity Fig. . D128 inThe immunoassay performed with the IgE 2F5 and the rZea m 12-E128D single mutant showed only a slight increase in recognition, barely above the background. However, the recognition of the rZea m 12 double mutant was significantly higher, reaching Abs values (405 nm) of approximately 50% of those observed for the binding of IgE 2F5 to rHev b 8  has reported natural and recombinant profilin Hev b 8 as an allergen able to trigger symptoms in latex-allergic patients. Profilins are ubiquitous proteins present in all eukaryotic cells; therefore, there is a high probability that individuals sensitized by one of the multiple isoforms of this protein will, in a subsequent exposure to other profilins, experience an allergic cross-reaction due to their high sequence identity, causing pollen-food syndrome35. In this study, we report the first crystal structures of the complex between a murine Fab/IgE , bound to the allergen profilin from natural rubber latex (Hev b 8), and the structure of the Fab/IgE free.The WHO/IUIS Allergen Nomenclature Sub‐Committee  shows that the Fab region of the murine IgE 2F5 exhibits flexibility at the elbow angle, comparable to that reported for Fabs from IgGsope Fig. .37; however, its effect on antigen binding is unknown.Our Fab/IgE 2F5-rHev b 8 structure at 3.04 Å resolution revealed an electron density in both maps (2Fo-Fc and Fo-Fc) Fig. , interpr39. One of the most significant challenges in structure-function studies of IgEs is to have a simple way to observe different conformations at the atomic level to predict the rules that govern the intensity of an allergic response. A recent paper on the structure of an intact IgE and its interaction with the anti-IgE antibody ligelizumab, obtained by electron microscopy (EM) and small-angle X-ray scattering (SAXS), suggested a major IgE conformation with a defined Fab and Fc organization10. However, there are no structural studies of IgE-allergen complexes. Only two allergens complexed with chimeric IgG constructs containing human IgE variable regions have been reported17. Even though we do not have the structure of the intact IgE, our Fab/IgE structures provide valuable information concerning different conformations of this molecule, probably due to crystal packing, as suggested by others23, but causing minimal changes at the paratope.Some IgE characterizations performed by molecular modeling, FRET, and electron microscopy have also shown dynamic segmental flexibility and angle restrictions in the IgE conformation41. According to our circular dichroism assays, obtained as a function of the temperature, rHev b 8 is an exceptionally stable panallergen. This profilin partially recovers its 3D structure but could presumably still trigger an allergic response after two annealing procedures since we detected 56% of the original IgE 2F5 recognition , the Fab’s KD is 200 times higher, indicating lower affinity.Functional Fabs from native, recombinant, or chimeric antibodies have been used in antibody structure characterization. The use of Fabs aims to decrease the intrinsic flexibility of whole antibodies. In our case, an intermediate interaction between Fab/IgE 2F5 and rHev b 8 was demonstrated using BLI experiments, considering that 1 M NaCl was present in the experiment. The Fab/IgE 2F5  nM Fig. . In cont42, IgE 2F5 recognition of the rZea m 12 mutants was significant compared to the recognition of rHev b 8, , that minimal changes in aminoacidic sequence could avoid or allow cross-recognition of profilins by IgE antibodies.The murine Fab/IgE 2F5 is a non-recombinant IgE obtained in response to immunization with rHev b 8 allergen. Therefore, VH and VL combination exhibits an authentic pairing of an IgE antibody. All CDR-H and CDR-L are essential for binding the surface of the profilin allergen. Further studies on the interaction of 2F5 (and other) anti-profilin antibodies with different profilins would help better define critical aspects of cross-reactivity among these significant allergens, which could have relevant implications in IgE engineering for diagnosis and research applications in allergy therapeutics.20. Briefly, female Balb/c mice were subcutaneously immunized every two weeks for two months with the recombinant allergen rHev b 8 adsorbed in alum (Al (OH)3). We selected the mouse with a positive IgE response to the allergen for cell fusion. Ten days after the fusion, the supernatants of hybridoma cells were tested for the presence of IgE or IgG antibodies that recognized rHev b 8 using ELISA. We isolated one clone secreting IgE anti-profilin antibody (mAb 2F5), and the hybridoma secreting this IgE was cloned by limiting dilution. The isotype of the monoclonal IgE 2F5 was determined using the mouse immunoglobulin isotyping kit .We described the methodology for obtaining the IgE 2F5 in our previous paper20 grown in RPMI-1640 medium supplemented with sodium pyruvate, l-glutamine, nonessential amino acids, antibiotics, and 3% of fetal bovine serum (FBS) in a humid atmosphere at 37 °C with 5% CO2 produced the antibody. The secreted antibody was purified from the culture supernatant after centrifugation of the cell culture at 2000 × g for 20 min, and the cell pellet was discarded; the supernatant was filtered through a 0.22 μm membrane and applied to an affinity column equilibrated with PBS buffer, containing rHev b 8 covalently bound to an Affi-gel resin . The antibody was eluted by changing pH with 0.2 M glycine-HCl buffer pH 2.8 and collected in 800 μL fractions in tubes containing 200 μL of 2.0 M Tris HCl buffer pH 8.0 to neutralize the pH immediately  were sent for sequencing of the Fv regions to Absolute Antibody Ltd , who provided the consensus sequences of the clone. Hybridoma cells43,  incubated for 36 h at 37 °C. Murine IgG 2D10, previously obtained in our group25, was used as a positive control for papain digestion. The negative control was IgE incubated for 36 h without papain. Reactions were stopped by the addition of iodoacetamide to a final concentration of 20 mM. These samples were then applied to SDS-PAGE gels under reducing and nonreducing conditions, along with papain and both IgE and IgG without treatment as molecular mass controls.We performed the IgE fragmentation tests under the conditions established by Haba and Nisonoff, 1991We used papain from Sigma , and according to the supplier the enzyme cleavage site in our antibody is R204-T-I-L-V-R209 ↓ P210-V-N-I213, located between the Cε1 and Cε2 domains.−1 cm−1.After papain hydrolysis, the IgE fragmentation mixture was applied to a Superdex 75 size exclusion column (SEC) in an AKTA-FPLC system at a 0.3 mL/min flow rate in 50 mM TRIS buffer, pH 8.4. The fractions displaying a protein band at 66 kDa in SDS-PAGE were concentrated in 10 000 MWCO diafiltration tubes  and subsequently applied to a Mono Q GL cation exchange column, with a NaCl gradient from 0 to 250 mM in 40 mL. Finally, the fractions containing Fab were concentrated and dialyzed against crystallization buffer containing 20 mM TRIS-HCl and 50 mM NaCl pH 8.4, and purity was verified using SDS-PAGE  transformed with the vectors pET-28c-rHevb8 or pET-28c-rZeam12. The transformed strains were inoculated in 50 mL of Luria Bertani liquid media (LB), supplemented with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol, and incubated at 37 °C overnight. Subsequently, the culture was scaled to 1 L under the same conditions and induced with 0.5 mM β-D-1-thiogalactopyranoside (IPTG) at 30 °C for 12 h when the optical density was 0.7, at 600 nm. After the induction, the cells were harvested by centrifugation at 3100 × g for 10 min at 4 °C.Overexpression of rHev b 8 and rZea m 12 profilins was performed following the protocol described by Mares-Mejía, et al., 2016TM, and all the rZea m 12 was cleaved using TEV protease (prepared in our lab). SDS-PAGE verified purity, and the rHev b 8 concentration was determined using  with a Misonix 3000 sonicator  using pulses of 10 s with power six and resting periods of 30 s until completing 10 min. The lysate was clarified by centrifuging at 3100 × g for 30 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm Millipore membrane and applied to a 5 mL HisTrap nickel affinity column (GE). Nonspecific interactions were eliminated by washing with 100 mL of 50 mM Tris HCl buffer, 300 mM NaCl, and 15 mM imidazole. Both recombinant proteins were eluted with 50 mM Tris HCl buffer, 300 mM NaCl, and 100 mM imidazole. Several milligrams of rHev b 8 whole construct was cleaved using EKMaxTM Pfx Super mix  and the following primers :iPCR was performed using AccuPrimeE128D mutationSEQUENCE 5′-TAC CTG ATC GAT CAG GGC TTC-3′COMPLEMENT5′-GAA GCC CTG ATC GAT CAG GTA-3′.E128D-G98N mutationSEQUENCE5′- C AAG AAA ACT AAC ATG TCC TTG-3′COMPLEMENT5′- CAA GGA CAT GTT AGT TTT CTT G-3′E. coli DH5α cells to be extracted, purified, and sequenced . Purified vectors were transformed into E. coli Rosetta cells (DE3) to be overexpressed as previously described.pET-28c-Zea m 12-E128D and pET-28c-Zea m 12-E128D-G98N plasmids were propagated in 2 × dmol−1). The baseline obtained using the buffer under identical conditions was subtracted from the final spectra. Three scans were averaged to obtain the final spectrum of the recombinant allergen.The rHev b 8 CD spectra were obtained in the far-UV region (195–260 nm) using a JASCO J-1500 spectropolarimeter and a 1 mm path-length quartz cuvette. The protein concentration was 0.18 mg/mL in PBS, and the experiments were performed at 25 °C. The signal is expressed in terms of molar ellipticity θ  or cooling (90–25 °C) at one °C/min with a Jasco PTC-510 Peltier temperature controller and mini-Jasco MCB-100 water circulation bath. The spectrum was analyzed using the BeStSel online softwarerHev b 8 thermal stability was evaluated by ELISA measuring the binding of IgE 2F5 to the different profilins. Plates of 96 wells were coated for two hours at 37 °C with 100 μL of 0.7 μM rHev b 8 or rZea m 12, in PBS buffer pH 7.4. Then, the wells were washed three times using PBS with 0.1% Tween 20 (washing buffer), blocked with 1% BSA in PBS, and incubated for two hours at 37 °C. The wells were washed three times, and 100 μL of IgE 2F5 diluted in PBS was added per well and incubated for one h at 37 °C. After incubation, the plate was washed three times with washing buffer, and 100 μL of HRP-labeled secondary antibody anti-mouse IgE Fcɛ specific , diluted in PBS (1:3000), was added per well, and then incubated for one h at 37 °C. The peroxidase reaction was developed using 100 μL/well ABTS  as the substrate and incubated for 20 min. Plates were read at 405 nm using a Cytation 3 plate reader . The average absorbance of four independent value experiments and the corresponding standard deviation were plotted.25. The gel was transferred to a PVDF membrane with a constant voltage (15 V) for 50 min. The PVDF membrane was blocked with 3% albumin, and 0.05% Tween 20 in PBS, washed three times using PBS with 0.1% Tween 20, and submerged in a two μg/μL IgE 2F5 solution overnight at 4 °C. The PVDF membrane was washed three times and then submerged in a secondary antibody anti-mouse IgE Fcɛ specific  (1:2000 dilution) solution for 2 h at 37 °C. The PVDF membrane was washed three times, and the peroxidase reaction was developed using Novex HRP, and chromogenic substrate (TMB)  for 15 minutes.For western blot experiments, ten µg of rHev b 8 was electrophoresed on a 15% SDS-PAGETo demonstrate cross-reactivity between rHev b 8 and rZea m 12, ELISA experiments were performed by coating the plate with 100 μL of 0.7 μM rHev b 8, DDDK-rHev b 8, rZea m 12, rZea m 12-E128D single mutant, or rZea m 12 -E128D-G98N double mutant, in PBS buffer pH 7.4. Phosphate-buffered saline (PBS) was used as a background control, DDDK-rHev b 8, and rHev b 8 were used as positive controls, and rZea m 12 was used as a negative control. We then followed the methodology previously described. The average of four independent determinations and the standard deviation were plotted.ka). The dissociation rate (kd) of the allergen-Fab complex was obtained in the last step. The data were processed using the Octet Data Analysis Software version 8.2 and fitted to 1:1. Besides, we used a BLI fitting considering nonspecific binding (NSB) through the Association-Dissociation model implemented in GraphPad Prism 833 and a 1:1 model GraphPad Prism 8 association-then-dissociation model to fit the processed data.Profilin rHev b 8 was biotinylated in PBS buffer pH 7.4, 0.05% Tween 20 and 1 M NaCl employing a 1:5 profilin: biotin ratio, and the excess of reactive ester groups were blocked using ethanolamine. Biotinylated profilin was dialyzed extensively to remove free biotin and ethanolamine using diafiltration tubes of 10,000 MWCO and immobilized to the streptavidin biosensor at a concentration of 125 nM. The assay was performed in a total volume of 200 μL using black bottom 96-well microplates  at 25 °C with orbital shaking at 1000 rpm. The immobilized profilin was titrated with different concentrations of the Fab/IgE 2F5 , BSA was used as the negative control, and new streptavidin biosensors were used for each experiment. All the experiments were performed using the Octet® RED96 System from FortéBio, controlled with the software Data Acquisition 8.2 . The BLI experiment baseline was PBS buffer pH 7.4, 0.05% Tween 20 and 1 M NaCl for 60 sec. The biotinylated allergen was then allowed to bind to the streptavidin sensor for 300 s, then washed with the same buffer to eliminate nonspecific binding. Next, the purified Fab/IgE 2F5 was bound to the allergen in the biosensor, and the association rate was measured . Using one of these crystals, we collected a dataset at 3.34 Å resolution employing a rotating anode generator MicroMax 007HF , with a Dectris-Pilatus 3 R/200K-A detector. The crystal detector distance was 70 cm, with omega increments of 0.2° and an exposure time of 200 s per image. The cryoprotective solution was prepared with the stock solution and 30% glycerol.TM pH 6.0, 0.1 M BIS-TRIS pH 6.5, 20% w/v Polyethylene glycol 3350. Data collection was performed using line 17-ID-1 of the National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory (BNL) in Upton, New York. We prepared the cryoprotective solution with the stock solution and 30% glycerol.After several months, we obtained an increasing number of crystals of the complex Fab/IgE 2F5 with the rHev b 8 DDDK-rHev b 8. We collected a dataset at 3.04 Å resolution from crystals that grew up in the condition containing 2% TacsimateFab/IgE 2F5 was dialyzed in 10 000 MWCO diafiltration tubes with ten washes of crystallization buffer II  using a dilution factor of 10. Subsequently, the Fab was concentrated to 3.3 mg/mL and screened using the PEG-Ion I and II crystallization kits  by vapor diffusion in the seated drop mode. Crystals appeared under various conditions after several months of incubation at 18 °C. The condition C1  produced suitable crystals to be diffracted, and a dataset at 3.75 Å resolution was collected using a rotating anode generator MicroMax 007HF  with a DECTRIS-PILATUS 3 R/200K-A detector. The crystal detector distance was 70 cm, with omega increments of 0.2° and an exposure time of 200 s per image. The cryoprotective solution was prepared with the stock solution and 30% glycerol.47 and HKL300048 software. Both crystals were orthorhombic and belonged to the space group (P212121). The structure of the complex Fab/IgE 2F5-DDDK-rHev b 8 at 3.04 Å was determined by molecular replacement with PHASER49 using rHev b 8 profilin (PDB 5FDS) and the anti-DNP Fab/IgE fragment (PBD: 1BAF) as search models. We performed iterative model building and refinement cycles with PHENIX Real-space refinement50, alternating manual model building with COOT51. The model was integrated onto the electron density maps 2Fo-Fc and Fo-Fc. Weight optimization of real-space refinement was performed during refinement. We used the coordinates of the complex Fab/IgE 2F5-rHev b 8 at 3.04 Å to determine the Fab/IgE 2F5-rHev b 8 structure using the crystal data at 3.34 Å resolution. For these models, the Ramachandran plots indicate 97.83% and 98.14% residues in favored region  for the 3.04 and 3.34 models, respectively. Data processing and refinement statistics for both models are provided in Table 31 and PDBsum28 were used to analyze the protein-protein interfaces.The data collected at 3.04 and 3.34 Å were indexed, integrated, and scaled using the XDS group P22121. The47, and the space group was verified with Aimless52. The crystal was monoclinic and belonged to the space group (I121). The Matthews coefficient suggested three molecules in the asymmetric unit. The structure of Fab/IgE 2F5 was determined by molecular replacement with PHASER49 using the Fab structure of the complex Fab/IgE 2F5-rHev b 8 at 3.04 Å as the search model. We performed iterative refinement cycles using PHENIX Real-space refinement50, alternating manual model building with COOT51. The B-factors were refined as implemented in Phenix . In the final steps of the refinement, we set the value of all atoms to 50 Å2. Weight optimization of real-space refinement was also used during refinement. The model was integrated into the electron density maps 2Fo-Fc and Fo-Fc. For this model, the Ramachandran Plot indicates 97.03% residues in the favored region, whereas 2.97% in the allowed region. All the structure figures were generated using Chimera53 and PyMOL54. We used the Praline server55 for sequence alignments.The data collected at 3.75 Å for the Fab/IgE 2F5 were scaled using XDSAtomic coordinates have been deposited in the Protein Data Bank under accession codes 7SBD (Complex at 3.04 resolution), 7SBG (Complex at 3.34 resolution), and 7SD2 (for the Fab).A gradient SDS-PAGE  was performed loading the Fab/IgE 2F5-rHev b 8 complex, IgE 2F5, Fab/IgG 2D10-rHev b 8 complex, IgG 2D10, Fab/IgG 1B4-rHev b 8 complex, IgG 1B4, profilin rHev b 8, horseradish peroxidase as a positive control and soybean trypsin inhibitor as a negative control. Glycoproteins were stained on gels with the periodic acid-Shiff reagent method was achieved using the Pierce Glycoprotein Staining Kit .We determined the molecular masses of recombinant rHev b 8 (whole construct), the Fab/IgE 2F5, and the complex Fab/IgE 2F5-rHev b 8 (whole construct) using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry . The standards for mass calibration were lysozyme  and bovine serum albumin (BSA) . The matrix used was a saturated solution of sinapinic acid in 30% (v/v) aqueous acetonitrile and 0.1% (v/v) trifluoroacetic acid. Samples were analyzed using FLEX ANALYSIS 3.0TM (Bruker) software.42. A P value < 0.05 was considered statistically significant. All summary statistics and analyses were performed using the Prism 8.0 software  (https://www.graphpad.com/). An asterisk identifies statistical significance and is denoted as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars represent standard deviation from the mean. Sample size and replicates for each experiment are listed in the figure legends.The statistical analysis of the ELISAs was performed using the one-way analysis of variance (ANOVA), followed by an all-pairwise multiple comparison procedure (Šídák method)Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


KaiC, a core protein of the cyanobacterial circadian clock, consists of an N-terminal CI domain and a C-terminal CII domain, and assembles into a double-ring hexamer upon binding with ATP. KaiC rhythmically phosphorylates and dephosphorylates its own two adjacent residues Ser431 and Thr432 at the CII domain with a period of ∼24 h through assembly and disassembly with the other clock proteins, KaiA and/or KaiB. In this study, to understand how KaiC alters its conformation as the source of circadian rhythm, we investigated structural changes of an inner-radius side of the CII ring using time-resolved Trp fluorescence spectroscopy. A KaiC mutant harboring a Trp fluorescence probe at a position of 419 exhibited a robust circadian rhythm with little temperature sensitivity in the presence of KaiA and KaiB. Our fluorescence observations show a remarkable environmental change at the inner-radius side of the CII ring during circadian oscillation. Crystallographic analysis revealed that a side chain of Trp at the position of 419 was oriented toward a region undergoing a helix–coil transition, which is considered to be a key event to allosterically regulate the CI ring that plays a crucial role in determining the cycle period. The present study provides a dynamical insight into how KaiC generates circadian oscillation. Since the discovery of a gene involved in the circadian clock in Drosophila , many clpression . Recent pression ,8, whichSynechococcus elongatus PCC 7942 has been extensively studied to elucidate the structures and functions of clock proteins [in vitro by mixing the three Kai-proteins with ATP [The circadian clock of a cyanobacterium proteins . The corwith ATP . KaiC, awith ATP . KaiC pewith ATP ,14 throuwith ATP . The poswith ATP ,19. The with ATP . Althougwith ATP ,22, it iWT) intrinsically has three Trp residues, one (W92) at the CI domain and two (W331 and W462) at the CII domain , we probed rhythmic structural changes at an inner-radius side of the CII ring during circadian oscillation. Through the present study, we propose the usefulness of Trp-based fluorescence spectroscopy for studying the circadian structural changes of the clock proteins in real time.In this study, we focused on an inner-radius side of the CII ring in KaiC as the research target based on our recent observation of KaiC crystal structures, in which an upstream region of the phosphorylation sites T416-H429) at the inner-radius side undergoes a structural change [ at the iF419W) to monitor the conformational change around the inner-radius side in solution by fluorescence spectroscopy. The F419W substitution had little impact on the overall structure of KaiC as evidenced by backbone RMSD of 0.42 Å between KaiCF419W and KaiCWT  at 30°C. The period length of the phosphorylation cycle was less dependent on temperatures and the Q10 value at 30°C was estimated to be 1.15 ± 0.02 (1.07 ± 0.03 for KaiCWT) from the Arrhenius plot as shown in According to the crystal structures of KaiC  Figure , the sidd KaiCWT . Moreovefunction . In the WT or KaiCF419W). Trp fluorescence spectra were measured using an excitation wavelength of 295 nm and the fluorescence intensity integrated from 320 to 370 nm (Fapp) was recorded every 30 min. As shown in the upper panels of Fapp values of solutions containing KaiCWT or KaiCF419W changed rhythmically; the oscillations lasted at least for 4 days without apparent damping. The amplitude of Fapp oscillation, the difference between the maximum and minimum florescence signal, was enhanced from 0.6 to 8.1 by F419W substitution. A significant increase in the amplitude indicates that the oscillation in Fapp of KaiCF419W largely reflects a rhythmic change in the environment surrounding the W419 site in solution. When the time-courses of Fapp values were compared with those of relative abundance in the four phosphorylation states , the amplitude of aiCF419W  might ineviously ,28, the n states . Fapp off KaiCWT .F value of each phosphorylation state (Fi) by assuming that Fapp(t) is represented as a linear summation of the contributions from four phosphorylation states as reported previously [F value of KaiCF419W at the SpT, pST and ST states was much larger than that of KaiCWT at the respective states. On the other hand, the F value of KaiCF419W at the pSpT state was comparable to that of KaiCWT  at each phosphorylation state by subtracting the F value of KaiCWT from that of KaiCF419W  of each phospho-mimicking mutant were extracted by subtracting F of each mutant without F419W substitution from that with the substitution  was shifted depending on the phosphorylation states . KaiCF419W showed robust circadian rhythm with the Q10 value of 1.15 ± 0.02  as observed by static fluorescence spectra of phospho-mimicking mutants (max of W419 fluorescence (∼5 nm) was wider than that of the intrinsic Trp residues (∼1 nm), including large contributing W462 [W419 fluorescence of the ST state was comparable to that of the SpT state, but was almost completely quenched in the transition from the SpT to pSpT states and then dequenched from the pSpT to ST via pST states , inset.  mutants . Notablying W462 . This sucis-trans isomerization of D145S146 peptide occurs during ATP-hydrolysis in the CI domain. In addition, the fluorescence emission from the three intrinsic Trp residues showed an obvious but slight change in response to the phosphorylation state, whereas it increased by about 5-fold upon dissociation into KaiC monomers concomitant with a red-shift in λmax by ∼4 nm. Although the global conformational changes of the CII ring upon S431 phosphorylation have been reported previously [In our previous report , a drasteviously ,34, the F419W-ST -tagged (pGEX-6P-1) form . The geneviously .2 and 1 mM ATP. For the auto-dephosphorylation process of KaiC, a solution containing KaiC alone was transferred from an ice bath to 30°C. The KaiC phosphorylation cycle was reconstructed in vitro by mixing KaiA (0.04 mg/ml), KaiB (0.04 mg/ml) and KaiC (0.2 mg/ml) as reported previously [Q10 value, the factor by which the cycle frequency  is accelerated by raising the temperature from 30°C to 40°C, was determined from the slope of the Arrhenius plot as described previously [All measurements were conducted in a buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCleviously ,18. Relaeviously . The pereviously .F419W was obtained using the vapor diffusion method. The purified KaiCF419W was concentrated up to 3.5 mg/ml in a solution of 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT and 1 mM ATP. The sample solution was mixed at a 1 : 1 ratio with a reservoir solution of 100 mM Tris–HCl (pH 7.0), 1 M KCl, 0.7 M sodium/potassium tartrate, 1.8 M sodium acetate, and 5 mM AMP-PNP. Because the reservoir solution itself exhibited the cryoprotectant effect, the crystal was picked up from the crystallization drop and directly soaked into the liquid nitrogen for the diffraction experiment conducted at the cryo-temperature.The crystal of KaiCX-ray diffraction data were collected on beamline BL44XU at SPring-8 . The crystal was mounted under a cryostream at 100 K during the X-ray radiation. Diffraction images were recorded using EIGER X 16M (DECTRIS) and processed with XDSGUI . Initial−1 at an excitation wavelength of 295 nm . The spectral bandwidth was set at 1.0 nm for excitation and 5.0 nm for emission. The observed spectra were normalized against both KaiC concentration and the fluorescence signal of an N-acetyl-l-tryptophan amide (NATA) standard solution with an absorbance of 0.05 at 280 nm. For static measurements, each sample was stored on ice, transferred to 30°C, and then incubated 10 min before measurements.Fluorescence measurements were carried out at 30°C and the temperature was controlled with a precision of 0.1°C by using a LTB-125 water-bath (AS ONE Corporation). Fluorescence emission spectra from Trp residues were collected every 1 nm with a 0.5 s response time and a scan speed of 240 nm miniA(t) is the relative phosphorylation-state abundance  pre-determined experimentally  to equation (1).The time-courses of fluorescence intensity during the auto-dephosphorylation process of KaiC alone were analyzed using the following equation ,28:1Fap