The mechanistic target of rapamycin (MTOR) is a giant protein kinase that, together with the accessory proteins Raptor and mLst8, forms a complex of over 1 MDa known as MTOR complex 1 (MTORC1). MTORC1, through its protein kinase activity, controls the accretion of cell mass through the regulation of gene transcription, mRNA translation, and protein turnover. MTORC1 is activated in an interdependent manner by insulin/growth factors and nutrients, especially amino acids, and is inhibited by stressors such as hypoxia and by the drug rapamycin. The action of insulin/growth factors converges on the small GTPase Rheb, which binds directly to the MTOR polypeptide in MTORC1 and, in its GTP-bound state, initiates kinase activation. Biochemical studies established that MTORC1 exists as a dimer of the MTOR/Raptor/mLst8 trimer, and progressive refinements in cryo-electron microscopy (cryo-EM) have enabled an increasingly clear picture of the architecture of MTORC1, culminating in a deep understanding of how MTORC1 interacts with and phosphorylates its best-known substrates—the eIF-4E binding protein/4E-BP, the p70 S6 kinase/S6K1B, and PRAS40/AKT1S1—and how this is inhibited by rapamycin. Most recently, Rheb-GTP has been shown to bind to MTORC1 in a cooperative manner at an allosteric site remote from the kinase domain that twists the latter into a catalytically competent configuration. Herein, we review the recent cryo-EM and associated biochemical studies of MTORC1 and seek to integrate these new results with the known physiology of MTORC1 regulation and signaling. MTORC1, composed of the MTOR protein kinase and the non-catalytic polypeptides Raptor and mLst83, is a central regulator of cell function through its broad control of anabolic and catabolic processes5. TORC1 controls polypeptide abundance through the regulation of gene transcription by all three RNA polymerases6, cap-dependent mRNA translation7, and protein degradation via autophagy and the proteasome8. Advances in structure determination—in particular, cryo-electron microscopy (cryo-EM)—have enabled considerable progress in understanding the structure of both MTORC114 and MTORC218. Here, we review the current understanding of MTORC1 structure and its implications for the regulation and signaling by this important protein kinase.The giant protein kinase mechanistic target of rapamycin (MTOR) operates in two physically distinct and independently regulated multi-protein complexes called MTOR complex 1 (MTORC1) and MTOR complex 2 (MTORC2)20. The amino-terminal 1,345 residues of the approximately 289 kDa MTOR polypeptide contain 32 HEAT repeats, each a 30- to 40-residue segment consisting of two antiparallel alpha-helices connected by a flexible linker22. The HEAT domains can be further subdivided into the N- and M-HEAT domains12. This is succeeded by the roughly 500-residue FAT (FRAP/ATM/TRRAP) domain, followed by the MTOR catalytic domain (MTOR is a member of the phosphatidylinositol 3-kinase (PI3K)-related kinases (PIKKs), along with ATM, ATR, DNA-PK, SMG1, and TRRAP (a pseudokinase), which share a catalytic domain that more closely resembles that of the phosphatidylinositol 3′ (lipid) kinases than that of the typical eukaryotic protein kinases domain .14; thus, inhibition by the FKBP12:rapamycin complex is not allosteric, as was long thought, but occurs by interference with substrate recruitment14. The 40 kDa mLst8/Gβl polypeptide is composed entirely of seven WD40 repeats in a beta propeller; despite its status as a core component of MTORC1, its function in the complex is unknown inasmuch as its deletion does not alter MTORC1 signaling23.The MTOR catalytic domain has a canonical two-lobed structure with key insertions in both the amino-terminal and the carboxy-terminal lobes. In the amino-terminal lobe, a long alpha-helix packs against the amino-terminal lobe, as seen in other PI3K family members. Following this helix is the FRB insertion, the site for FKBP12:rapamycin binding. In the carboxy-terminal lobe, a conserved, roughly 35-residue segment, FATC, seen only in the PIKKs that contain a FAT domain, stabilizes the activation loop and is completely integrated into the carboxy-terminal domain. Another segment in the carboxy-terminal lobe, referred to as LBE, forms a site for mLst8 binding. An insertion in the carboxy-terminal lobe plugs a pocket beneath helix alpha D (in the PKA nomenclature), a surface often used for substrate binding, leaving little room immediately proximal to the phosphorylation site for the form of sequence recognition interactions seen in protein kinases like PKA. The FRB domain extends over the catalytic cleft from the amino-terminal lobe, as do the FATC and LBE from the carboxy-terminal lobe, thereby creating a deeply recessed catalytic cleft. Modeling the FKBP12:rapamycin complex onto the FRB segment indicates that the latter severely limits access to the catalytic cleft. Moreover, the side of the FRB facing the catalytic cleft serves as a secondary substrate-binding site14. MTORC1 operates as a dimer of the MTOR, mLst8, and Raptor heterotrimer9, mediated by an antiparallel arrangement of the MTOR HEAT domains, which extend from the FAT alpha solenoid14 noid14 . The N-H4. The model generally proposed for TORC1 activation in mammalian systems envisions two parallel pathways that converge at the lysosome. Growth factors and cytokines promote the activation of MTORC1 by inhibiting the tuberous sclerosis complex, a complex of TSC1, TSC2, and TBC1D17 that is the GTPase activator protein (GAP) for the ras-like small GTPase Rheb25. Rheb binds directly to the MTOR polypeptide and, in its GTP-bound form, is required for TORC1 (but not TORC2) activation26. The kinases controlled by PI3K (Akt) and Ras-GTP  phosphorylate TSC2 and the Iκb kinases phosphorylate TSC1 so as to impair, by various mechanisms, TSC GAP function and thereby promote Rheb GTP charging25. AAs act through an independent pathway that converges on the Rag GTPase heterodimer28, which in its activated state binds to the Raptor subunit of TORC1 and conveys TORC1 to the cytoplasmic surface of the lysosome29; there, MTOR is activated by interaction with Rheb-GTP. Withdrawal of AAs, especially leucine and arginine, markedly inhibits MTORC1 signaling despite maximal growth factor signaling30. Conversely, overexpression of Rheb, which swamps endogenous TSC GAP activity, enables MTORC1 activation even in the absence of all AAs31. Thus, a critical step in MTORC1 activation by insulin, growth factors, and AAs is the binding of Rheb-GTP to the MTOR polypeptide in MTORC1 at the surface of the lysosome32.MTORC1 serves as a positive regulator of cell mass and proliferative capability in all eukaryotic cells, and much effort has been directed toward elucidating the regulation of TORC1 activity. In single-cell eukaryotes, TORC1 is activated primarily by nutrients, which are both the substrate and the stimulus for cell growth and proliferation. In metazoans, MTORC1 is activated by growth factors and cytokines and in an interdependent manner by nutrients, especially amino acids (AAs)33. Thomaset al.34 described the ability of Rab1-GTP to promote TORC1 activation at the Golgi in a Rheb-dependent manner. InCaenorhabditis elegans, the TSC is entirely lacking and CeTORC1 is regulated independently of the InsR/IGFR Daf235 except for transcriptional regulation of CeRaptor/Daf15 abundance36. In lower eukaryotes, whereas TORC1 activation inSchizosaccharomyces pombe is SpRheb dependent37, TORC1 regulation inSaccharomyces cerevisiae is entirely independent of ScRheb and sustained activation does not require the Rag GTPase Gtr1/Gtr2 and requires Gln rather than Leu; nevertheless, activation occurs at the vacuolar surface38.S. cerevisiae contains about 200 TORC1 dimers per cell, distributed diffusely around the cytosolic face of the vacuole. Upon removal of extracellular glucose (but not AAs or ammonium), TORC1, while remaining associated with the vacuole, undergoes a rapid inactivation, accompanied by a Gtr1/Gtr2-mediated reorganization into a large hollow cylinder composed of 100 or more dimers39. The packing of the TORC1 dimers in these assemblies is such that the central Raptor HEAT/armadillo repeat domain is apposed to the TOR FRB segment in a manner that resembles the binding of the inhibitory FKBP12:rapamycin complex; in fact, rapamycin interferes with the assembly of the cylinders. Based on these and other features, these cylindrical TORC1 assemblies have been designated “TOR organized in inhibited domains” or TOROIDs. Although the diverse mechanisms of TORC1 regulation ultimately will each require elucidation, here we focus on MTORC1 interaction with Rheb and with the preferred substrate PRAS40.Multiple variants and exceptions to this model have been described; for example, in mammalian cells, whereas Leu acts via Rag recruitment of TORC1 to the lysosome, Gln has been observed to recruit TORC1 to the lysosome through Arf1 in a Rag-independent manner40; the Raptor binding motif in PRAS40 is slightly variant (FVMDE)42. The integrity of their TOS motifs and the binding of these substrates to Raptor are necessary for their phosphorylation by MTORC1, bothin vitro and in the cell45. However, at least one physiologic MTORC1 substrate, the Igf2 mRNA-binding protein, IGF2BP2/IMP2, does not bind Raptor but binds to MTOR directly and is phosphorylated by MTORC1in vivo andin vitro independently of Raptor46. A systematic analysis of the increasing number of MTORC1 substrates for the Raptor dependence of their phosphorylation is awaited. A second unusual feature of MTORC1 is that its phosphorylation site selection is relatively broad; all Ser/Thr sites on 4E-BP, on the carboxy-terminal non-catalytic tail of S6K1B, and on IMP2 are followed immediately by a Pro residue, whereas the critical regulatory MTORC1 phosphorylation site on S6K1B, Thr389/412, is situated in the highly hydrophobic motif FLGFTYVA. Surprisingly, one report describes the ability of MTORC2 to catalyze tyrosine phosphorylation of the insulin and IGF1 receptors47.The MTORC1 signaling output, as defined by its Ser/Thr kinase catalytic function, displays several unusual features. First, the presence of a substrate-binding subunit  separate from the kinase polypeptide itself is distinctly uncommon. Raptor binds the substrates S6K1B and 4E-BP through their TOS  motif 48. PRAS40 binds strongly to Raptor and, in contrast to the canonical physiologic TORC1 substrates 4E-BP or S6K1B, is commonly retrieved in stable association with MTORC1. Overexpression of PRAS40 strongly inhibits the phosphorylation of both 4E-BP and S6K150, which themselves show cross-competition for phosphorylation by MTORC151, suggesting that access to Raptor may be limiting for substrate phosphorylation52. PRAS40 remains bound to Raptor despite its MTOR-catalyzed phosphorylation unless it is also phosphorylated by Akt at a carboxy-terminal site, Thr246, which enables its binding to 14-3-3 and release from Raptor50. This behavior, together with the usual residency of PRAS40 in the unstimulated MTORC1 complex, led to the proposal that PRAS40 may serve as an Akt controlled regulator of substrate access to MTORC150, acting in concert with Akt inhibition of TSC and activation of Rheb to promote optimal MTORC1 activation. However, support for this plausible and attractive hypothesis is scant; despite its ubiquitous expression and in contrast to inactivation of TSC1 or TSC2, inactivation of theAKT1S1/PRAS40 gene in mice54 or Drosophila55 does not result in global phenotypes indicative of enhanced MTORC1 signaling, although tissue-specific effects are described55. Depletion of PRAS40 in cell culture has been reported to promote50, impair57, or not alter58 insulin/growth factor-stimulated MTORC1 activity. The robust activation of MTORC1 by Rheb overexpression58 or treatment with phorbol esters (via Ras-GTP)59 occurs without any displacement of PRAS40 from MTORC1. Although its physiologic functions remain obscure, PRAS40 is clearly a preferred MTORC1 substrate and an excellent model for how MTOR recognizes Raptor-bound substrates.The PRAS40 polypeptide is multiply phosphorylated by MTORC114: the PRAS40 TOS motif and the adjacent N-terminal residue binds to a pocket in Raptor formed by a cleft between the caspase-like domain and the succeeding solenoid structure. A set of arginine residues in the TOS binding pocket generates a positive electrostatic potential that favors acidic residues in flanking regions proximal to the consensus sequence. The site on Raptor occupied by the PRAS40 TOS motif is essentially superimposable on that occupied by the 4E-BP and S6K1 TOS motifs. Raptor localizes the TOS site about 65 Å from the active site of the kinase, increasing the local concentration of the substrate. PRAS40 also binds to two other sites within MTORC1. A PRAS40 long amphipathic alpha-helix (residues 212–232) makes an extensive interface with the FRB segment of the MTOR catalytic domain, positioning the substrate near the catalytic cleft; S6K1 and 4E-BP each use a region about 15 residues C-terminal to their phosphorylation sites to interact in a similar manner with the FRB segment, and mutation of the FRB-interacting sequences in S6K1 and 4E-BP markedly impairs their phosphorylation. In addition, PRAS40 interacts with a WD40 domain of mLST8 through a beta-strand interaction that is not observed with S6K1 or 4E-BP; this third site of PRAS40 interaction within the complex may account for the greater overall affinity for PRAS40 as compared with S6K1 and 4E-BP. How these interactions facilitate the processive phosphorylation of substrates by MTOR is unclear.Cryo-EM structures show three potential PRAS40 interaction sites in MTORC1et al.14, the catalytic domain in “apo” MTORC1  exhibits a wide catalytic cleft, prohibiting effective phosphate transfer from ATP bound at the P-site to polypeptide substrates. This conformation is enforced by the FAT domain, which clamps to both the amino- and carboxy-terminal lobes of the catalytic domain. The individual catalytic residues are positioned in a primed, active orientation; however, in contrast to, for example, PKA or PI3K  in MTORC1 in response to cell stimulation. Unlike many conventional protein kinases, the MTOR activation loop is not subject to phosphorylation and the orientation of the critical catalytic segments of the MTOR kinase domain in apo-MTORC1 is already in an active configuration, requiring only the Rheb-induced narrowing of the catalytic cleft to promote kinase activity. Reports detecting insulin activation of the kinase activity of MTORC1 assayedin vitro appear to reflect increased access of the added Raptor-dependent substrate (4E-BP or S6K1)52; no differences are evident using a Raptor-independent substrate or salt washes to deplete PRAS40 from the complexes58. It has not been possible to retrieve endogenous MTOR in complex with endogenous Rheb after cell disruption  has been observed35). It can be argued that the very weak affinity of Rheb for MTOR results in a loss of the Rheb-MTOR interaction upon cell extraction; however, this returns to the question of how the Rheb activation mechanism demonstrated by Yanget al.14 relates to the mechanism operative in the cell and whether a continuing association of Rheb with MTOR is required for the maintenance of MTORC1 activity in the cell. Rheb is a membrane-bound protein  that is found predominantly in the Golgi67; although a small fraction of Rheb plausibly becomes apposed with MTORC1 at the lysosome to initiate activation67, MTORC1 thereafter operates in essentially every cellular compartment, including the nucleus68. Fluorescence lifetime imaging of overexpressed Rheb, MTOR, and Raptor, each fused to fluorescent proteins, did detect the presence of a Rheb-MTOR complex in both the cytoplasm and the nucleus (the latter devoid of Raptor)69; as yet, however, the presence of endogenous Rheb in the nucleus and its localization therein await confirmation. Moreover, Rheb membrane association suggests a specific orientation for MTORC1 position relative to the membrane, which would restrict cooperative activation only at the membrane. The mechanism by which MTORC1 activity is sustained within all cell compartments and the role of Rheb remain critical, unanswered questions.Conceivably, there may be additional interactions or modifications (or both) of MTORC1 activity involved in Rheb activation, as is the case, for example, for the Ras-GTP regulation of the Raf kinases; the initial high-affinity Ras-GTP/Raf interactionMTORC1 exists as an extremely elaborate large holoenzyme. Why go to all the trouble? The structure may highlight three important properties of MTORC1 functions:1. Adaptor binding. The interface formed by the MTORC1 dimer provides a platform for Raptor. This same site is used by MTORC2 for RICTOR binding, building in a modularity often found in signaling assemblies.2. Cooperative, allosteric activation. The mechanism for Rheb activation via HEAT/FAT domain re-organization is cooperative and is coupled through the dimeric interface mediated by the HEAT domains. The dimeric holoenzyme is required for the sigmoidal response. Could there be other modulators of MTORC1 activity that act through other allosteric sites?3. Localization. The Rheb-dependent activation presumably occurs at a membrane surface. The location of the Rheb binding sites, if engaged simultaneously, would define a specific orientation of MTORC1 at the membrane. How this positioning influences its engagement with other upstream and downstream factors is one of the many unexplored questions that remain.


Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a key regulator of myocardial contractility, and dephosphorylation of cMyBP-C is associated with heart failure. However, the molecular mechanisms underlying contractile regulation by cMyBP-C phosphorylation are poorly understood. We describe the kinase specificity of the multiple phosphorylation sites on cMyBP-C and show that they are interdependent and have distinct effects on the structure of the thin and thick filaments. The results lead to a model of regulation by cMyBP-C phosphorylation through altered affinity of cMyBP-C’s N terminus for thin and thick filaments, as well as their structures and associated regulatory states. Impairment of these mechanisms is likely to underlie the functional effects of mutations in filament proteins associated with cardiomyopathy. The heart’s response to varying demands of the body is regulated by signaling pathways that activate protein kinases which phosphorylate sarcomeric proteins. Although phosphorylation of cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator of myocardial contractility, little is known about its mechanism of action. Here, we used protein kinase A (PKA) and Cε (PKCε), as well as ribosomal S6 kinase II (RSK2), which have different specificities for cMyBP-C’s multiple phosphorylation sites, to show that individual sites are not independent, and that phosphorylation of cMyBP-C is controlled by positive and negative regulatory coupling between those sites. PKA phosphorylation of cMyBP-C’s N terminus on 3 conserved serine residues is hierarchical and antagonizes phosphorylation by PKCε, and vice versa. In contrast, RSK2 phosphorylation of cMyBP-C accelerates PKA phosphorylation. We used cMyBP-C’s regulatory N-terminal domains in defined phosphorylation states for protein–protein interaction studies with isolated cardiac native thin filaments and the S2 domain of cardiac myosin to show that site-specific phosphorylation of this region of cMyBP-C controls its interaction with both the actin-containing thin and myosin-containing thick filaments. We also used fluorescence probes on the myosin-associated regulatory light chain in the thick filaments and on troponin C in the thin filaments to monitor structural changes in the myofilaments of intact heart muscle cells associated with activation of myocardial contraction by the N-terminal region of cMyBP-C in its different phosphorylation states. Our results suggest that cMyBP-C acts as a sarcomeric integrator of multiple signaling pathways that determines downstream physiological function. Contraction of cardiac muscle is initiated by activation of the actin-containing thin filaments, but is modulated by structural changes in the myosin-containing thick filaments. Calcium binding to troponin induces an azimuthal movement of tropomyosin on the surface of the thin filaments which allows myosin head domains from the neighboring thick filaments to strongly attach to actin . SubsequHeart muscle contractility is also regulated by posttranslational modifications of sarcomeric proteins, including phosphorylation of the regulatory components of the thick filaments . PhosphocMyBP-C is localized to 9 transverse stripes in the central region of each half-thick filament, called the C-zone, via interactions of its C-terminal anchoring region with the myosin tails and titin A, closelBoth the inhibitory and activating interactions of cMyBP-C are believed to be controlled by its phosphorylation state. The cardiac specific “m-motif” between domains C1 and C2 contains a series of conserved serine residues that are phosphorylated by several protein kinases in vivo and in vitro  Fig. 1AAFig. 1A.However, the detailed function and mechanism underlying cMyBP-C phosphorylation at individual sites remain poorly understood, likely due to the complexity of its interaction with other sarcomeric components, which is further compounded by the multiplicity of signaling pathways acting upon cMyBP-C and its associated phosphorylation states . Current21The aim of the present work was to understand the molecular mechanism of cMyBP-C phosphorylation and its structural and functional effects on both thin and thick filament-based regulation in heart muscle cells. We investigated the relationship of individual cMyBP-C phosphorylation sites and potential cross-talk using several protein kinases known to phosphorylate cMyBP-C. We utilized cMyBP-C fragments with well-characterized phosphorylation states to show that site-specific phosphorylation has distinct effects on its interaction with and regulation of the thin and thick filaments, combining in vitro biochemical binding assays with in situ structural measurements in intact heart muscle cells. The results show that cMyBP-C acts as a sarcomeric integrator of different signaling pathways to determine downstream physiological effects, and that the functional effects of cMyBP-C phosphorylation can only be understood by combining thin and thick filament-based mechanisms into an integrated model of contractile regulation.To elucidate the regulatory function of cMyBP-C phosphorylation, we phosphorylated a recombinant fragment of rat cMyBP-C containing domains C1, the phosphorylatable m-motif, and C2 C1mC2; A with thSI Appendix, Table S1). Tetrakis-phosphorylated C1mC2 was only observed after prolonged incubation with PKA at 30 °C, suggesting that the fourth site is a poor substrate for PKA.Incubation of C1mC2 for 30 to 60 min with PKA resulted in a mixture of unphosphorylated, monophosphorylated, bis-phosphorylated, and tris-phosphorylated protein, which could be clearly separated by both Phos-tag and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) B. IndiviSI Appendix, Fig. S1) . Analysis of the IEC fraction corresponding to monophosphorylated C1mC2 (C1mC2-1P) revealed specific phosphorylation on a single serine residue, S288, in agreement with previous studies suggesting that S288 in the cardiac-specific insertion is the initial PKA phosphorylation site , suggesting that S313 is phosphorylated after S288 and S279. In contrast, phosphorylation of S308 by PKA was only observed after prolonged incubation, suggesting that this serine is a poor substrate for PKA in vitro. Thus, our results suggest that PKA phosphorylation of the cardiac-specific m-motif follows a concerted hierarchical mechanism in the sequence of S288, followed by S279, followed by S313 .Phosphorylated amino acid residues in each IEC fraction were identified by proteolytic digestion followed by phospho-peptide enrichment and matrix-assisted laser desorption ionization (MALDI) MS and ESI-MS  compared with wild type . S279A- and S313A-substituted C1mC2 showed intermediate levels of phosphorylation, although S279A had a stronger inhibitory effect than S313A , in agreement with the hierarchical phosphorylation sequence proposed above.To further test this conclusion, we prepared serine-to-alanine (S-A) substitutions of each individual PKA site in C1mC2, and analyzed their phosphorylation profiles by Phos-tag–SDS/PAGE E. Total Next, we analyzed the effects of S-A substitutions on phosphate incorporation by PKA at each individual phosphorylation site F. ConsisAlthough PKA is considered to be the primary kinase acting upon cMyBP-C in vivo, several other kinases have been shown to be able to phosphorylate specific residues within the cardiac-specific m-motif . MoreoveSI Appendix, Fig. S3). RSK2 primarily phosphorylated rat C1mC2 at S288 in vitro, although we observed phosphorylation of other residues at higher enzyme-to-substrate ratios or after prolonged incubation. In contrast, α-adrenergic stimulation leads to an increase in cMyBP-C phosphorylation primarily via stimulation of protein kinase Cε (PKCε) . Phosphorylation of S288 was not a prerequisite for phosphorylation of S308 by PKCε as shown previously  has been suggested to specifically phosphorylate serine residues in the cardiac-specific m-motif insertion in response to activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway , and we ε (PKCε)  [and proε (PKCε) ] activiteviously .i/mol of C1mC2  D and E. of C1mC2 F. AlthouIn summary, the multiple cMyBP-C phosphorylation sites are not independent, and exhibit positive and negative regulatory coupling with partially overlapping specificity for multiple kinases.ds) of 20.7 ± 3.5 μmol/L  and >400 μmol/L, respectively  , in good agreement with the results obtained for recombinant rat C1mC2.To characterize the effects of site-specific cMyBP-C phosphorylation on its proposed interactions with both the myosin-containing thick and actin-containing thin filaments , 20, we d to ∼30 μmol/L . In contrast, both PKA bis-phosphorylation (S288 and S279) and tris-phosphorylation  completely abolished S2Δ binding, indicating that either bis-phosphorylation or phosphorylation of S279 per se controls the cMyBP-C–myosin S2 interaction. We addressed this question by phosphorylating C1mC2-S288A with PKA and isolating the monophosphorylated species (C1mC2-S288A-1P) using IEC. Both unphosphorylated and monophosphorylated C1mC2-S288A bind myosin S2Δ with a Kd similar to that measured for the wild-type protein (Kd of ∼20 μmol/L), suggesting that bis-phosphorylation is necessary and sufficient to abolish cMyBP-C–myosin S2 interaction . To further test this conclusion, we sequentially phosphorylated C1mC2 with RSK2 and PKCε, and measured the affinity of the bis-phosphorylated C1mC2 (S288 and S308) for myosin S2Δ . The RSK2/PKCε bis-phosphorylated C1mC2 did not bind to myosin S2Δ, further supporting the hypothesis that bis-phosphorylation per se, independent of the phosphorylation site combination, abolishes cMyBP-C–myosin S2 interaction. These results are consistent with the largely ionic nature of cMyBP-C’s main interaction site in the m-motif and myosin S2  of ∼1, indicating stoichiometric binding of C1mC2 to actin . Addition of calcium (pCa 4.5) had no effect on C1mC2 binding to NTFs as indicated by identical Kd and Bmax values . Strikingly, both PKA monophosphorylation (S288) and bis-phosphorylation (S288 and S279), as well as monophosphorylation of S308 by PKCε, showed no change in the affinity of C1mC2 for NTFs (Kd ∼ 20 μmol/L), but decreased Bmax to ∼0.5, suggesting a lower binding capacity of C1mC2 in the partially phosphorylated states. As before, full calcium activation of NTFs had no additional effect on either Kd or Bmax. However, PKA tris-phosphorylation (3P) significantly decreased C1mC2’s affinity for NTF with an estimated Kd of ∼60 μmol/L independent of [Ca2+]. Addition of calcium increased Bmax from ∼0.5 to ∼1 for C1mC2-3P, suggesting an interplay between tris-phosphorylation of C1mC2, thin filament binding, and calcium activation (We measured the affinity of C1mC2 in its different phosphorylation states for isolated bovine cardiac NTFs using high-velocity cosedimentation. Unphosphorylated C1mC2 (0P) binds NTFs in the absence of calcium (pCa 9) in a saturable manner with a Ktivation .These results suggest that cMyBP-C phosphorylation regulates contractility partly via differential modulation of its affinity for myosin and actin binding sites, so that phosphorylation leads to a redistribution of cMyBP-C’s N-terminal domains from their myosin binding sites in the thick filaments to their actin binding sites in the thin filaments. Monophosphorylation weakens and bis-phosphorylation abolishes thick filament binding, and only tris-phosphorylation affects C1mC2 binding to regulated thin filaments.2+ (pCa 9) with a half-maximal effective concentration (EC50) of ∼20 μmol/L  to monitor structural changes in the thin filaments of demembranated ventricular muscle cells associated with the activating effect of C1mC2 described previously . Unphospof C1mC2 B, althouPKA phosphorylation of S288 reduced the activating effect of 40 μmol/L C1mC2 on active force to ∼30% of that measured during control conditions B, purpleP2>, which is significantly larger than that observed for calcium activation alone (∼120%) (2+.The effects on isometric force described above are mirrored by those on the thin filament structure as monitored by the cTnC-E probe orientation. C1mC2 “superactivates” the thin filament as indicated by a change in the order parameter < (∼120%) C. PKA mo (∼120%) C, yellowThese results are in stark contrast to the NTF binding data described above, suggesting that although phosphorylation of cMyBP-C has only minor effects on its binding to the thin filament, it significantly alters thin filament regulation. Recent electron microscopy studies demonstrated that N-terminal domains of cMyBP-C bind polymorphically to isolated actin filaments, and that only a subset of binding modes can directly interfere with tropomyosin’s position and induce the ON state of the thin filament , 34. TheP2> from this probe increases upon calcium activation. In contrast to its effect on thin filament structure described above, C1mC2 incubation leads to a partial activation of the thick filament structure corresponding to ∼70% of that measured during control activations  . BSR-cRLSI Appendix, Table S2).Monophosphorylation of either S288 by PKA or S308 by PKCε showed no significant reduction in the activating effect of 40 μmol/L C1mC2 on the thick filament structure as reported by the BC probe orientation D, in conThe comparison of the effects of C1mC2 in its different phosphorylation states on the myosin head conformation with the MST binding data described above further suggests that C1mC2 has a direct activating effect on the thick filament and that the activating effect is, in turn, controlled by its phosphorylation-dependent affinity for myosin S2Δ.We further investigated the functional consequences of site-specific C1mC2 phosphorylation on actomyosin interactions by measuring the NTF-stimulated adenosinetriphosphatase (ATPase) activity of isolated bovine myosin S1 in the presence of C1mC2 in its different phosphorylation states using a colorimetric assay A.50 of ∼1 μmol/L and a Hill coefficient of ∼1.5, suggesting cooperative activation of NTFs by C1mC2. In contrast, [C1mC2] > 2 μmol/L inhibits the ATPase activity with a half-maximal inhibitory concentration (IC50) of ∼3 μmol/L. PKA monophosphorylation  further supports the conclusion that C1mC2 interacts with NTF in different binding modes and that phosphorylation controls the distribution between states with inhibitory and activating effects. Surprisingly, [C1mC2-3P] < 2 μmol/L increased the NTF-stimulated myosin S1 ATPase in the presence of calcium (pCa 4), consistent with its increased binding capacity for NTFs in presence of Ca2+ described above and recent evidence for antagonistic effects of phosphorylation and Ca2+ on cMyBP-C structure (The direct comparison with the NTF binding data (tructure .i) the distribution of cMyBP-C’s N-terminal domains (NcMyBP-C) between binding sites in the thin and thick filaments and (ii) the structure and associated regulatory state of the filaments.Multiple conserved phosphorylation sites have been identified in close proximity to each other within cMyBP-C’s regulatory m-motif . Moreoveds in the micromolar range . All of the effective concentrations are significantly larger than the measured micromolar Kds, suggesting that the fraction of “free” or unbound cMyBP-C in the filament lattice is very low, and that the majority of NcMyBP-Cs are bound to either actin or myosin . Moreover, although C1mC2 binds NTFs and myosin S2Δ with similar affinity, the higher local concentration of available actin binding sites predicts that a larger fraction will be bound to the thin filaments. This conclusion is consistent with electron microscopy reconstructions of resting skeletal muscle demonstrating links between thick and thin filaments with an axial periodicity of ∼43 nm, as expected for MyBP-C  would first activate thin filament regulatory units opposite the C-zone, allowing individual myosin head domains to strongly attach to actin. However, most myosin heads would remain OFF due to inhibitory interactions of NcMyBP-C with myosin S2. As more and more thin filament regulatory units in the C-zone are switched ON, the fraction of actin-attached cross-bridges increases, and a combination of structural changes in the myosin head domains and cMyBP-C would trigger the activation of additional myosin heads. At higher [Ca2+]i, the rest of the thin filaments would become activated . The functional consequence is a faster systolic activation of contraction, because myosin heads are no longer constrained by cMyBP-C and can bind to activated thin filament regulatory units. According to our model, even a modest reduction in the affinity of the m-motif for myosin S2 via PKA monophosphorylation is sufficient to cause reorientation of a significant fraction of myosin-bound cMyBP-Cs (∼30%) toward their actin binding sites. Although those represent a small fraction of cMyBP-C molecules, we have previously presented evidence that the regulatory transition in the thick filaments is highly cooperative, so that activation of a small fraction of myosin heads (<20%) can activate the whole thick filament structure . According to this model, even moderate changes in basal levels of cMyBP-C phosphorylation would have significant functional consequences for the myocardium.In contrast, phosphorylation of S308 does not abolish the activating effect of C1mC2 on thin filament or force development, but similarly reduces its affinity for myosin S2Δ. Thus, phosphorylation of S308 in cMyBP-C is predicted to partially activate the thick filament C-zone without the associated deactivation of the thin filament structure B. The coCamKII has been shown to be an important regulator of cMyBP-C function, and, recently, CamKII-mediated phosphorylation of S308 has been implicated in the positive force-frequency relation of cardiac muscle , suggestThe phosphorylation-dependent interactions of cMyBP-C have important implications for the physiology and pathophysiology of contractile regulation in the heart, and the current results show that cMyBP-C functions as an integrator of multiple signaling elements that mediate context-specific functions of the myocardium in health and disease. Dephosphorylation of cMyBP-C has been frequently observed during heart failure , likely Of particular interest in the heart failure setting is the RSK2-mediated phosphorylation of S288, which partially mimics the structural and functional effects of β-adrenergic stimulation and primes S279 for phosphorylation by PKA D and po, and ourThe concept of modulating the distribution of cMyBP-C’s N-terminal domains between inhibitory binding sites in the thick filaments and activating binding sites in the thin filaments has wider implications for the regulation of cardiac contractility. Other signaling pathways such as RLC phosphorylation or length-dependent activation, the cellular analog of the Frank–Starling law of the heart, might act through a similar mechanism by disrupting cMyBP-C–myosin interactions and favoring binding of the N-terminal domains of cMyBP-C to the thin filament. From the perspective of the well-known mechanisms of cardiac muscle regulation, the current results therefore require a paradigm shift that integrates both thin and thick filament-based mechanisms into a single model of contractile regulation, with a key role for cMyBP-C.SI Appendix, Supplementary Information Methods.Protein production and phosphorylation, preparation of cardiac trabeculae, protein exchange protocols, and fluorescence polarization experiments were performed according to published protocols. Details of materials and methods are provided in Supplementary File


Although geological and modelling evidence indicate that the last glacial inception in North America was in NE Canada, little is known about the glacial response of the nearby western Greenland Ice Sheet (GIS) during the glacial advance of marine oxygen isotope stage 4 (MIS4). Our multi-proxy study of a marine sediment core collected about 60 km southwest of the Outer Hellefisk Moraines demonstrates that in the southern Davis Strait region the most extreme Greenland shelf glaciation of the last glacial cycle occurred during MIS 4, with another prominent glacial advance at 37–33 kyr BP. During those periods the GIS likely reached the Outer Hellefisk Moraines in this area. Except for these two periods, our data suggest significant advection of relatively warm Irminger Sea Water by the West Greenland Current since MIS 4. This advection likely limited the extent of the MIS2 glaciation on the SW Greenland shelf. Decreased precipitation over southwestern Greenland predicted by atmospheric models as a downstream effect of a much larger MIS2 Laurentide Ice Sheet may have played an additional role. This interglacial-to-glacial transition at the end of MIS 5e featured prominent changes in oceanographic circulation in the subpolar North Atlantic2, which were characterized by progressive expansion of cold water masses and associated southward advances of the Arctic Front as well as ice sheet expansion during marine isotope stages (MIS) 5b and 4. A rapid expansion of the ice sheet in the early glacial is also indicated by numerical ice sheet model simulations3. Moreover, model-based simulations further suggest that the glacial expansion in the Baffin Bay – Labrador Sea region most likely started in the mountain range of Baffin Island4. Ice sheet extent culminated during the Last Glacial Maximum , i.e. during MIS 2.At the end of the last interglacial (Marine Isotope Stage (MIS) 5e) approximately 115,000 years ago , the astronomically-driven decline of northern summer insolation and resulting atmospheric and ocean feedback processes led towards the first step in glacial ice sheet formation in the North American region5. Yet, few details are known about the extension of the MIS 4 (71–57 kyr) ice sheets. It has, however, been reported that during MIS 4, an extensive outer shelf glaciation occurred on the western Siberian shelf7, while northern Siberian mountain glaciation was also large8. Both for MIS 4 and the preceding cold MIS5b substage (86.2 kyr model age) the size and location of the early glacial ice sheets could be reconstructed by a combination of geological information and numerical modelling3. The latter results indicate that the entire north-eastern North American continental edge was glaciated during MIS4, but that it was not until later, i.e. under MIS2, that the ice sheet expanded into the continental interior. Within the present context it is worth noting that an apparent major collapse of the GIS at the MIS 4 termination presumably led to massive calving of North Greenland glaciers and probably contributed to widespread IRD deposition in the Arctic Ocean at the beginning of MIS 37.Many studies have outlined the extent of Quaternary glaciations worldwide and in particular the LGM9, suggesting strong variability in the ice sheet extent. Due to a lack of accurate dating, most of the glacial features on the West Greenland shelf have been assumed to have an age corresponding to the LGM. A shelf edge extension of the GIS during the LGM has been suggested by a number of studies13, although a circum-Greenland GIS shelf-edge extension is still a matter of debate14. Moreover, there is notably little information available about the response of the GIS margin during the early stage of the last (Weichselian) glaciation, i.e. during MIS415, although the GIS extent during MIS 4 is generally believed to have been smaller than during MIS 2. Most the coastal regions of Greenland were deglaciated during MIS 5e, and glacial expansion onto the shelf may not have commenced until MIS 49.Little information on the previous glacial history is available for West Greenland due to subsequent erosion of pre-existing glacial features. Bedrock exposure data indicate ice-free conditions in coastal regions of Sisimiut, Uummannaq and Upernavik for about 55% of the last 1 million years17. It is thus very likely that the overall changes in Atlantic Meridional Overturning Circulation (AMOC), and not the least its resulting changes in the warm-water flux to West Greenland, may have played a role in deciding extend of expansion of the GIS onto the shelf.The West Greenland margin is today strongly influenced by the oceanic conditions. The advection of warm, Atlantic-sourced water transported by the West Greenland Current (WGC) is believed to play an important role as one of the controlling factors for the expansion and retreat of marine-terminating glaciersIn this study we aim to test for potential periods of extensive shelf glaciations of the GIS during the last glacial cycle, with particular focus on MIS 4 and MIS 2, more in particular for investigating whether the GIS was indeed more extensive during MIS 2 than during MIS 4, as generally assumed. We will further focus on the possible link between shelf glaciation events and changes in the ocean current regime in the north-eastern Labrador Sea Fig. , testing19  and a LGM (MIS2) origin20 have provisionally been proposed. In 2003, the 503 cm long sediment gravity core TTR13-AT-479G (hereafter 479 G) was retrieved from a water depth of 1033 m in the southern Davis Strait near the southwest Greenland shelf edge , about 60 km southwest of the Outer Hellefisk Moraine  layer of cold, low-salinity Polar Water derived from the East Greenland Current and melt water discharge from local South Greenland outlet glaciers. Hence, bottom-water conditions at the site are today influenced primarily by the relatively warm WGC, while surface-water conditions are cooler.Oceanographically, our study site is today located within the lower depth stratum of the West Greenland Current (WGC) realm Fig. , a boundThe sediment in gravity core 479 G consists of olive-green to olive-grey silty and sandy clay interrupted by layers of sand and gravel (>1 mm); some levels show a light pink colour in discrete bands . However, Ca may also originate from detrital carbonate, which is an important component of IRD in the study regioni24 Fig. , and off18O records of core 479 G  provide a firm chronology in the upper about 130 cm of core 479 G  with no late glacial to Holocene sediments preserved. As the core top did not show any substantial loss of sediment during coring, this suggests that Holocene erosion or non-deposition has apparently has been typical for this coring site, likely due to high bottom-water energy.Calibrated AMS 9 G Figs ; for det31 is not possible. Thus, we depend on a range of data, i.e. stable isotope data . Consequently a MIS 4 depositional environment is inferred for the bottom of the core, which is barren of foraminifera  Eventera Fig. , suggest18O values is between about 250 and 320 cm core depth for the benthic and 270 and 340 cm core depth for the planktic foraminiferal isotopes. The latter core segment is further characterised by benthic foraminiferal fauna assemblages indicative of maximum Atlantic water influence  may be identified with relative certainty as it is present within the range of 14C datings and it has the characteristics of a prominent detrital carbonate content combined with a clear IRD signal .In the oxygen isotope record of core 479 G . This allows us to identify the periods GI14 to GI08 in our data set and to use the chronology of Andersen del Fig. .Cassidulina neoteretis45)  the site experienced a distinct increase in overall abundance of both planktic and benthic foraminifera, and in benthic species associated with subsurface Atlantic water masses and high food availability occurred Fig. . CombineIslandiella norcrossi .Warmer conditions with prevailingly subsurface Atlantic water masses persisted until ca. 45 kyr BP (ca. 270 cm), when the influx of Atlantic water gradually decreased and colder, more Arctic conditions characterised by Polar Water are observed  conditions again became harsh for foraminifera, resulting in their near-absence, possibly due to extreme sea-ice conditions and extensive sediment supply from expanding glacier ice. Return of the, albeit more limited, influence of subsurface Atlantic-sourced water masses shortly after 33 kyr BP suggest a change to more open water conditions  events, significant changes have occurred in Labrador Sea circulation36. The latter study reports enhanced advection of relatively warm intermediate water into the southeastern Labrador Sea during Heinrich events, whereas isotope data and benthic faunal distributions suggest inflow of subsurface Atlantic-derived water into the deep Labrador Sea basin between the Heinrich events36. Further indications for a possible link between changes in advection of warm (Atlantic) water and regional ice-rafting in the Davis Strait region have been found by Andrews et al.22, which supports the scenario of enhanced subsurface ocean heat transport leading to glacial ice sheet destabilization and subsequent large-scale ice-rafting proposed by Moros et al.49, and also suggested by other studies offshore Greenland50. Although IRD events in the Baffin Bay – Labrador Sea may not always be directly correlated to the Heinrich events of the North Atlantic22, several other sediment core records from the Labrador Sea have revealed all major Heinrich events and several Dansgaard-Oeschger cycles51.Evidence has been provided for large variability of water mass formation and distribution in the subpolar western North Atlantic during the late Quaternary52. According to the sediment lithology and structure, it is not a till, but was likely deposited relatively close to, but not right next to, a fairly stable glacier margin. This interval was immediately followed by the major IRD event of H6, which suggests that immediately prior to MIS 3, i.e. during MIS 4, the SW Greenland Ice Sheet had extended far to the southwest and a major break-up of the ice sheet occurred during H6 and at the MIS4/3 boundary  may signal a more general collapse of the Greenland Ice Sheet margins at the MIS 4 termination, which at the beginning of MIS 3 contributed to widespread IRD deposition in the Arctic Oceanues Fig.  indicateues Fig. , assigne18O and δ13C values in the early MIS 3 . This is in accordance with the general cooling also recognised in the Greenland ice core records  deposition, as also recorded in core 479 G. Thus, for the various stages of the Weichselian glaciation we found evidence for a gradual change in glacial Northwest Atlantic circulation patterns and in severity of southwest Greenland glaciations.Following the extensive advection of these warmer (ISW) water masses by the WGC in the early MIS 3, likely linked to a strong AMOC seen in the North Atlantic region in particular during the early part of MIS 35 kyr BP 70 cm. Th37.The foraminiferal record of core 479 G Fig.  confirms55, who found evidence of seasonally ice-covered surface conditions prevailing in the north-eastern Labrador Sea between 31,000 and 12,600 BP. Enhanced thermohaline circulation in the Labrador-Irminger Sea basin was already reported by Fagel et al.56 to have started with an increasing Deep Western Boundary Undercurrent activity from the southwest Greenland rise immediately after the LGM. This scenario is supported by a study of magnetic grain sizes in a core from Eirík Drift south of Greenland, which shows enhanced bottom-water circulation peaking shortly after 19,000 cal yrs BP57.The transport of foraminiferal shelf species to the deeper-water surroundings of the 479 G core site was more or less continuous during the latter part of MIS 3 and LGM  shelf advance. Brine formation due to seasonal freezing would have been an important process that promoted advection of warm Atlantic (subsurface) water masses into the north-western Atlantic59. Moreover, benthic foraminiferal fauna studies by Rasmussen et al.36 indicate advection of Atlantic-derived water masses into the north-eastern Labrador Sea not only between Heinrich events, but also during them.Enhanced circulation of (warmer) subsurface ISW since the LGM may have played an important role for the deglacial retreat of the southwestern Greenland Ice Sheet dated as early as about 18,000 cal. yrs BP7 as well as with extensive land-ward ice extent in Siberia60. Apart from the bottom section of core 479 G representing extreme glacial conditions during MIS 4, another period characterised by glacial conditions offshore Southwest Greenland that was more severe than under the LGM is noted for the core record around 170–140 m subbottom depth  icebergs drifting within the North Atlantic subpolar gyre, presumably originating from a southern Greenland ice shelf calving at, or beyond, the continental shelf edge62, i.e. seaward of the Outer Hellefisk Moraines. Thus, a decreasing trend in southwest Greenland shelf glaciation may be observed since MIS6. Our findings are further thought to have implications for the possible age of origin of the Outer Hellefisk Moraines, which according to our results may be attributed to the MIS4 shelf glaciation rather than to the MIS2 (LGM) glacial stage, at least for the southern Davis Strait region.Previous studies of southwest Greenland shelf glaciations report a total of only five extreme shelf edge glaciations over the last ca 4.5 million years, of which the youngest is assigned to the MIS6 Saale glaciation48. Such a shift in North Atlantic ocean heat transport may have been accompanied by a corresponding decrease of warm-water transport by the Norwegian Atlantic Current, and may be an important factor, when explaining differences in the extent of maximum glaciation between NW Europe and Scandinavia and SW Greenland, when comparing MIS 2 and 4. When comparing the difference of the MIS 2 and MIS 4 southwest Greenland shelf glaciations, another important factor may have been the difference in North American ice sheet size and location during MIS 2 and MIS 43 as well as their respective effect on atmospheric circulation. For the much larger MIS2 ice sheet, atmospheric modelling studies63 indicate a regional decrease in precipitation over the southwest Greenland region, which should imply lower snow accumulation rates over SW Greenland and consequently a decreasing glacier growth and ice sheet shelf expansion. Further to the north in the Baffin Bay region, the Greenland Ice Sheet had also significantly expanded during MIS4, but here the MIS 2 glaciation seems to have been more extensive64. It should be noted that data are yet insufficient to evaluate whether the entire Outer Hellefisk Moraines complex extending further north is in fact of the same age as found for the area of our study. Nevertheless, we tentatively conclude that the relatively weak influx of Atlantic-sourced water during MIS 4 may have allowed the GIS to extent relatively far to the shelf edge also in surrounding regions. This in contrast to MIS 2, when our data point to a stronger Atlantic water flux.The difference between the MIS4 and MIS2 glaciation can be explained by gradually increasing Atlantic-derived (ISW) water mass advection and WGC activity in the course of the last glaciation, and may reflect a fundamental change in the AMOC and the North Atlantic circulation pattern since the last interglacial (MIS 5e), when deep-water formation in the Labrador Sea was found to be absentBased on the multiproxy analyses of a marine sediment core from the northernmost Labrador Sea, we here present new evidence of a significant extension of the Greenland Ice sheet (GIS) in the early Weichselian (MIS 4) in the Davis Strait region. Our results suggest that the ice sheet expanded further onto the West Greenland shelf than during the Last Glacial Maximum , and it retreated fairly abruptly at the onset of early MIS 3 warming. During MIS 4 the GIS ice margin reached close to the shelf edge, but did not exceed the shelf edge during the time period covered in our study . The nearby sector of the Outer Hellefisk Moraine is thus likely of MIS 4 age.During much of the last 60 kyr (starting in early MIS 3) Atlantic-sourced water has penetrated into the Labrador Sea at intermediate depths, although the strength of Atlantic-water influx has varied significantly over time and was clearly weaker during the second part of MIS 3 and during MIS 2 than during the vigorous AMOC of the early MIS 3. One additional larger ice sheet expansion likely occurred between just after Greenland Interstadial (GI) GI08 (ca. 37–33 kyr BP). During our >60 kyr long record, there is thus a strong inverse relationship between the advection of the relatively warm Atlandic-sourced water to the West Greenland margin and the shelf-ward expansion of the GIS. This result highlights not only that regional ice sheet extent may well be uncoupled from the global ice volume, but also illustrates the fast response of the Greenland Ice Sheet to climate and ocean change.14C-datings from the top 130 cm of core 479 G were performed at the Aarhus AMS 14C Dating Centre, Aarhus University, and the Leibniz Laboratory for Radiometric Dating and Stable Isotope Research, Kiel University (Table Neogloboquadrina pachyderma (left-coiled). One sample of mixed benthic foraminifera was also dated  in the age model for this top section of the core  , Texel at a 1-cm resolution.Multi-element geochemical data Fig. , S2 was Magnetic measurements were made at Lund University, Sweden, on sediment subsamples packed into cubes with 2.2 cm dimensions. Magnetic susceptibility (MS) was measured using a Geofyzica Brno (now Agico) KLY2 kappabridge. Anhysteretic remanent magnetization (ARM) was induced in each sample with a Molspin alternating field demagnetiser, using a direct current (DC) bias field of 0.1 milliTesla (mT) that was superimposed on a peak AF of 100 mT. Saturation Isothermal Remanent Magnetization (SIRM) was induced in a field of 1 T by a Redcliffe 700 BSM pulse magnetizer. The induced remanences (ARM and SIRM) were measured with a Molspin Minispin Fig. . The MS Grain-size analyses were carried out through wet-sieving of the sediment through sieves with mesh sizes of 0.063, 0.1 and 1.0 mm as part of the foraminiferal laboratory analyses. The IRD-content was calculated as the weight % of the sediment fraction > 1 mm, thus mainly identifying the larger IRD grains Fig. . Choice For foraminiferal analyses Fig. ; Fig. S4Neogloboquadrina pachyderma  Ehrenberg and benthic species Cassidulina neoteretis Seidenkrantz and Elphidium clavatum Cushman (Fig. 67. An overlap between the benthic isotopes measured on the two species, made it possible to create a combined benthic stable isotope curve. Values of E. clavatum were standardised to C. neoteretis by subtracting 0.02‰ from oxygen isotope and adding 1.14‰ to carbon isotope values of E. clavatum. All values are calibrated to the PDB scale.Oxygen and carbon isotope measurements of the planktic species man Fig.  were perSupplementary materialSupplementary data 1


This report may pave the way towards the development and optimisation of novel small molecule allosteric Caspase-6 inhibitors and illustrates that functional characterisation of rare natural variants holds promise for the identification of allosteric sites on other therapeutic targets in drug discovery.Caspase-6 is a cysteine protease that plays essential roles in programmed cell death, axonal degeneration, and development. The excess neuronal activity of Caspase-6 is associated with Alzheimer disease neuropathology and age-dependent cognitive impairment. Caspase-6 inhibition is a promising strategy to stop early stage neurodegenerative events, yet finding potent and selective Caspase-6 inhibitors has been a challenging task due to the overlapping structural and functional similarities between caspase family members. Here, we investigated how four rare non-synonymous missense single-nucleotide polymorphisms (SNPs), resulting in amino acid substitutions outside human Caspase-6 active site, affect enzyme structure and catalytic efficiency. Three investigated SNPs were found to align with a putative allosteric pocket with low sequence conservation among human caspases. Virtual screening of 57,700 compounds against the putative Caspase-6 allosteric pocket, followed by  Although apoptotic caspases are traditionally classified as initiators  and effectors , data from Caspase-6 (Casp6) has challenged this classification. In addition to being activated by initiator and effector caspases, Casp6 can be activated by a unique intramolecular self-cleavage mechanism due to its longer intersubunit linker2. Moreover, the peptide substrate specificity of Casp6 is distinct from Caspase-3 (Casp3) and Caspase-7 (Casp7)5. Furthermore, inhibitors of apoptosis proteins that suppress Casp3 and Casp7 activity do not suppress Casp6 activity7. Finally, Casp3 or Casp7 activation in human embryonic kidney cells induces apoptosis whereas Casp6 activation does not9. These findings indicate that Casp6 displays unique structural features, substrate preference, and function compared to the other effector caspases.Caspases belong to a highly conserved family of cysteine proteases that have essential roles in programmed cell death, inflammation, differentiation, neuronal development and axon pruning12. Neuronal activation of Casp6 is associated with an increased production of amyloid beta peptide and caspase cleavage of Golgi-localised γ-ear-containing ARF binding protein 3 (GGA3), an inhibitor of the beta secretase15. In addition, Casp6 cleaves numerous substrates such as amyloid precursor protein18, tau10, α-tubulin19, valosin-containing protein20; a facilitator of misfolded ubiquitinated protein degradation, and actin-regulating postsynaptic density proteins spinophilin, α-actinin-1 and -4, and drebrin19, that may lead to neuronal dysfunction. Furthermore, Casp6 has been implicated in age-dependent cognitive impairment since levels of active Casp6 in the entorhinal cortex, the first region to be affected by AD21, inversely correlate with global cognitive, episodic, and semantic memory scores in individuals within normal cognitive range22. Knock-in mice that express a self-activated form of human Casp6 in the CA1 region of the hippocampus display age-dependent spatial and episodic memory impairment, in the absence of NFTs and amyloid plaques23. Furthermore, in nerve growth factor deprived mouse primary neuron cultures26, activation of Casp6 is associated with axonal degeneration, while Casp3 activation induces cell death. In wild type or mutant amyloid precursor protein-transfected human primary CNS neurons, Casp6 is activated in absence of Casp3 activation and leads to neuritic degeneration but not cell death27, consistent with observations in AD brains that Casp6 activity is present in neurons that do not have an apoptotic morphology10. Therefore, Casp6 represents a novel early therapeutic target in age-dependent cognitive impairment and AD.Excess neuronal active Casp6 is associated with Alzheimer disease (AD) neuropathology and age-dependent cognitive impairment. Whereas active Casp6 is undetected in normal brains, it is highly abundant in post-mortem AD brains. Active Casp6 and Tau cleaved by Casp6 are present in neurofibrillary tangles (NFT), neuropil threads and neuritic plaques in the brain of mild, moderate, severe and very severe sporadic AD and in familial AD individuals28. Due to the role of Casp6 in B cell activation and plasma cell differentiation, these mice display increased antigen-stimulated production of antibodies; thus, Casp6 inhibition in AD patients may increase their immunity29. In humans, pro-Casp6 is not significantly expressed in foetal or adult brains30. Casp6 expression in peripheral tissues declines with age, but Casp6 expression remains relatively high in the gastrointestinal system30 raising concern that Casp6 inhibition may promote cancer. However, Casp6 involvement in tumour progression has been excluded in an inflammation-associated model of colon carcinogenesis31. Hence, Casp6 inhibition looks promising as a potential treatment for age-dependent cognitive impairment and AD.Casp6 is a viable drug target. Casp6 null mice develop normally without any noticeable apoptotic defects32, lamin A33, Huntingtin (Htt)34, and synthetic caspase peptide substrates35. Most of these inhibitors contain an electrophilic warhead, aldehyde, fluoromethylketone or methyl vinyl sulfone36, which reacts with a catalytic cysteine. In addition, aza peptide epoxides37, Michael acceptors38, non-peptide inhibitors, sulfon amide isatin Michael acceptors39, 1,2,3-triazoles coupled to 2,3,5,6-tetrafluorophenoxy methyl ketone40, methylene blue and its derivatives41, have been shown to inhibit Casp6. Although most of these inhibitors display low nanomolar to micromolar potency against Casp6, some are toxic in mammals, have low blood brain barrier permeability, or lack selectivity. The catalytic site is highly conserved among the caspases, which challenges the development of highly selective Casp6 active site inhibitors.Many competitive Casp6 inhibitors have been reported. Substrate mimetic peptide inhibitors were derived from the Casp6 cleavage site in transcription factor activator protein 2 isoform alpha42. Casp6 inhibition via zinc binding at exosite E 43, and phosphorylation of S257 at exosite D by ARK5 kinase46 represent two examples of natural Casp6 allosteric regulation. However, only exosite E has low conservation across the caspase family constituting an attractive allosteric site for Casp6 specific drug design. Despite the growing knowledge in the field of caspase allosteric regulation47, very few synthetic allosteric Casp6 inhibitors have been developed to date. A noncompetitive small peptide inhibitor identified through phage display screen against pro-Casp6 selectively inhibits Casp6 activity by trapping Casp6 in a tetrameric zymogen-like form48 but has poor cell penetration properties. Small molecule Casp6 inhibitors that bind at the dimer interface and stabilise the zymogen conformation49 do not bind to active Casp6. In addition, selective uncompetitive Casp6 inhibitors have been reported, but their action depends upon binding of an artificial peptide substrate limiting their therapeutic potential50. Active Casp6 inhibition is desirable because it accumulates before the onset of AD symptoms11. To our knowledge, no synthetic small molecule noncompetitive allosteric inhibitors of active Casp6 have been reported.A more promising approach is to target non-conserved allosteric sitesin silico screen of a diverse small molecules collection against this allosteric site identified two hits, S10G (IC50 = 4.2 µM) and C13 (IC50 = 13.2 µM) that inhibited Casp6 activity in vitro through a noncompetitive mechanism. Our data serves as a starting point for the development of a small molecule allosteric Casp6 inhibitors and also illustrates that screening of rare natural variants holds promise for the identification of allosteric sites on other therapeutic targets in drug discovery.By investigating the activity of four rare natural Casp6 variants  and employing X-ray crystallography along with molecular modelling, we have identified a putative allosteric pocket on mature active Casp6. An CASP6 affect Casp6 activity and structure. Following our previous work, which described rare natural Casp6-R65W and Casp6-G66R variants with significantly impaired activity51, we examined four additional missense rare variants with single amino acid substitutions located remotely from Casp6 active site: Casp6-A34E, Casp6-E35K, Casp6-A109T, and Casp6-T182S , small subunit (SS), and a minor amount of large subunit (LS)  Fig. . Casp6 vLS) Fig.  whereas 52 did not reveal any significant changes in the active sites of these two variants , stabilising the dimeric Casp6 structure ses Fig. . This poses Fig. . Becauseely Fig. , furtherTo identify candidate inhibitors of Casp6 activity, we performed a virtual screen by docking of small organic compounds targeting the putative allosteric site of the Ac-VEID-AFC-bound Casp6-WT structure. A total of 57,700 diverse molecules, originating from curated Chembridge and Sigma commercial libraries were screened. Subsequently to docking calculations, the poses of the best ranked molecules based on predicted binding energy were visually inspected to select the candidates that showed both favourable interactions within the putative allosteric site and ligand conformation of low energy. Finally, to ensure chemotype diversity a structural clustering was performed on the best candidates and a selection of 40 compounds was acquired Tables  and S4.in silico hits, a fluorescence-based Ac-VEID-AFC cleavage assay was used to measure recombinant active Casp6 reaction rate in the absence or presence of 100 µM concentration of compounds. A 30% inhibition cut off was arbitrarily chosen for further investigation of putative inhibitors. Eleven of the forty compounds (27.5%) screened showed inhibition of Casp6 . Interestingly, compound S10P, which is devoid of the critical pyrimidinetrione scaffold known as being a PAINS53, retained some activity with an IC50 of 80 µM.To probe a preliminary structure-activity relationship (SAR) of the two most active hits, S10 and C13, a few commercially available analogues Table  were tesIn the C13 series, seven analogues were tested, most of them exploring various replacements of the di-hydrobenzofuran right hand side-chain. While two of them (C13F and C13G) retained the activity seen with C13, most of them were found significantly less potent  of S10G was 1.8 µM, which was approximately 3-fold lower than C13 (Ki = 5.3 µM)  Fig. . Moreoveode Fig. .Figure 4tert-butyl group (C13E) were too short to optimally fill all subpockets of the allosteric site occupied by C13 and showed low potency against Casp6 . Casp6-K284A underwent complete processing at the TETD23 site but, in contrast to Casp6-WT, it only partially self-cleaved at the TEVD193 site generating ΔCasp6, LS-L and SS fragments  model inhibition  was not significantly different from the Ki for Casp6-WT-LS-SS (7.5 µM), whereas Ki for Casp6-K284A-LS-SS (11.8 µM) was 1.6-fold higher compared to the Ki for Casp6-WT-LS-SS , a conserved region also found in the putative allosteric pocket of Casp6, revealed a network of interactions, which couple changes in the allosteric H3CL site to both active sites of the Casp3 dimer56. The localised changes in the H3CL region were shown to propagate through the helix-3 to the connected β-strand containing catalytic histidine as well as affect the active site loop bundle of the opposite protomer56. The authors of the latter study made an excellent insight that the H3CL region in Casp6 is connected to the helix-3, part of which undergoes conformational change into β-strands when Casp6 transits from inactive extended helical to active canonical conformation58. This suggests that the putative Casp6 allosteric pocket may indeed have a more central role in regulating Casp6 activity, and perhaps an important biological ligand for this allosteric site remains to be uncovered since Casp6 is not known to be phosphorylated at the S150 site identical to the one in Casp3.Located more than 30 Å away from either active site of the Casp6 dimer, the N-terminal loop E35 is found in the neighbourhood of K36, which together with E244 and H287 forms Zn45. Small molecule inhibitors bind procaspase-6 allosteric site at the dimer interface and interact with residues in the L2 loop stabilising Casp6 zymogen-like conformation49. Likewise, small molecule covalent inhibitors bind at the analogous allosteric site at the dimer interface of Casp159, 560, 3 and 761, and lock L2′ loop in inactive zymogen-like conformation in Casp7, preventing substrate binding61. Our data indicate that Casp6 L2′ loop L200A mutant almost completely abolishes Casp6 self-processing and catalytic efficiency, consistent with the previous report for the analogous I213A mutation in the L2′ loop of Casp762, which revealed an essential role of L2′ loop in providing a platform for the proper arrangement of the substrate-binding groove in caspases. Interestingly, this was the first study62 to propose that small-molecule binding to the area occupied by I213 of L2′ loop in Casp7, which overlaps with the putative allosteric pocket of Casp6, could provide a novel strategy for modulating caspase activity.The important structural components of the putative Casp6 allosteric pocket are L2 and L2′ loops. Upon cleavage of the intersubunit linker, the released C-terminal region of the large subunit, and the N-terminal region of the small subunit, become L2, which contains catalytic C163, and L2′ loops, respectively. In a fully mature, active-site liganded conformation of Casp6, a region of the L2 loop of one protomer interacts in the putative allosteric pocket with a segment of the L2′ loop of the opposite protomer, which could represent a putative interprotomer coupling between the postulated allosteric pocket and the active site in the Casp6 dimer. Casp6 allosteric inhibition is often achieved through the conformational changes in the L2′ and L2 loops. Phosphorylation of Casp6 at the S257 results in a steric clash with L2′ loop, which folds over the dimer interface as seen in the apo mature form of Casp6, resulting in misalignment of the substrate binding loopsin vitro evaluation of Casp6 inhibition identified two Casp6 hit inhibitors, S10G and C13. Both compounds display low micromolar Ki and a noncompetitive mode of Casp6 inhibition, suggesting that they bind to an allosteric rather than a catalytic site, which validates our approach. Although our study lacks experimental evidence supporting the direct binding of C13 and S10G to the postulated allosteric pocket, the preliminary SAR of C13, where the core of the C13 chemotype has remained constant, in part supports the predicted C13 binding model in the Casp6 putative allosteric pocket. Modifications at the dihydro-benzofuran end have highlighted the importance of the shape of the pocket delimited by L200. Similarly, a change at the other end of the C13 molecule indicates that the furan ring is likely involved in critical binding interactions as predicted by modelling. It is necessary to assess additional analogues to validate the model, especially in light of the fact that alanine mutagenesis of two Casp6 allosteric pocket residues, L200 and K284, which are predicted to make key interactions with C13 according to our in silico model, did not abolish Casp6 inhibition by C13. Analogues bearing amide and carboxy isosteres, as well as various linkers attaching both ends and the central phenyl ring would be critical to verify the importance of these functionalities for hydrogen bonding or lipophilic interactions. In addition, the fact that Casp6 putative allosteric pocket is mainly composed of flexible loops, complicates accurate predictions of ligand binding using computational tools. Hence, additional C13 in silico binding models have to be experimentally evaluated and methods such as X-ray crystallography could be used to validate C13 binding to the postulated Casp6 allosteric site.Virtual screening of small molecules against active human Casp6 putative allosteric pocket followed by 53, and may act as a promiscuous compound, hence, one should consider H/DX-MS results with caution. The observed S10G potency for Casp6, however, is not entirely dependent on the presence of pyrimidinetrione structure since the S10P analogue lacking this structure still inhibited Casp6.The identification of S10G and Casp6 potential interaction sites using H/DX-MS was inconclusive. S10G contains a pyrimidinetrione PAINS structureThis study illustrates challenges faced in the early stage drug discovery and serves as a starting point for the future optimisation of our identified hits towards potent Casp6 allosteric inhibitors.https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=839).Casp6 missense variants were identified from the dbSNP short genetic variation NCBI database , Sigma-Aldrich  TimTec Inc , and Vitas-M Laboratory Ltd , and solubilised in 100% DMSO at 10 mM stock concentration.63 encoding full-length human Casp6 (Addgene plasmid 11823) was distributed by Dr. Guy Salvesen . The pET23b-Casp6(WT)p20/p10-His plasmid36 for E. coli codon optimized co-expression of Casp6-WT large and small subunits and pET23b( + ) plasmid encoding Casp6-C163A8 were previously described.Plasmid pET23b-Casp6-HisSingle mutations were introduced into pET23b-Casp6-His plasmid using QuikChange Site Directed Mutagenesis Kit  with the following primers and corresponding reverse sequence: A34E: 5′-AAA AGA GAA ATG TTT GAT CCG GAA GAA AAG TAC AAA ATG GAC CAC-3′, E35K: 5′-GAG AAA TGT TTG ATC CGG CAA AAA AGT ACA AAA TGG ACC AC-3′, A109T: 5′-TGT CAA CTG TTA GCC ACA CAG ATG CCG ATT GCT TT-3′, T182S: 5′-GGA TGT AGT AGA TAA TCA GTC AGA GAA GTT GGA CAC CAA-3′, L200A: 5′-CCG TTT ACA CGG CCC CTG CTG GAG C-3′, K284A: 5′-AAT GCT AAC TGC CAA GCT GCA TTT C-3′. Mutations were introduced into pET23b-Casp6(WT)p20/p10-His plasmid with the following primers and corresponding reverse sequence: L200A: 5′-GTT TAC ACC GCG CCG GCG GGC-3′, K284A: 5′-CAT GCT GAC CGC GAA ACT GCA CT TC-3′. Plasmids were verified by Sanger sequencing .E. coli BL21(DE3)pLysS strain  and purified51. Purity was evaluated by SDS-PAGE and Coomassie blue staining, and protein concentration was measured using Quick Start Bradford 1x Dye Reagent (Bio-Rad Laboratories).Plasmids encoding full-length Casp6-WT and variants, large and small subunits of Casp6-WT and variants, and Casp6-C163A were expressed in 64. Casp6 activity was measured in Stennicke’s buffer  pH 7.2, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonic acid, 10% sucrose)65 using 40 µM Ac-VEID-AFC 64 and 10-250 nM active site titrated Casp6. Reactions were initiated by Ac-VEID-AFC addition and followed for 20 min at 37 °C in a black clear bottom 96-well microplate  in Synergy H4 Hybrid instrument  with excitation/emission at 380/505 nm. Released AFC was calculated from AFC standard curve (0-25 µM). Casp6 VEIDase activity (released pmol AFC/min) was calculated from the initial linear reaction part. Ac-VEID-AFC hydrolysis initial velocities were measured with 10 nM active-site titrated Casp6 and 1–300 nM Ac-VEID-AFC. Initial velocities v versus substrate concentration [S] were fit to a rectangular hyperbola using Michaelis-Menten equation in GraphPad Prism 5 .Casp6 active sites were quantified using zVAD-FMK -fluoromethylketone, MP Biomedicals, Santa Ana, CA, USA)174PLDVVD179 p20 subunit10, 1:250 mouse monoclonal IgG1 clone B93-4 against human Casp6 271GKKQVPCFASMLTKK285 p10 subunit , and 1:1000 mouse monoclonal IgG1 SC81653 antibody against human pro-Casp6 24–293 aa . Antibodies were diluted in Tris-buffered saline with 0.1% Tween-20 and 5% non-fat dry milk. Proteins were detected with 1:5000 secondary anti-mouse  and anti-rabbit  antibodies conjugated to horseradish peroxidase using ECL western blotting detection reagent , and Carestream Biomax MR films .25 ng purified Casp6 were separated by SDS-PAGE on 15% gel, transferred to polyvinylidene fluoride membrane in Trans-Blot Turbo Transfer system (Bio-Rad Laboratories) and probed with 1:10,000 rabbit polyclonal neoepitope antiserum 10630 against active human Casp6 66. The structures were solved by molecular replacement with Phaser67 in Phenix68 using the crystal structure of wild-type Casp6 (PDB: 4FXO) as a search model, and re-built manually in Coot69. Refinement was carried out by phenix.refine70, with non-crystallographic symmetry restraints applied for the A109T mutant structure. Crystallographic data collection and structure refinement statistics are presented in Supplementary Table Fully processed Casp6 mutants E35K and A109T were crystallised in 100 mM sodium acetate pH 4.6 with 2 M sodium chloride. Crystals were grown by hanging drop vapour diffusion at 22 °C and were flash-frozen. X-ray diffraction data was collected at 100 K on beamline F1 with an ADSC Q-270 detector at the Cornell High Energy Synchrotron Source (MacCHESS). Data was processed by HKL200071. Molecular graphics were rendered with PyMOL (Version 1.7.1).The MOE package (Molecular Operating Environment (MOE), 201; Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7) was used for structural analysis, protein protonation (Protonate 3D), automated active site prediction  and hit compounds binding mode analysis. The ConSurf server was used for analysing sequence conservation of the members of human caspase family, as well as the mapping of this information onto the Casp6 crystal structurewww.sigmaaldrich.com and www.chembridge.com, respectively). In-house script was used to filter out compounds with undesired substructures. The retained 57,681 compounds were canonicalized and their three-dimensional coordinates were generated using Chemaxon Standardizer, JChem 5.1.2 (www.chemaxon.com). Protonation at pH 7.4 was performed on Ac-VEID-AFC-bound Casp6-WT structure (PDB: 3OD5) using the Protonate 3D module in MOE with the Amber12:EHT forcefield. The histidine H41 that lines the allosteric site was attributed the HSD protonation state. The AutoDockTools interface72 was used to prepare the Ac-VEID-AFC-bound Casp6-WT protonated structure into PDQT format. The docking grid was defined as a 16 × 16 × 22-Å box around the entire putative allosteric site when docking the compounds library as well as the follow up C13 analogues. Although the C13 compound purchased and tested is a racemic mixture , the model derived from docking strongly suggests the R-form to be most likely the isomer to optimally bind in the putative site. For blind docking of S10G compound  and the Chembridge Premium Set  were acquired from their commercial provider  was added to 200 nM Casp6 in SB with 100 μM compound, and Ac-VEID-pNA cleavage rate was measured at 37 °C for 30 min in clear 96-well plate  using Synergy H4 plate reader at 405 nm. Absorbance was converted to the released pNA amount based on a standard curve of 0–250 µM pNA. Cleavage rates were calculated from the linear assay phase and were normalised to 1% DMSO control. In Casp6-C163A cleavage assay, 0.5 ng/µl Casp6-WT was preincubated with 100 µM inhibitor, 5 µM Q-VD-OPh or 1% DMSO in SB , followed by transfer on ice, addition of 0.5 µg/µl Casp6-C163A, and incubation . Reactions were stopped by adding SDS-PAGE sample buffer  and heating . Samples (2 µl) were analysed on 15% SDS-PAGE gels, proteins were stained with Coomassie blue, and gels were de-stained with 10% acetic acid. The intensities of protein bands were quantified from scanned gels using ImageJ. The band intensity of Casp6-C163A without a prodomain was plotted against the time and the Casp6-C163A cleavage rates were calculated from the slopes of linear plots. The % activity of Casp6-WT in the presence of inhibitors was calculated relative to DMSO control.In fluorescence-based Casp6 activity assay, 10 µM Ac-VEID-AFC was added to 20 nM Casp6 in SB with 100 μM compound and 1% dimethyl sulfoxide (DMSO), and reaction rate was measured for 20 min at 37 °C as above. Compound potency was calculated by normalising Ac-VEID-AFC cleavage rate to 1% DMSO control. In colorimetric Casp6 activity assay, 200 µM Ac-VEID-50 values were determined against 20 nM Casp6 active sites in fluorescence-based Casp6 activity assay with 10 µM Ac-VEID-AFC and 1 nM-500 µM inhibitor. IC50 was calculated with GraphPad Prism 5 by a nonlinear regression fit for log inhibitor vs relative Casp6 activity (% DMSO control) using a Hill slope of – 1, and top and bottom constraints of 100 and 0, respectively.ICKM and Vmax values were determined through nonlinear regression analysis of Michaelis-Menten plots (v vs. [S]) using GraphPad Prism 5. The inhibition mode was determined from the pattern of converging Lineweaver-Burk plots (1/v vs. 1/[S])54. The dissociation constant (Ki) for Casp6-inhibitor complex was determined from the x-axis intercept  in the plot of Lineweaver-Burk slopes vs. inhibitor concentration74. Alternatively, inhibition model and Ki were determined by global fit of kinetic data to the competitive, noncompetitive, noncompetitive (mixed) or uncompetitive inhibition models using respective equations in GraphPad Prism 5. IC50 curves for S10G and C13 against Casp6 were generated using the fluorescence-based activity assay with 5–75 µM Ac-VEID-AFC.Compounds, S10G , and C13 , were added to 20 nM active site titrated Casp6 and enzyme activity was measured with eight different Ac-VEID-AFC substrate concentrations (5.0 μM–200 μM) using fluorescence-based Casp6 activity assay as above. The apparent http://www.ebi.ac.uk/Tools/msa/clustalo/) using UniProt accession numbers for human caspases: P29466, P42575, P42574, P49662, P51878, P55212, P55210, Q14790, P55211, Q92851, Q6UXS9, P31944, and Casp6 from different species: P55212, K7B3Z2, Q3T0P5, S9XS51, A0A0B8S0B0, O35397, O08738, L5K505, S7N3D7, A0A0Q4ALF8, O93415, A0A0Q3PLE0, Q9IB66, V9L2A1, Q9I8S9, Q52KK6, G0XQH6, Q9NHF9, K1R808.Multiple sequence alignments were generated with Clustal Omega tool (75. HDX was initiated by diluting 25 µM Casp6 stock solution with or without S10G (125 µM) into the D2O-based buffer 76 using 1:9 dilution ratio. For Casp6 in the presence of S10G, stock solution of Casp6 with S10G was diluted by D2O-based buffer containing S10G. The HDX incubation period and temperature were set to 10, 60, 900, 2400 s and 25 °C, respectively. HDX was quenched with chilled quenching buffer  using 1:2 dilution ratio. Quenched solutions were flash frozen in MeOH containing dry ice, and samples were stored at −80 °C until use. For the undeuterated control, initial dilution was made in H2O buffer. Prior to ultra-high performance liquid chromatography (UHPLC)-MS analysis, the deuterated Casp6 was digested in an on-line immobilised pepsin column prepared in house. Resulting peptides were loaded onto a C18 analytical column  equipped to an Agilent 1290 Infinity II UHPLC system. Peptides for each sample were separated using a 5–40% linear gradient of acetonitrile containing 0.1% formic acid for 8 min at a 65 µl/min flow rate. To minimize back-exchange, the columns, solvent delivery lines, injector and other accessories were placed in an ice bath. The C18 column was directly connected to the electrospray ionization source of the LTQ Orbitrap XL (Thermo Fisher Scientific), and mass spectra of peptides were acquired in positive-ion mode for m/z 200–2000. Duplicate measurements were performed for each time point. Identification of peptides was carried out in separate experiments by tandem MS (MS/MS) analysis in data-dependent acquisition mode, using collision-induced dissociation. All MS/MS spectra were analysed using Proteome Discoverer 1.4 (Thermo Fisher Scientific). Peptide searching results were further manually inspected, and only those verifiable were used in HDX analysis. The deuteration (%) as a function of incubation time was determined using HDExaminer 2.3 . The first two amino acid residues in peptides were excluded from the analysis.HDX-MS was carried out as describedSupplementary information file


The emerging view that the West Antarctic ice sheet is in the early stage of collapse owes as much to paleoclimatology as to contemporary observations. Our knowledge of the ice sheets has grown rapidly, thanks to advances in remote sensing and to dedicated field campaigns, which have provided an unprecedented view of the surface, interior, and bed, as well as the conditions in the surrounding oceans and atmosphere. It was discovered in the mid 1990s that there is rapid melting under the ice shelf of Pine Island Glacier2 one of the major outlet glaciers that drains West Antarctica into the sea. Shortly thereafter, observations showed that Pine Island Glacier and other outlet glaciers were retreating and thinning4. It is now clear that most of the margin of the West Antarctic ice sheet – and parts of East Antarctica as well – is undergoing rapid retreat5, and that the proximal cause is the erosion of ice shelves by warm ocean water6, amplified by intrinsic instabilities in glacier dynamics7.The stability of the Antarctic ice sheet in the face of anthropogenic climate change is the central question in polar science. The retreat of glaciers and loss of ice shelves on the Antarctic Peninsula is well-documented8, who 40 years ago suggested that anthropogenic climate warming would destabilize ice shelves, leading to ice sheet collapse. Recent observations certainly support Mercer’s understanding of the crucial role of ice shelves. Yet a solicitation of researchers’ views conducted as recently as 2013 concluded that expert opinion is both “uncertain and undecided” on whether recent trends in Antarctic ice sheet behavior are owing to natural variability, or are a response to climate change9. The reason for this ambiguity is simple: the record of direct observations of Antarctic ice sheet change is insufficient in length. The earliest application10 of satellite radar interferometry to obtain flow velocities for the Antarctic ice sheet was in 1993, and most other observational products, such as the satellite altimetry critical for evaluating thinning rates, are even more recent. Modern geophysical observations of the ice sheet thus comprise a period less than the standard textbook requirement for a representative climatology – 30 years – against which trends can be evaluated. This is surely too short a time to have observed the Antarctic ice sheet, characterized by intrinsic variability on even longer timescales than the atmosphere, and expect a definitive answer. Unless we are willing to wait another few decades or more – by which time the answer may well be clear, but no longer as useful – we need to incorporate information from paleoclimatology.Our contemporary understanding of the state of the West Antarctic ice sheet is often traced to the prescience of Mercer11 had established that sea level was several meters higher than present during the last interglacial period (~132–116 thousand years ago), and it was soon recognized that the Antarctic ice sheet must have been considerably smaller12. Sediment cores drilled beneath the present-day ice sheet have since shown that the West Antarctic ice sheet collapsed at least once in the last million years13. Sea level during the last interglacial rose rapidly to between 5 and 9 m higher than present, a finding that is difficult to explain without a major contribution from Antarctica14. Such evidence remains the strongest indication of ice sheet sensitivity to climate change.Mercer’s insight that the West Antarctic ice sheet is vulnerable was motivated by emerging paleoclimate evidence. Data from deep sea sediment cores15. Simulations with this model physics cause West Antarctic ice sheet collapse during the last interglacial, provided that strong warming of the Southern Ocean is assumed as a boundary condition16. Evidence for such warming can indeed be found in the paleoclimate record17. The same model physics, under projected anthropogenic climate forcing, leads to rapid deglaciation of West Antarctica16. Thus, evidence of the past response of the ice sheet to paleoclimate informs our understanding of its future.Modern numerical ice-sheet modeling is also informed by paleoclimate evidence. Simulations of the future behavior of the ice sheet rely on estimates of past ice sheet configuration and past climate forcing  for initialization and validation. Difficulties in matching the output of model simulations to geological constraints on paleo-ice sheet configuration and sea level have demanded the implementation of new model physics: a more-efficient mechanism for ice-shelf calving than had been conventionally assumed19 leading to the suggestion that this event was an important forcing of glacier retreat in the Amundsen Sea20. Sediment cores21 collected from beneath the Pine Island Glacier ice shelf have since shown that flooding of the continental shelf by warm Circumpolar Deep Water began around the 1940s. Data like these will be critical for determining when – and if – climate changes in Antarctica reach the point where, as Mercer put it, “a dangerous trend is under way.” To date, a definitive answer is available only for the Antarctic Peninsula, but the result from an ice core there is dramatic: summer melt now exceeds anything in the past millennium22.Paleoclimate data also provide context for the ice sheet changes being observed today Fig. . Increas23. For such techniques to yield reliable information about the conditions most relevant to the ice dynamics, additional records will be needed from regions closer to the center of action. Cores from the numerous ice domes along the Amundsen and Bellingshausen Sea coasts of West Antarctica would be particularly valuable in this respect, though the logistical difficulties of working in this remote region have thus far prevented initiatives to obtain such cores from moving forward.Paleoclimate data, of course, have limitations compared with instrumental climate observations and modern geophysical imaging of ice sheet structure. Yet, the spatial coverage of ice cores is far greater than that of weather stations, and there is potential to use such records even more effectively, through the blending of proxy data with information from climate models to produce longer-term climate reanalysis products24. Another promising idea is to use borehole drilling to obtain samples of bedrock from beneath the current ice sheet, and then use cosmogenic nuclide measurements to determine bedrock exposure history, yielding information about times when the ice sheet was smaller25.Somewhat remarkably, it still remains to be resolved whether the West Antarctic ice sheet collapsed during the last interglacial period. This uncertainty presents an important challenge that can only be answered definitively with paleoclimate and paleo-glaciological data. One approach is to obtain spatial reconstructions of climate using deep ice core records. A significantly smaller ice sheet would be associated with changes in weather patterns which should be detectable in ice cores from strategically-located sites such as Hercules Dome, inland of the Transantarctic Mountains in East Antarctica, adjacent to the West Antarctic ice sheet. Climate model results suggest that the climate at Hercules Dome would be especially sensitive to atmospheric circulation changes associated with ice-sheet collapse27. Certainly, the current retreat of the margins of the Antarctic ice sheet is remarkable, and expert opinion on whether such trends are worrisome has probably moved somewhat from “uncertain” towards “likely” in the last few years. Observations from paleoclimate have played a powerful role in this emerging view, by providing critical boundary conditions for ice sheet models and elucidating the conditions under which ice sheet collapse may have occurred in the past. Paleoclimate data also provide an important role in balancing the interpretation of modern observations against the longer-term context in which ongoing ice sheet changes are occurring. Such complementary information will continue to be necessary as the ice sheet continues to change in a warming world.Some researchers have concluded that the West Antarctic ice sheet is already in the early stages of an irreversible collapse