The Ca2+/calmodulin-dependent protein phosphatase calcineurin (CN), a heterodimer composed of a catalytic subunit A and an essential regulatory subunit B, plays critical functions in various cellular processes such as cardiac hypertrophy and T cell activation. It is the target of the most widely used immunosuppressants for transplantation, tacrolimus (FK506) and cyclosporin A. However, the structure of a large part of the CNA regulatory region remains to be determined, and there has been considerable debate concerning the regulation of CN activity. Here, we report the crystal structure of full-length CN (β isoform), which revealed a novel autoinhibitory segment (AIS) in addition to the well-known autoinhibitory domain (AID). The AIS nestles in a hydrophobic intersubunit groove, which overlaps the recognition site for substrates and immunosuppressant-immunophilin complexes. Indeed, disruption of this AIS interaction results in partial stimulation of CN activity. More importantly, our biochemical studies demonstrate that calmodulin does not remove AID from the active site, but only regulates the orientation of AID with respect to the catalytic core, causing incomplete activation of CN. Our findings challenge the current model for CN activation, and provide a better understanding of molecular mechanisms of CN activity regulation.

The HIN domain of myeloid nuclear differentiation antigen (MNDA) was expressed and purified as a monomer using E. coli JM109 as host. The protein interacted with double-stranded DNA at a Kd of 3.15 μM and did not recognize the termini of double-stranded DNA. Isothermal titration calorimetry indicated that the interaction between the protein and double-stranded DNA is mainly mediated by electrostatic attractions and hydrogen bonding. We developed a model to analyze the potential DNA binding site of the MNDA HIN domain. Based on the model, molecular docking and mutation studies suggest that the double-stranded DNA binding site of the protein is different from other HIN–DNA structures. This work facilitates the design of specific drugs against pathogens detected by human MNDA.

The atypical PKC isoforms (ζ and ı) play essential roles in regulating various cellular processes. Both the hetero-interaction between PKCζ and p62 through their N-terminal PB1 domains and the homo-oligomerization of p62 via its PB1 domain are critical for the activation of NF-κB signaling; however, the molecular mechanisms concerning the formation and regulation of these homotypic complexes remain unclear. Here we determined the crystal structure of PKCζ-PB1 in complex with a monomeric p62-PB1 mutant, where the massive electrostatic interactions between the acidic OPCA motif of PKCζ-PB1 and the basic surface of p62-PB1, as well as additional hydrogen bonds, ensure the formation of a stable and specific complex. The PKCζ-p62 interaction is interfered with the modification of a specific Cys of PKCζ by the antiarthritis drug aurothiomalate, though all four cysteine residues in the PKCζ-PB1 domain can be modified in in vitro assay. In addition, detailed structural and biochemical analyses demonstrate that the PB1 domains of aPKCs belong to the type I group, which can depolymerize the high-molecular-weight p62 aggregates into homo-oligomers of lower order. These data together unravel the molecular mechanisms of the homo-or hetero-interactions between p62 and PKCζ and provide the basis for designing inhibitors of NF-κB signaling.

The AMP-activated protein kinase (AMPK) is the key energy sensor in response to various stresses that decrease cellular ATP levels, including low glucose, hypoxia, ischemia and heat shock. Recently, evidence has suggested that AMPK also plays critical roles in coupling the cellular bio-energetic state to various physiological processes, such as tumor suppression, cell polarity, life span, circadian clock, gene transcription and autophagy. Thus, AMPK represents a potential drug target for metabolic disorders and cancer treatment1,2.AMPK is a heterotrimeric protein kinase composed of one catalytic (α) and two regulatory (β and γ) subunits (Figure 1A). The α-subunit contains an N-terminal kinase domain, followed by an autoinhibitory domain (AID), a regulatory linker region (α-linker) and a C-terminal β/γ-subunit interacting domain (α-CTD). Phosphorylation of a conserved threonine in the kinase domain (Thr172 in rat α1) by upstream protein kinases is the prime activation step of AMPK, while the adjacent AID directly binds to the kinase domain and inhibits its catalytic activity3. The heterotrimeric core structures of the yeast and mammalian AMPKs, including the entire γ-subunit and the interacting fragments of the α- and β-subunits, have revealed that the C-terminal domain of the β-subunit (β-CTD) serves as a bridge between the α-CTD and the γ-subunit and that the γ-subunit adopts a pseudosymmetric conformation comprising four tandem cystathionine β-synthase (CBS1-4) motifs for adenine nucleotide binding4. Binding of AMP and ADP to the γ-subunit has activating effects in protecting AMPK against dephosphorylation and/or promoting its phosphorylation5,6. In addition, raising the cellular AMP level will allosterically enhance the activity of mammalian AMPK that has been primarily activated by upstream kinases. Therefore, a central issue pertaining to the regulation of AMPK lies in how AMP binding to the γ-subunit ultimately regulates the kinase activity in the α-subunit.Although the γ-subunit contains four potential nucleotide binding sites (1-4), site 2 in mammalian AMPKs possesses a key Asp-to-Arg substitution and remains unoccupied even in the presence of high concentrations of AMP or ATP4,7. The other three sites (1, 3 and 4) on the γ-subunit appear to reversibly bind three AMP or two ATP molecules, which results in substantial conformational changes and distinct activities of AMPK7. Our structure-based mutagenesis studies have demonstrated that two of the three nucleotide-binding sites, γ-site 3 and γ-site 4, are important for AMPK allosteric activation. Xiao et al.8 reported the structure of an active AMPK heterotrimer, comprising the kinase domain, AID and the regulatory α-linker of the α-subunit in addition to the core fragment, which revealed additional inter-subunit interactions involving the activation loop of the kinase domain and a small segment within the α-linker. However, in the same study, it was concluded that mammalian AMPK does not contain an AID domain, inconsistent with our results3. We found that the amino acid sequence of the AID and α-linker region in that AMPK structure was incorrectly assigned due to the poor electron density9. We have thus rebuilt the model and determined the structures of two isolated mammalian AID domains, which affirms the universal presence of AID (Figure 1B and Supplementary information, Figure S1). More importantly, we identified the α-RIM (regulatory subunit interacting motif) sandwiched in between γ-site 3 and the induced β-loop (Supplementary information, Figure S2), and we also determined the essential role of α-RIM in AMPK allosteric regulation upon AMP binding9.Intriguingly, the α-linker segment prior to the α-RIM reaches the other side of the induced β-loop and then packs against the non-bound site 2 on the γ-subunit (Figure 1B). This α- and γ-subunit interface is mediated by a novel α-subunit motif containing the last turn of AID helix α3 and the following flexible loop (rat α1 332-342). According to the linear sequence of the α-subunit, we hereafter refer to this new segment as α-RIM1 and rename the previously identified α-RIM as α-RIM2. At the interface between α-RIM1 and γ-site 2, the aromatic residues αPhe340 and αTyr341 interact with γLys173, γPhe174 and γLeu177 from the γ-Helix7; in turn, the hydrophobic γPhe178 inserts between αIle333 and αMet334 (Figure 1C). In addition, the highly conserved γArg170, which is believed to be the main determinant of the non-bound site 2, further strengthens this interface by participating in multiple hydrophilic interactions with the main chains of αPhe340 in α-RIM1 and γLeu40 and γPro42 from an N-terminal segment of the γ-subunit, while the main chains of γAsp39, γThr43 and γSer44 from this γ-subunit segment form hydrogen bonds with both the side and main chains of αTyr341 in α-RIM1. Therefore, both the γ-Helix7 at site 2 and the spatially adjacent γ-subunit segment (rat γ1 39-44) play important roles in recruiting the newly identified α-RIM1. On close inspection, we also found additional hydrophobic interactions involving the previous α-RIM2 (Supplementary information, Figure S3). The αPro365 and αPhe366, which follow the two important charged residues αGlu362 and αArg3639, nestle into a hydrophobic pocket formed by six residues at γ-site 3 (γPhe243, γIle246, γAsn247, γAla250, γHis267 and γVal64). Notably, the aromatic ring of γPhe243, the residue preceding the indispensable and highly conserved γAsp244, also stabilizes the ribose ring of AMP bound at site 3. Taken together, two tandem α-subunit motifs, the α-RIM1 and the previously defined α-RIM2, bind to site 2 and site 3 on the γ-subunit, respectively.To assess the importance of the aforementioned inter-subunit interactions, we generated a series of point mutations on the rat α1β1γ1-holoenzyme and examined their effects on AMPK allosteric activation. In the presence of 200 μM AMP, the activity of the wild-type AMPK was stimulated by ~2-fold (Figure 1D). In contrast, mutating the key α-RIM1 residues largely abolished the AMP dependence as observed in the mutants in which the hydrophobic pairs of interacting residues were replaced with charged residues (αF340D/Y341D and αI333D/M334D). Similarly, the γ-site 2 mutants (γR170A and γF178D) no longer responded to the increase in AMP concentration. We also simultaneously substituted the two hydrophobic residues in the α-RIM2, and the AMP dependence of the αP365D/F366D mutant was completely abolished as previously observed in other α-RIM2 mutants, including αE362A9. The independence of these mutant AMPKs on changes in AMP concentration clearly demonstrates that both α-RIM1 and α-RIM2 play important roles in the allosteric regulation of AMPK activity.Significantly, in the vertebrate AMPK homologs, 7 out of the 8 important interacting residues in the new α-RIM1 and previous α-RIM2 are identical, and the remaining one is highly conserved (Supplementary information, Figure S1). The interacting γ-subunit residues are also strictly conserved across vertebrates (Supplementary information, Figure S4). Therefore, various AMPK αβγ-heterotrimers may adopt similar inter-subunit interactions and be activated via a conserved molecular mechanism. By contrast, some of these residues in the α-RIM1/2 and γ-sites 2 and 3 are not conserved in invertebrate AMPKs, such as those from Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae and Schizosaccharomyces pombe. This provides a possible explanation for why only vertebrate AMPKs can be allosterically activated by AMP.The next question is how AMPK senses the change in the cellular AMP/ATP levels. We have shown that the mammalian AMPK core structures in complex with ATP or AMP adopt distinct conformations, in that the γ-site 3 is free of any nucleotide in the ATP-bound state but bound by AMP in the AMP-bound state7. This active AMPK structure in complex with two AMP molecules at γ-sites 3 and 4 adopts an almost identical conformation to the AMP-bound core structure (Supplementary information, Figure S2), but it exhibits substantial conformational changes around γ-site 3 compared to the ATP-bound structure (Figure 1E). In the ATP-bound structure, the α-CTD and β-CTD, as well as the first β-strand of the γ-subunit that forms an inter-subunit β-sheet with the β-CTD, collectively move upwards, which would abolish the multiple hydrogen bonds between the main chains of the γ-subunit N-terminal segment and the αTyr341 of α-RIM1 (Figure 1E and Supplementary information, Figure S5A). The side chains of γArg170 and γLys173 at γ-site 2 also shift away from α-RIM1, which may disrupt both the hydrophilic and hydrophobic interactions with αPhe340. In addition, the γ-Helix7 tilts ~5° and forms an additional turn at the C-terminus, and the aromatic ring of γPhe178 thereby rotates almost 180° and moves away from the hydrophobic side chains on the AID helix α3. Hence, when the γ-subunit is bound by ATP, most of the interactions between the α-RIM1 and the γ-subunit would also be disrupted. Upon ATP binding, the most significant shift (up to 6 Å) occurs in γ-Helix10, which contains the highly conserved Asp244 as well as four of the six residues accommodating the hydrophobic αPro365 and αPhe366 in α-RIM2 (Supplementary information, Figures S5B and S3). The spatially adjacent γ-Helix11 and γ-Helix3 also move away from α-RIM2; specifically, the imidazole side chain of γHis267 rotates ~180° and shifts > 5 Å. In addition, the ε-amino group of γLys169 is pushed away from the key αGlu362 by the dramatically changed γHis297. Therefore, the γ-site 3 in the ATP-bound state is distorted and unable to recruit α-RIM2. In contrast, AMP binding to the γ-subunit induces substantial changes and enables recruitment of both α-RIM1 and α-RIM2 to the γ-subunit.Then, why and how would the binding of two RIM motifs ultimately enhance AMPK activity in the α-subunit? We suggested previously that the γ-sites 3 and 4 of mammalian AMPKs are indispensable for AMP-induced allosteric regulation and that the γ-site 1 has little effect7. It is natural that α-RIM2 plays an important role in the AMP regulation of AMPK activity because α-RIM2 packs against the essential γ-site 3 in its AMP-bound state9. Here we demonstrate that α-RIM1, though surprisingly bound to the nucleotide-free γ-site 2, has a significant effect on AMPK regulation. As α-RIM1 partially overlaps with the AID domain, binding of α-RIM1 to the γ-site 2 would recruit the AID domain to the proximity of the γ-subunit. More importantly, all the AID side chains that were shown to interact with the kinase domain in the autoinhibited AMPK structure3 are facing the γ-subunit, and binding of the kinase domain to AID in the active AMPK conformation would thus cause severe steric clash between the kinase domain and the γ-subunit (Supplementary information, Figure S6). As revealed in our rebuilt structure of the active AMPK, binding of α-RIM1 and α-RIM2 to the AMP-bound γ-subunit forces the kinase domain to dissociate from AID and pack against the β-CTD, thereby relieving the inhibitory function of AID.A full understanding of the regulation of AMPK upon energy stress may facilitate the design of novel therapeutics for metabolic diseases. Together with our previous studies, the structural and mutational analyses demonstrate that the three regulatory elements in the α-subunit, AID, α-RIM1 and α-RIM2, are indispensable for the allosteric activation of AMPK, leading to a more complete understanding of the allosteric regulation of mammalian AMPK (Figure 1F). When the cellular AMP concentration is low, α-RIM1 and α-RIM2 do not bind to the ATP-bound γ-subunit. AID directly binds to the kinase domain, thus leading to the autoinhibition of AMPK activity. Under energy stress (increased AMP levels), the γ-subunit is bound by AMP and undergoes substantial conformational changes. The α-RIM1 and α-RIM2 are recruited to γ-site 2 and γ-site 3, respectively. The AID domain is thus pulled into the proximity of γ-site 2 and dissociates from the kinase domain. The free kinase domain then binds to the β-CTD, and the heterotrimeric AMPK holoenzyme adopts a more compact, fully activated conformation10. Following this model, molecules that can stabilize the interactions between α-RIM1/2 and γ-sites 2/3 or disrupt those between AID and the kinase domain would retain AMPK in a stimulated state.This work was supported by the National Natural Science Foundation of China (31130062), the National Key Basic Research Program of China (2013CB530603) and Tsinghua University (20121080028).(Supplementary information is linked to the online version of the paper on the Cell Research website.)

The mitogen-activated protein kinase (MAPK) p38α is a key regulator in many cellular processes, whose activity is tightly regulated by upstream kinases, phosphatases and other regulators. Transforming growth factor-β activated kinase 1 (TAK1) is an upstream kinase in p38α signaling, and its full activation requires a specific activator, the TAK1-binding protein (TAB1). TAB1 was also shown to be an inducer of p38α’s autophosphorylation and/or a substrate driving the feedback control of p38α signaling. Here we determined the complex structure of the unphosphorylated p38α and a docking peptide of TAB1, which shows that the TAB1 peptide binds to the classical MAPK docking groove and induces long-range conformational changes on p38α. Our structural and biochemical analyses suggest that TAB1 is a reasonable substrate of p38α, yet the interaction between the docking peptide and p38α may not be sufficient to trigger trans-autophosphorylation of p38α.

A critical challenge to the fragment-based drug discovery (FBDD) is its low-throughput nature due to the necessity of biophysical method-based fragment screening. Herein, a method of pharmacophore-linked fragment virtual screening (PFVS) was successfully developed. Its application yielded the first picomolar-range Qo site inhibitors of the cytochrome bc1 complex, an important membrane protein for drug and fungicide discovery. Compared with the original hit compound 4 (Ki = 881.80 nM, porcine bc1), the most potent compound 4f displayed 20 507-fold improved binding affinity (Ki = 43.00 pM). Compound 4f was proved to be a noncompetitive inhibitor with respect to the substrate cytochrome c, but a competitive inhibitor with respect to the substrate ubiquinol. Additionally, we determined the crystal structure of compound 4e (Ki = 83.00 pM) bound to the chicken bc1 at 2.70 Å resolution, providing a molecular basis for understanding its ultrapotency. To our knowledge, this study is the first application of the FBDD method in the discovery of picomolar inhibitors of a membrane protein. This work demonstrates that the novel PFVS approach is a high-throughput drug discovery method, independent of biophysical screening techniques.

The cytochrome bc1 complex (EC 1.10.2.2, bc1) is one of the most promising targets for new drugs and agricultural fungicides. Among the existing bc1 complex inhibitors specifically binding to the Qo site, oxazolidinedione derivatives have attracted great attention. With the aim to understand the substituent effects of oxazolidinedione derivatives on the inhibition activity against the bc1 complex, a series of new oxazolidinedione derivatives were designed, synthesized, and biologically evaluated. The further inhibitory kinetics studies against porcine succinate–cytochrome c reductase (SCR) revealed that the representative compound 8d and famoxadone are both non-competitive inhibitors with respect to the substrate cytochrome c, but competitive inhibitors with respect to substrate decylubiquinol (DBH2). In addition, compound 8d and famoxadone showed, respectively, 35-fold and 15-fold greater inhibitory activity against the porcine SCR than the porcine bc1 complex, indicating that these two inhibitors not only inhibited the activity of the bc1 complex, but possibly affect the interaction between the complex II and the bc1 complex. To our knowledge, this is the first report that famoxadone and its analogs have effects on the interaction between the complex II and the bc1 complex.Graphical abstract

Thermolysin-like proteases (TLPs), a large group of zinc metalloproteases, are synthesized as inactive precursors. TLPs with a long propeptide (∼200 residues) undergo maturation following autoprocessing through an elusive molecular mechanism. We report the first two crystal structures for the autoprocessed complexes of a typical TLP, MCP-02. In the autoprocessed complex, Ala205 shifts upward by 33 Å from the previously covalently linked residue, His204, indicating that, following autocleavage of the peptide bond between His204 and Ala205, a large conformational change from the zymogen to the autoprocessed complex occurs. The eight N-terminal residues (residues Ala205-Gly212) of the catalytic domain form a new β-strand, nestling into two other β-strands. Simultaneously, the apparent Tm of the autoprocessed complex increases 20 °C compared to that of the zymogen. The stepwise degradation of the propeptide begins with two sequential cuttings at Ser49-Val50 and Gly57-Leu58, which lead to the disassembly of the propeptide and the formation of mature MCP-02. Our findings give new insights into the molecular mechanism of TLP maturation.

Cytochrome bc1 complex (EC 1.10.2.2, bc1), an essential component of the cellular respiratory chain and the photosynthetic apparatus in photosynthetic bacteria, has been identified as a promising target for new drugs and agricultural fungicides. X-ray diffraction structures of the free bc1 complex and its complexes with various inhibitors revealed that the phenyl group of Phe274 in the binding pocket exhibited significant conformational flexibility upon different inhibitors binding to optimize respective π−π interactions, whereas the side chains of other hydrophobic residues showed conformational stability. Therefore, in the present study, a strategy of optimizing the π−π interaction with conformationally flexible residues was proposed to design and discover new bc1 inhibitors with a higher potency. Eight new compounds were designed and synthesized, among which compound 5c, with a Ki value of 570 pM, was identified as the most promising drug or fungicide candidate, significantly more potent than the commercially available bc1 inhibitors, including azoxystrobin (AZ), kresoxim-methyl (KM), and pyraclostrobin (PY). To our knowledge, this is the first bc1 inhibitor discovered from structure-based design with a potency of subnanomolar Ki value. For all of the compounds synthesized and assayed, the calculated binding free energies correlated reasonably well with the binding free energies derived from the experimental Ki values, with a correlation coefficient of r2 = 0.89. The further inhibitory kinetics studies revealed that 5c is a noncompetitive inhibitor with respect to substrate cytochrome c, but it is a competitive inhibitor with respect to substrate ubiquinol. Due to its subnanomolar Ki potency and slow dissociation rate constant (k−0 = 0.00358 s−1), 5c could be used as a specific probe for further elucidation of the mechanism of bc1 function and as a new lead compound for future drug discovery.

AMP-activated protein kinase (AMPK) senses cellular energy status to maintain a balance between ATP production and consumption, and has important roles in regulating cell growth and proliferation. Here, crystal structures of kinase and autoinhibitory domains from yeast AMPK subunits, together with biochemical data, reveal a mechanism for AMPK autoinhibition and suggest a model for allosteric activation by AMP.

The decapentaplegic(Dpp), a member of the TGF-β superfamily, plays a pivotal role in the control of proliferation, global patterning and induction of specific cell fates during Drosophila development. Mother against Dpp(Mad) is the founding member of the conserved Smad protein family which specifically transduces the intracellular TGF-β signaling cascade. Here we report the 2.80 Å structure of the MH2 domain of Mad(Mad-MH2) that was readily superposed to the mammal Smad-MH2 structures. This unphosphorylated Mad-MH2 forms a symmetric homotrimer in crystals, consistent with the result of the size-exclusion chromatography that Mad-MH2 exhibited a propensity for concentration-dependent oligomerization prior to phosphorylation. Structural analysis revealed that the formation of homotrimeric Mad-MH2 is mainly mediated by contacts involving the extreme C-terminal SSVS motif, and is strengthened by phosphorylation of the last two Ser residues which was confirmed by the gel filtration analysis of the pseudophosphorylated Mad-MH2(DVD). Intriguingly, the homotrimer within an asymmetric unit only possesses two ordered C-terminal tails, reminiscent of the arrangement of the R-Smad/Smad4 complexes, indicating that the subunit with a flexible SSXS motif would be readily replaced by Co-Smad to form a functional heterotrimer.

AMP-activated protein kinase (AMPK) is a heterotrimeric complex composed of α catalytic subunit, β scaffolding subunit, and γ regulatory subunit with critical roles in maintaining cellular energy homeostasis. However, the molecular architecture of the intact complex and the allostery associated with the adenosine binding-induced regulation of kinase activity remain unclear. Here, we determine the three-dimensional reconstruction and subunit organization of the full-length rat AMPK (α1β1γ1) through single-particle electron-microscopy. By comparing the structures of AMPK in ATP- and AMP-bound states, we are able to visualize the sequential conformational changes underlying kinase activation that transmits from the adenosine binding sites in the γ subunit to the kinase domain of the α subunit. These results not only make substantial revision to the current model of AMPK assembly, but also highlight a central role of the linker sequence of the α subunit in mediating the allostery of AMPK.Graphical AbstractDownload high-res image (266KB)Download full-size imageHighlights► The molecular architecture of full-length AMPK is determined by single-particle EM ► Sequential conformational changes underlying AMPK activation are observed ► A three-state model for AMPK structure and regulation is proposed ► A central role of the linker sequence of α subunit in AMPK allostery is highlighted