Phosphoinositide-dependent kinase-1 orthologues from five eukaryotes are activated by the hydrophobic motif in AGC kinases

Phosphoinositide-dependent kinase-1 (PDK1) mediates activation of many AGC kinases by docking onto a phosphorylated hydrophobic motif located C-terminal of the catalytic domain in the AGC kinase. The interaction shifts PDK1 into a conformation with increased catalytic activity and leads to autophosphorylation of PDK1. We demonstrate here that addition of a hydrophobic motif peptide increases the catalytic activity of PDK1 orthologues from Homo sapiens, Aplysia californica, Arabidopsis thaliana, Schizosaccharomyces pombe (ksg1), and Saccharomyces cerevisiae (Pkh1 and Pkh2) 2- to 12-fold. Furthermore, the hydrophobic motif peptide increases autophosphorylation of PDK1 from Homo sapiens, S. pombe, and S. cerevisiae (Phk2). Our results suggest that PDK1 interaction and activation by the hydrophobic motif of AGC kinases is a central mechanism in PDK1 function, which is conserved during eukaryotic evolution.     

Maize Ribosome-Inactivating Protein Uses Lys158–Lys161 to Interact with Ribosomal Protein P2 and the Strength of Interaction Is Correlated to the Biological Activities

Ribosome-inactivating proteins (RIPs) inactivate prokaryotic or eukaryotic ribosomes by removing a single adenine in the large ribosomal RNA. Here we show maize RIP (MOD), an atypical RIP with an internal inactivation loop, interacts with the ribosomal stalk protein P2 via Lys158–Lys161, which is located in the N-terminal domain and at the base of its internal loop. Due to subtle differences in the structure of maize RIP, hydrophobic interaction with the ‘FGLFD’ motif of P2 is not as evidenced in MOD-P2 interaction. As a result, interaction of P2 with MOD was weaker than those with trichosanthin and shiga toxin A as reflected by the dissociation constants (K_D) of their interaction, which are 1037.50±65.75 µM, 611.70±28.13 µM and 194.84±9.47 µM respectively.Despite MOD and TCS target at the same ribosomal protein P2, MOD was found 48 and 10 folds less potent than trichosanthin in ribosome depurination and cytotoxicity to 293T cells respectively, implicating the strength of interaction between RIPs and ribosomal proteins is important for the biological activity of RIPs. Our work illustrates the flexibility on the docking of RIPs on ribosomal proteins for targeting the sarcin-ricin loop and the importance of protein-protein interaction for ribosome-inactivating activity.     

SNAP-25 substrate peptide (residues 180-183) binds to but bypasses cleavage by catalytically active Clostridium botulinum neurotoxin E

Clostridium botulinum neurotoxins are the most potent toxins to humans. The recognition and cleavage of SNAREs are prime evente in exhibiting their toxicity. We report here the crystal structure of the catalytically active full-length botulinum serotype E catalytic domain (BoNT E) in complex with SNAP-25 (a SNARE protein) substrate peptide Arg(180)-Ile(181)-Met(182)-Glu(183) (P1-P3'). It is remarkable that the peptide spanning the scissile bond binds to but bypasses cleavage by the enzyme and inhibits the catalysis fairly with K(i) approximately 69 microm. The inhibitory peptide occupies the active site of BoNT E and shows well defined electron density. The catalytic zinc and the conserved key residue Tyr(350) of the enzyme facilitate the docking of Arg(180) (P1) by interacting with its carbonyl oxygen that displaces the nucleophilic water. The general base Glu(212) side chain interacts with the main chain amino group of P1 and P1'. Conserved Arg(347) of BoNT E stabilizes the proper docking of the Ile(181) (P1') main chain, whereas the hydrophobic pockets stabilize the side chains of Ile(181) (P1') and Met(182) (P2'), and the 250 loop stabilizes Glu(183) (P3'). Structural and functional analysis revealed an important role for the P1' residue and S1' pocket in driving substrate recognition and docking at the active site. This study is the first of its kind and rationalizes the substrate cleavage strategy of BoNT E. Also, our complex structure opens up an excellent opportunity of structure-based drug design for this fast acting and extremely toxic high priority BoNT E.     

Structure of the human Parkin ligase domain in an autoinhibited state

Mutations in the protein Parkin are associated with Parkinson's disease (PD), the second most common neurodegenerative disease in men. Parkin is an E3 ubiquitin (Ub) ligase of the structurally uncharacterized RING‐in‐between‐RING(IBR)‐RING (RBR) family, which, in an HECT‐like fashion, forms a catalytic thioester intermediate with Ub. We here report the crystal structure of human Parkin spanning the Unique Parkin domain (UPD, also annotated as RING0) and RBR domains, revealing a tightly packed structure with unanticipated domain interfaces. The UPD adopts a novel elongated Zn‐binding fold, while RING2 resembles an IBR domain. Two key interactions keep Parkin in an autoinhibited conformation. A linker that connects the IBR with the RING2 over a 50‐Å distance blocks the conserved E2～Ub binding site of RING1. RING2 forms a hydrophobic interface with the UPD, burying the catalytic Cys431, which is part of a conserved catalytic triad. Opening of intra‐domain interfaces activates Parkin, and enables Ub‐based suicide probes to modify Cys431. The structure further reveals a putative phospho‐peptide docking site in the UPD, and explains many PD‐causing mutations.     

Association of the disordered C-terminus of CDC34 with a catalytically bound ubiquitin

Cell division cycle protein 34 (CDC34) is a key E2 ubiquitin (Ub)-conjugating enzyme responsible for the polyubiquitination of proteins controlling the G1/S stages of cell division. The acidic C-terminus of the enzyme is required for this function, although there is little structural information providing details for a mechanism. One logical time point involving the C-terminus is the CDC34-Ub thiolester complex that precedes Ub transfer to a substrate. To examine this, we used a CDC34-Ub disulfide complex that structurally mimics the thiolester intermediate. NMR spectroscopy was used to show that the CDC34 C-terminus is disordered but can intramolecularly interact with the catalytically bound Ub. Using chemical shift perturbation analysis, we mapped two interacting regions on the surface of Ub in the CDC34-Ub complex. The first site comprises a hydrophobic patch (typical of other Ub complexes) that associates with the CDC34 catalytic domain. A novel second site, dependent on the C-terminus of CDC34, comprises a lysine-rich surface (K6, K11, K29, and K33) on the opposite face of Ub. Further, NMR experiments show that this interaction is described by two slowly exchanging states—a compact conformation where the C-terminus of CDC34 interacts with bound Ub and an extended structure where the C-terminus is released. This work provides the first structural details that show how the C-terminus of CDC34 might direct a thiolester-bound Ub to control polyubiquitin chain formation.     

Shiga-like toxin inhibition of FLICE-like inhibitory protein expression sensitizes endothelial cells to bacterial lipopolysaccharide-induced apoptosis

Shiga-like toxin (SLT) has been implicated in the pathogenesis of hemolytic uremic syndrome and its attendant endothelial cell (EC) injury. Key serotypes of Escherichia coli produce SLT-1 in addition to another highly pro-inflammatory molecule, lipopolysaccharide (LPS). It has previously been established that SLT-1 induces EC apoptosis and that LPS enhances this effect. LPS alone has no affect on human EC viability, and the mechanism for this enhancement remains unknown. In the present report, we demonstrate that SLT-1 sensitizes EC to LPS-induced apoptosis. Pretreatment with SLT-1 sensitized EC to LPS-induced apoptosis, whereas pretreatment with LPS did not influence SLT-1-induced apoptosis. SLT-1 exposure resulted in decreased expression of FLICE-like inhibitory protein (FLIP), an anti-apoptotic protein that has previously been shown to block LPS-induced apoptosis. This SLT-1-mediated decrease in FLIP expression preceded the onset of apoptosis elicited by SLT-1 alone or in combination with LPS. SLT-1-mediated decrements in FLIP expression correlated in a dose- and time-dependent manner with sensitization to LPS-induced apoptosis. Finally, transient or stable overexpression of FLIP protected against LPS enhancement of SLT-1-induced apoptosis, and this protection corresponded with sustained expression of FLIP. Together, these data suggest that SLT-1 sensitizes EC to LPS-induced apoptosis by inhibiting FLIP expression.     

Solution structure of the dimerization domain of the eukaryotic stalk P1/P2 complex reveals the structural organization of eukaryotic stalk complex

The lateral ribosomal stalk is responsible for the kingdom-specific binding of translation factors and activation of GTP hydrolysis during protein synthesis. The eukaryotic stalk is composed of three acidic ribosomal proteins P0, P1 and P2. P0 binds two copies of P1/P2 hetero-dimers to form a pentameric P-complex. The structure of the eukaryotic stalk is currently not known. To provide a better understanding on the structural organization of eukaryotic stalk, we have determined the solution structure of the N-terminal dimerization domain (NTD) of P1/P2 hetero-dimer. Helix-1, -2 and -4 from each of the NTD-P1 and NTD-P2 form the dimeric interface that buries 2200 A(2) of solvent accessible surface area. In contrast to the symmetric P2 homo-dimer, P1/P2 hetero-dimer is asymmetric. Three conserved hydrophobic residues on the surface of NTD-P1 are replaced by charged residues in NTD-P2. Moreover, NTD-P1 has an extra turn in helix-1, which forms extensive intermolecular interactions with helix-1 and -4 of NTD-P2. Truncation of this extra turn of P1 abolished the formation of P1/P2 hetero-dimer. Systematic truncation studies suggest that P0 contains two spine-helices that each binds one copy of P1/P2 hetero-dimer. Modeling studies suggest that a large hydrophobic cavity, which can accommodate the loop between the spine-helices of P0, can be found on NTD-P1 but not on NTD-P2 when the helix-4 adopts an 'open' conformation. Based on the asymmetric properties of NTD-P1/NTD-P2, a structural model of the eukaryotic P-complex with P2/P1:P1/P2 topology is proposed.     

Conserved amino acids at the C-terminus of rat phospholipase D1 are essential for enzymatic activity.

Rat brain phospholipase D1 (rPLD1) has two highly conserved motifs [H(X)K(X)4D, denoted HKD] located at the N-terminal and C-terminal halves, which are required for activity. Association of the two halves is essential for rPLD1 activity, which probably brings the two HKD domains together to form a catalytic center. In the present study, we find that an intact C-terminus is also essential for the catalytic activity of rPLD1. Serial deletion of the last four amino acids, EVWT, which are conserved in all mammalian PLD isoforms, abolished the catalytic activity of rPLD1. This loss of catalytic activity was not due to a lack of association of the N-terminal and C-terminal halves. Mutations of the last three amino acids showed that substitutions with charged or less hydrophobic amino acids all reduced PLD activity. For example, mutations of Thr1036 and Val1034 to Asp or Lys caused marked inactivation, whereas mutation to other amino acids had less effect. Mutation of Trp1035 to Leu, Ala, His or Tyr caused complete inactivation, whereas mutation of Glu1033 to Ala enhanced activity. The size of the amino acids at the C-terminus also affected the catalytic activity of PLD, reduced activity being observed with conservative mutations within the EVWT sequence (such as T/S, V/L or W/F). The enzyme was also inactivated by the addition of Ala or Val to the C-terminus of this sequence. Interestingly, the inactive C-terminal mutants could be complemented by cotransfection with a wild-type C-terminal half to restore PLD activity in vivo. These data demonstrate that the integrity of the C-terminus of rPLD1 is essential for its catalytic activity. Important features are the hydrophobicity, charge and size of the four conserved C-terminal amino acids. It is proposed that these play important roles in maintaining a functional catalytic structure by interacting with a specific domain within rPLD1.     

Preliminary structural studies of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi, a part of the P0/P1/P2 complex

The Trypanosoma cruzi ribosomal P0 protein (TcP0) is part of the ribosomal stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. The TcP0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by Trypanosoma cruzi infection. The structural properties of TcP0 have been explored by circular dichroism, tryptophan fluorescence and limited proteolysis experiments. These studies were complemented by secondary structure consensus prediction analysis. The results suggest that the tertiary structure of TcP0 could be described as a compact, stable, trypsin-resistant, 200 residues long N-terminal domain belonging to the alpha/beta class and a more flexible, degradable, helical, 123 residues long C-terminal domain which could be involved in the formation of an unusual hydrophobic zipper with the ribosomal P1/P2 proteins to form the P0/P1/P2 complex.     

The extraction and purification of a peptide from rat insulinoma tissue

A peptide was extracted and purified from rat insulinoma tissue which, although similar, was not identical to normal rat C peptides. The purity of the peptide, called rat insulinoma peptide (RIP), was investigated using polyacrylamide gel electrophoresis, isoelectric focusing and high-performance liquid chromatography. It appears to contain two peptides similar to each other but differing in their isoelectric points. The peptides as assessed by fast atom bombardment mass spectrometry have molecular masses in the region of 1982 Da, given a chain length of approx. 22 amino-acid residues. Evidence obtained using an established rat C peptides radioimmunoassay suggests that RIP shares a common C-terminus with rat C peptides. The antiserum produced to RIP was used to develop a radioimmunoassay using a tracer prepared by iodinating purified tyrosylated RIP.     

Electrostatic properties of the structure of the docking and dimerization domain of protein kinase A IIalpha.

The structure of the N-terminal docking and dimerization domain of the type IIalpha regulatory subunit (RIIalpha D/D) of protein kinase A (PKA) forms a noncovalent stand-alone X-type four-helix bundle structural motif, consisting of two helix-loop-helix monomers. RIIalpha D/D possesses a strong hydrophobic core and two distinct, exposed faces. A hydrophobic face with a groove is the site of protein-protein interactions necessary for subcellular localization. A highly charged face, opposite to the former, may be involved in regulation of protein-protein interactions as a result of changes in phosphorylation state of the regulatory subunit. Although recent studies have addressed the hydrophobic character of packing of RIIalpha D/D and revealed the function of the hydrophobic face as the binding site to A-kinase anchoring proteins (AKAPs), little attention has been paid to the charges involved in structure and function. To examine the electrostatic character of the structure of RIIalpha D/D we have predicted mean apparent pKa values, based on Poisson-Boltzmann electrostatic calculations, using an ensemble of calculated dimer structures. We propose that the helix promoting sequence Glu34-X-X-X-Arg38 stabilizes the second helix of each monomer, through the formation of a (i, i +4) side chain salt bridge. We show that a weak inter-helical hydrogen bond between Tyr35-Glu19 of each monomer contributes to tertiary packing and may be responsible for discriminating from alternative quaternary packing of the two monomers. We also show that an inter-monomer hydrogen bond between Asp30-Arg40 contributes to quaternary packing. We propose that the charged face comprising of Asp27-Asp30-Glu34-Arg38-Arg40-Glu41-Arg43-Arg44 may be necessary to provide flexibility or stability in the region between the C-terminus and the interdomain/autoinhibitory sequence of RIIalpha, depending on the activation state of PKA. We also discuss the structural requirements necessary for the formation of a stacked (rather than intertwined) dimer, which has consequences for the orientation of the functionally important and distinct faces.     

Substrate specificity of a maize ribosome-inactivating protein differs across diverse taxa.

The superfamily of ribosome-inactivating proteins (RIPs) consists of toxins that catalytically inactivate ribosomes at a universally conserved region of the large ribosomal RNA. RIPs carry out a single N-glycosidation event that alters the binding site of the translational elongational factor eEF1A and causes a cessation of protein synthesis that leads to subsequent cell death. Maize RIP1 is a kernel-specific RIP with the unusual property of being produced as a zymogen, proRIP1. ProRIP1 accumulates during seed development and becomes active during germination when cellular proteases remove acidic residues from a central domain and both termini. These deletions also result in RIP activation in vitro. However, the effectiveness of RIP1 activity against target ribosomes remains species-dependent. To determine the potential efficiency of maize RIP1 as a plant defense protein, we used quantitative RNA gel blots to detect products of RIP activity against intact ribosomal substrates from various species. We determined the enzyme specificity of recombinant maize proRIP1 (rproRIP1), papain-activated rproRIP1 and MOD1 (an active deletion mutant of rproRIP1) against ribosomal substrates with differing levels of RIP sensitivity. The rproRIP1 had no detectable enzymatic activity against ribosomes from any of the species assayed. The papain-activated rproRIP1 was more active than MOD1 against ribosomes from either rabbit or the corn pathogen, Aspergillus flavus, but the difference was much more marked when rabbit ribosomes were used as a substrate. The papain-activated rproRIP1 was much more active against rabbit ribosomes than homologous Zea mays ribosomes and had no detectable effect on Escherichia coli ribosomes.     

Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses

Trichosanthin (TCS) is a type I ribosome-inactivating protein that inactivates ribosome by enzymatically depurinating the A⁴³²⁴ at the α-sarcin/ricin loop of 28S rRNA. We have shown in this and previous studies that TCS interacts with human acidic ribosomal proteins P0, P1 and P2, which constitute the lateral stalk of eukaryotic ribosome. Deletion mutagenesis showed that TCS interacts with the C-terminal tail of P2, the sequences of which are conserved in P0, P1 and P2. The P2-binding site on TCS was mapped to the C-terminal domain by chemical shift perturbation experiments. Scanning charge-to-alanine mutagenesis has shown that K173, R174 and K177 in the C-terminal domain of TCS are involved in interacting with the P2, presumably through forming charge–charge interactions to the conserved DDD motif at the C-terminal tail of P2. A triple-alanine variant K173A/R174A/K177A of TCS, which fails to bind P2 and ribosomal stalk in vitro, was found to be 18-fold less active in inhibiting translation in rabbit reticulocyte lysate, suggesting that interaction with P-proteins is required for full activity of TCS. In an analogy to the role of stalk proteins in binding elongation factors, we propose that interaction with acidic ribosomal stalk proteins help TCS to locate its RNA substrate.     

Structural analysis of papain-like NlpC/P60 superfamily enzymes with a circularly permuted topology reveals potential lipid binding sites

NlpC/P60 superfamily papain-like enzymes play important roles in all kingdoms of life. Two members of this superfamily, LRAT-like and YaeF/YiiX-like families, were predicted to contain a catalytic domain that is circularly permuted such that the catalytic cysteine is located near the C-terminus, instead of at the N-terminus. These permuted enzymes are widespread in virus, pathogenic bacteria, and eukaryotes. We determined the crystal structure of a member of the YaeF/YiiX-like family from Bacillus cereus in complex with lysine. The structure, which adopts a ligand-induced, "closed" conformation, confirms the circular permutation of catalytic residues. A comparative analysis of other related protein structures within the NlpC/P60 superfamily is presented. Permutated NlpC/P60 enzymes contain a similar conserved core and arrangement of catalytic residues, including a Cys/His-containing triad and an additional conserved tyrosine. More surprisingly, permuted enzymes have a hydrophobic S1 binding pocket that is distinct from previously characterized enzymes in the family, indicative of novel substrate specificity. Further analysis of a structural homolog, YiiX (PDB 2if6) identified a fatty acid in the conserved hydrophobic pocket, thus providing additional insights into possible function of these novel enzymes.     

The RIP-like kinase, RIP3, induces apoptosis and NF-kappaB nuclear translocation and localizes to mitochondria

A RIP-like protein, RIP3, has recently been reported that contains an N-terminal kinase domain and a novel C-terminal domain that promotes apoptosis. These experiments further characterize RIP3-mediated apoptosis and NF-kappaB activation. Northern blots indicate that rip3 mRNA displays a restricted pattern of expression including regions of the adult central nervous system. The rip3 gene was localized by fluorescent in situ hybridization to human chromosome 14q11.2, a region frequently altered in several types of neoplasia. RIP3-mediated apoptosis was inhibited by Bcl-2, Bcl-x(L), dominant-negative FADD, as well as the general caspase inhibitor Z-VAD. Further dissection of caspase involvement in RIP3-induced apoptosis indicated inhibition by the more specific inhibitors Z-DEVD (caspase-3, -6, -7, -8, and -10) and Z-VDVAD (caspase-2). However, caspase-1, -6, -8 and -9 inhibitors had little or no effect on RIP3-mediated apoptosis. Mutational analysis of RIP3 revealed that the C-terminus of RIP3 contributed to its apoptotic activity. This region is similar, but distinct, to the death domain found in many pro-apoptotic receptors and adapter proteins, including FAS, FADD, TNFR1, and RIP. Furthermore, point mutations of RIP3 at amino acids conserved among death domains, abrogated its apoptotic activity. RIP3 was localized by immunofluorescence to the mitochondrion and may play a key role in the mitochondrial disruptions often associated with apoptosis.     

Pentameric organization of the ribosomal stalk accelerates recruitment of ricin a chain to the ribosome for depurination

Ribosome inactivating proteins (RIPs) depurinate a universally conserved adenine in the α-sarcin/ricin loop (SRL) and inhibit protein synthesis at the translation elongation step. We previously showed that ribosomal stalk is required for depurination of the SRL by ricin toxin A chain (RTA). The interaction between RTA and ribosomes was characterized by a two-step binding model, where the stalk structure could be considered as an important interacting element. Here, using purified yeast ribosomal stalk complexes assembled in vivo, we show a direct interaction between RTA and the isolated stalk complex. Detailed kinetic analysis of these interactions in real time using surface plasmon resonance (SPR) indicated that there is only one type of interaction between RTA and the ribosomal stalk, which represents one of the two binding steps of the interaction with ribosomes. Interactions of RTA with the isolated stalk were relatively insensitive to salt, indicating that nonelectrostatic interactions were dominant. We compared the interaction of RTA with the full pentameric stalk complex containing two pairs of P1/P2 proteins with its interaction with the trimeric stalk complexes containing only one pair of P1/P2 and found that the rate of association of RTA with the pentamer was higher than with either trimer. These results demonstrate that the stalk is the main landing platform for RTA on the ribosome and that pentameric organization of the stalk accelerates recruitment of RTA to the ribosome for depurination. Our results suggest that multiple copies of the stalk proteins might also increase the scavenging ability of the ribosome for the translational GTPases.     

Importance of a flexible hinge near the motor domain in kinesin-driven motility.

Conventional kinesin is a molecular motor consisting of an N-terminal catalytic motor domain, an extended stalk and a small globular C-terminus. Whereas the structure and function of the catalytic motor domain has been investigated, little is known about the function of domains outside the globular head. A short coiled-coil region adjacent to the motor domain, termed the neck, is known to be important for dimerization and may be required for kinesin processivity. We now provide evidence that a helix-disrupting hinge region (hinge 1) that separates the neck from the first extended coiled-coil of the stalk plays an essential role in basic motor activity. A fast fungal kinesin from Syncephalastrum racemosum was used for these studies. Deletion, substitution by a coiled-coil and truncation of the hinge 1 region all reduce motor speed and uncouple ATP turnover from gliding velocity. Insertion of hinge 1 regions from two conventional kinesins, Nkin and DmKHC, fully restores motor activity, whereas insertion of putative flexible linkers of other proteins does not, suggesting that hinge 1 regions of conventional kinesins can functionally replace each other. We suggest that this region is essential for kinesin movement in its promotion of chemo-mechanical coupling of the two heads and therefore the functional motor domain should be redefined to include not only the catalytic head but also the adjacent neck and hinge 1 domains.     

Mutation of the toxin binding site of PP-1c: comparison with PP-2B

The catalytic cores of PP-1c and PP-2B (calcineurin) are structurally conserved. However, PP-2B is resistant to inhibition by toxins of the okadaic acid and cyclic peptide classes, while PP-1c is potently inhibited. Molecular docking of the structure of microcystin-LR onto the catalytic core of PP-2B identified residues that may be responsible for blocking access of toxins to the catalytic site. Amino acids in PP-1c were substituted with these PP-2B residues to investigate their contribution to PP-2B toxin resistance. Mutants of PP-1c were also produced to test the importance of hydrophobic interactions to toxin binding. Our results suggest that different classes of toxin inhibitors interact with the same hydrophobic side chains of PP-1c through different mechanisms. Substitution of amino acids in PP-1c with PP-2B residues demonstrated no highly significant changes in toxin inhibition. We hypothesize that an interaction outside the catalytic core causing the L7 loop of PP-2B to block the catalytic site may be responsible for PP-2B resistance to toxins.