Turn Motif Research Articles

Regulation of the Interaction between Protein Kinase C-related Protein Kinase 2 (PRK2) and Its Upstream Kinase, 3-Phosphoinositide-dependent Protein Kinase 1 (PDK1)

The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do not apply for other AGC kinases.

Regulation of protein kinase Cδ downregulation by protein kinase Cε and mammalian target of rapamycin complex 2

Phosphorylation and dephosphorylation of PKCs can regulate their activity, stability and function. We have previously shown that downregulation of PKCδ by tumor promoting phorbol esters was compromised when HeLa cells acquired resistance to cisplatin (HeLa/CP). In the present study, we have used these cells to understand the mechanism of PKCδ downregulation. A brief treatment of HeLa cells with phorbol 12,13-dibutyrate (PDBu) induced phosphorylation of PKCδ at the activation loop (Thr505), turn motif (Ser643), hydrophobic motif (Ser662) and Tyr-311 sites to a greater extent in HeLa/CP cells compared to HeLa cells. Prolonged treatment with PDBu led to downregulation of PKCδ in HeLa but not in HeLa/CP cells. The PKC inhibitor Gö 6983 inhibited PDBu-induced downregulation of PKCδ, decreased Thr505 phosphorylation and increased PKCδ tyrosine phosphorylation at Tyr-311 site. However, knockdown of c-Abl, c-Src, Fyn and Lyn had little effect on PKCδ downregulation and Tyr311 phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor Ly294002 and mTOR inhibitor rapamycin restored the ability of PDBu to downregulate PKCδ in HeLa/CP cells. Knockdown of mTOR and rictor but not raptor facilitated PKCδ downregulation. Depletion of PKCε also enhanced PKCδ downregulation by PDBu. These results suggest that downregulation of PKCδ is regulated by PKCε and mammalian target of rapamycin complex 2 (mTORC2).

The β-d-Glucose Scaffold as a β-Turn Mimetic

Activity and selectivity are typically the first considerations when designing a drug. However, absorption, distribution, metabolism, excretion, and toxicity (ADMET) are equally important considerations. Peptides can provide a combination of potent binding and exquisite selectivity, as evidenced by their pervasive use as enzymes, hormones, and signaling agents within living systems. In particular, peptidic turn motifs are key elements of molecular recognition. They may be found at the exposed surfaces of globular proteins, where they are available for binding interactions with other peptides and small molecules. However, despite these advantages, peptides often make poor drugs. The amide backbone is subject to rapid enzymatic proteolysis, resulting in short half-lives. Furthermore, the ability of the amide backbone to hydrogen bond with water restricts its ability to cross membranes and, consequentially, results in poor oral bioavailability. Accordingly, the development of nonpeptidic scaffolds that mimic peptidic turn motifs represents a promising means of converting peptidic agents into more drugable molecules. In this Account, we describe the design and synthesis of beta-turn mimetics that use a beta-D-glucose scaffold, the first use of a sugar scaffold for this purpose. Somatostatin (SRIF) is a small protein (14 amino acid residues) human hormone; a shorter (6 amino acid residues) synthetic peptide, L-363,301, is a fully peptidal agonist. These two cyclic peptides share the beta-turn motif comprising Phe(7)-Trp(8)-Lys(9)-Thr(10) (d-Trp(8) in the case of L-363,301), of which the tryptophan and lysine residues in the i + 1 and i + 2 positions, respectively, are critical for binding. In 1988, we initiated a program that tested and validated the then-novel proposition that the beta-D-glucose scaffold can mimic the beta-turn in L-363,301. The beta-D-glucose scaffold proved to be an attractive mimic of a beta-turn in part because it permits the convenient attachment of amino acid side chains via facile etherification reactions, rather than carbon-carbon bond formations; it is also an inexpensive starting material with well-defined stereochemistry. From the beginning, biological assays were used alongside physical measurements to assess the relevance of the design. Our first two synthetic targets, compounds 6 and 7, bound the SRIF receptors on benchmark (AtT-20) cells, albeit weakly, consistent with the objective of the design. Subsequently, a better ligand (8) and two congeners were found to be agonists at the SRIF receptors, providing convincing evidence that the peptide backbone is not required for receptor binding or signal transduction. The unexpectedly high level of receptor affinity of selected analogs, as well as the fortuitous discovery that our peptidomimetics were active against several chemically distinct receptors, led us to hypothesize that these monosaccharides could access multiple potential binding modes. Our later studies of this sugar scaffold confirmed this property, which we termed pseudosymmetry, whereby multiple similar but nonidentical motifs are displayed within a single analog. We propose the presence of pseudosymmetry to be an element of privilege and an advantage for lead discovery.

A chimeric mechanism for polyvalent trans‐phosphorylation of PKA by PDK1

Phosphorylation on the activation loop of AGC kinases is typically mediated by PDK1. The precise mechanism for this in-trans phosphorylation is unknown; however, docking of a hydrophobic (HF) motif in the C-tail of the substrate kinase onto the N-lobe of PDK1 is likely an essential step. Using a peptide array of PKA to identify other PDK1-interacting sites, we discovered a second AGC-conserved motif in the C-tail that interacts with PDK1. Since this motif [FD(X)(1-2)Y/F] lies in the active site tether region and in PKA contributes to ATP binding, we call it the Adenosine binding (Ade) motif. The Ade motif is conserved as a PDK1-interacting site in Akt and PRK2, and we predict it will be a PDK1-interacting site for most AGC kinases. In PKA, the HF motif is only recognized when the turn motif Ser338 is phosphorylated, possibly serving as a phosphorylation "switch" that regulates how the Ade and HF motifs interact with PDK1. These results demonstrate that the extended AGC C-tail serves as a polyvalent element that trans-regulates PDK1 for catalysis. Modeling of the PKA C-tail onto PDK1 structure creates two chimeric sites; the ATP binding pocket, which is completed by the Ade motif, and the C-helix, which is positioned by the HF motif. Together, they demonstrate substrate-assisted catalysis involving two kinases that have co-evolved as symbiotic partners. The highly regulated turn motifs are the most variable part of the AGC C-tail. Elucidating the highly regulated cis and trans functions of the AGC tail is a significant future challenge.

Rescue of atypical protein kinase C in epithelia by the cytoskeleton and Hsp70 family chaperones

Atypical PKC (PKC iota) is a key organizer of cellular asymmetry. Sequential extractions of intestinal cells showed a pool of enzymatically active PKC iota and the chaperone Hsp70.1 attached to the apical cytoskeleton. Pull-down experiments using purified and recombinant proteins showed a complex of Hsp70 and atypical PKC on filamentous keratins. Transgenic animals overexpressing keratin 8 displayed delocalization of Hsp70 and atypical PKC. Two different keratin-null mouse models, as well as keratin-8 knockdown cells in tissue culture, also showed redistribution of Hsp70 and a sharp decrease in the active form of atypical PKC, which was also reduced by Hsp70 knockdown. An in-vitro turn motif rephosphorylation assay indicated that PKC iota is dephosphorylated by prolonged activity. The Triton-soluble fraction could rephosphorylate PKC iota only when supplemented with the cytoskeletal pellet or filamentous highly purified keratins, a function abolished by immunodepletion of Hsp70 but rescued by recombinant Hsp70. We conclude that both filamentous keratins and Hsp70 are required for the rescue rephosphorylation of mature atypical PKC, regulating the subcellular distribution and steady-state levels of active PKC iota.

Mechanism of Activation and Functional Role of Protein Kinase Cη in Human Platelets

The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCeta using pharmacological and gene knock-out approaches. nPKCeta was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKCeta. Similarly, ADP failed to activate nPKCeta in platelets isolated from P2Y1 and G(q) knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCeta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCeta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(IIb)beta3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a alpha(IIb)beta3 receptor antagonist, nPKCeta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cgamma, a catalytic subunit of serine/threonine phosphatase, alpha(IIb)beta3 failed to dephosphorylate nPKCeta. Thus, we conclude that ADP activates nPKCeta via P2Y1 receptor and is subsequently dephosphorylated by PP1gamma phosphatase activated by alpha(IIb)beta3 integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKCeta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCeta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.

Structure of dystrophia myotonica protein kinase

Dystrophia myotonica protein kinase (DMPK) is a serine/threonine kinase composed of a kinase domain and a coiled-coil domain involved in the multimerization. The crystal structure of the kinase domain of DMPK bound to the inhibitor bisindolylmaleimide VIII (BIM-8) revealed a dimeric enzyme associated by a conserved dimerization domain. The affinity of dimerisation suggested that the kinase domain alone is insufficient for dimerisation in vivo and that the coiled-coil domains are required for stable dimer formation. The kinase domain is in an active conformation, with a fully-ordered and correctly positioned alphaC helix, and catalytic residues in a conformation competent for catalysis. The conserved hydrophobic motif at the C-terminal extension of the kinase domain is bound to the N-terminal lobe of the kinase domain, despite being unphosphorylated. Differences in the arrangement of the C-terminal extension compared to the closely related Rho-associated kinases include an altered PXXP motif, a different conformation and binding arrangement for the turn motif, and a different location for the conserved NFD motif. The BIM-8 inhibitor occupies the ATP site and has similar binding mode as observed in PDK1.

The Chaperones Hsp90 and Cdc37 Mediate the Maturation and Stabilization of Protein Kinase C through a Conserved PXXP Motif in the C-terminal Tail*♦

The life cycle of protein kinase C (PKC) is tightly controlled by mechanisms that mature the enzyme, sustain the activation-competent enzyme, and degrade the enzyme. Here we show that a conserved PXXP motif (Kannan, N., Haste, N., Taylor, S. S., and Neuwald, A. F. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 1272-1277), in the C-terminal tail of AGC (c-AMP-dependent protein kinase/protein kinase G/protein kinase C) kinases, controls the processing phosphorylation of conventional and novel PKC isozymes, a required step in the maturation of the enzyme into a signaling-competent species. Mutation of both Pro-616 and Pro-619 to Ala in the conventional PKC betaII abolishes the phosphorylation and activity of the kinase. Co-immunoprecipitation studies reveal that conventional and novel, but not atypical, PKC isozymes bind the chaperones Hsp90 and Cdc37 through a PXXP-dependent mechanism. Inhibitors of Hsp90 and Cdc37 significantly reduce the rate of processing phosphorylation of PKC. Of the two C-terminal sites processed by phosphorylation, the hydrophobic motif, but not the turn motif, is regulated by Hsp90. Overlay of purified Hsp90 onto a peptide array containing peptides covering the catalytic domain of PKC betaII identified regions surrounding the PXXP segment, but not the PXXP motif itself, as major binding determinants for Hsp90. These Hsp90-binding regions, however, are tethered to the C-terminal tail via a "molecular clamp" formed between the PXXP motif and a conserved Tyr (Tyr-446) in the alphaE-helix. Disruption of the clamp by mutation of the Tyr to Ala recapitulates the phosphorylation defect of mutating the PXXP motif. These data are consistent with a model in which a molecular clamp created by the PXXP motif in the C-terminal tail and determinants in the alphaE-helix of the catalytic domain allows the chaperones Hsp90 and Cdc37 to bind newly synthesized PKC, a required event in the processing of PKC by phosphorylation.

The C-terminus of PRK2/PKNγ is required for optimal activation by RhoA in a GTP-dependent manner

PRK2/PKNγ is a Rho effector and a member of the protein kinase C superfamily of serine/threonine kinases. Here, we explore the structure–function relationship between various motifs in the C-terminal half of PRK2 and its kinase activity and regulation. We report that two threonine residues at conserved phosphoacceptor position in the activation loop and the turn motif are essential for the catalytic activity of PRK2, but the phosphomimetic Asp-978 at hydrophobic motif is dispensable for kinase catalytic competence. Moreover, the PRK2-Δ958 mutant with the turn motif truncated still interacts with 3-phosphoinositide-dependent kinase-1 (PDK-1). Thus, both the intact hydrophobic motif and the turn motif in PRK2 are dispensable for the binding of PDK-1. We also found that while the last seven amino acid residues at the C-terminus of PRK2 are not required for the activation of the kinase by RhoA in vitro, however, the extreme C-terminal segment is critical for the full activation of PRK2 by RhoA in cells in a GTP-dependent manner. Our data suggest that the extreme C-terminus of PRK2 may represent a potential drug target for effector-specific pharmacological intervention of Rho-medicated biological processes.

Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling.

Protein kinase C (PKC) is involved in a wide array of cellular processes such as cell proliferation, differentiation and apoptosis. Phosphorylation of both turn motif (TM) and hydrophobic motif (HM) are important for PKC function. Here, we show that the mammalian target of rapamycin complex 2 (mTORC2) has an important function in phosphorylation of both TM and HM in all conventional PKCs, novel PKCepsilon as well as Akt. Ablation of mTORC2 components (Rictor, Sin1 or mTOR) abolished phosphorylation on the TM of both PKCalpha and Akt and HM of Akt and decreased HM phosphorylation of PKCalpha. Interestingly, the mTORC2-dependent TM phosphorylation is essential for PKCalpha maturation, stability and signalling. Our study demonstrates that mTORC2 is involved in post-translational processing of PKC by facilitating TM and HM phosphorylation and reveals a novel function of mTORC2 in cellular regulation.

The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C

The target of rapamycin (TOR), as part of the rapamycin-sensitive TOR complex 1 (TORC1), regulates various aspects of protein synthesis. Whether TOR functions in this process as part of TORC2 remains to be elucidated. Here, we demonstrate that mTOR, SIN1 and rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl-terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain. Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination-mediated proteasome degradation. Finally, we demonstrate that mTORC2 independently controls the Akt TM and HM sites in vivo and can directly phosphorylate both sites in vitro. Our studies uncover a novel function of the TOR pathway in regulating protein folding and stability, processes that are most likely linked to the functions of TOR in protein synthesis.

Phosphorylation of the Human Full-Length Protein Kinase Cι

Atypical protein kinases C, including protein kinase Ciota (PKCiota), play critical roles in signaling pathways that control cell growth, differentiation and survival. This qualifies them as attractive targets for development of novel therapeutics for the treatment of various human diseases. In this study, the full-length PKCiota was expressed in Sf9 insect cells, purified, and digested with trypsin and endoproteinase Asp-N, and its phosphorylation analyzed by liquid chromatography-high accuracy mass spectrometry. This strategy mapped 97% of the PKCiota protein sequence and revealed seven new Ser/Thr phosphorylation sites, in addition to the two previously known, pThr403 in the activation loop and pThr555 in the turn motif of the kinase domain. Most of the newly identified phosphorylation sites had low estimated occupancies (below 2%). Two phosphorylation sites were located in domain connecting amino acid sequence stretches (pSer217 and pSer237/pSer238) and may contribute to an improved stability and solubility of the protein. The most interesting new phosphorylation site was detected in a well-accessible loop of the PB1 domain (pSer35/pSer37) and may be involved in the interactions of the PB1 domain with different partners in the relevant signaling pathways.

Crystal structure of the Clostridium limosum C3 exoenzyme

C3-like toxins ADP-ribosylate and inactivate Rho GTPases. Seven C3-like ADP-ribosyltransferases produced by Clostridium botulinum, Clostridium limosum, Bacillus cereus and Staphylococcus aureus were identified and two representatives – C3bot from C. botulinum and C3stau2 from S. aureus – were crystallized. Here we present the 1.8 Å structure of C. limosum C3 transferase C3lim and compare it to the structures of other family members. In contrast to the structure of apo-C3bot, the canonical ADP-ribosylating turn turn motif is observed in a primed conformation, ready for NAD binding. This suggests an impact on the binding mode of NAD and on the transferase reaction. The crystal structure explains why auto-ADP-ribosylation of C3lim at Arg41 interferes with the ADP-ribosyltransferase activity of the toxin.

The Phosphatase PHLPP Controls the Cellular Levels of Protein Kinase C

The life cycle of protein kinase C (PKC) is controlled by multiple phosphorylation and dephosphorylation steps. The maturation of PKC requires three ordered phosphorylations, one at the activation loop and two at COOH-terminal sites, the turn motif and the hydrophobic motif, to yield a stable and signaling-competent enzyme. Dephosphorylation of the enzyme leads to protein degradation. We have recently discovered a novel family of protein phosphatases named PH domain leucine-rich repeat protein phosphatase (PHLPP) whose members terminate Akt signaling by dephosphorylating the hydrophobic motif on Akt. Here we show that the two PHLPP isoforms, PHLPP1 and PHLPP2, also dephosphorylate the hydrophobic motif on PKC betaII, an event that shunts PKC to the detergent-insoluble fraction, effectively terminating its life cycle. Deletion mutagenesis reveals that the PH domain is necessary for the effective dephosphorylation of PKC betaII by PHLPP in cells, whereas the PDZ-binding motif, required for Akt regulation, is dispensable. The phorbol ester-mediated dephosphorylation of the hydrophobic site, but not the turn motif or activation loop, is insensitive to okadaic acid, consistent with PHLPP, a PP2C family member, controlling the hydrophobic site. In addition, knockdown of PHLPP expression reduces the rate of phorbol ester-triggered dephosphorylation of the hydrophobic motif, but not turn motif, of PKC alpha. Last, we show that depletion of PHLPP in colon cancer and normal breast epithelial cells results in an increase in conventional and novel PKC levels. These data reveal that PHLPP controls the cellular levels of PKC by specifically dephosphorylating the hydrophobic motif, thus destabilizing the enzyme and promoting its degradation.

A Logical OR Redundancy within the Asx-Pro-Asx-Gly Type I β-Turn Motif

Turn secondary structure is essential to the formation of globular protein architecture. Turn structures are, however, much more complex than either α-helix or β-sheet, and the thermodynamics and folding kinetics are poorly understood. Type I β-turns are the most common type of reverse turn, and they exhibit a statistical consensus sequence of Asx-Pro-Asx-Gly (where Asx is Asp or Asn). A comprehensive series of individual and combined Asx mutations has been constructed within three separate type I 3:5 G1 bulge β-turns in human fibroblast growth factor-1, and their effects on structure, stability, and folding have been determined. The results show a fundamental logical OR relationship between the Asx residues in the motif, involving H-bond interactions with main-chain amides within the turn. These interactions can be modulated by additional interactions with residues adjacent to the turn at positions i+4 and i+6. The results show that the Asx residues in the turn motif make a substantial contribution to the overall stability of the protein, and the Asx logical OR relationship defines a redundant system that can compensate for deleterious point mutations. The results also show that the stability of the turn is unlikely to be the prime determinant of formation of turn structure in the folding transition state.

Discrete functional motifs reside within the cytoplasmic tail of αV integrin subunit

Previous studies have demonstrated that cell-permeable cytoplasmic tail (CT) alpha(II)beta peptides can modulate the activation of alpha(IIb)b(3). As alpha(V) CT contains an alpha(II)beta homologous region, a series of cell-permeable alpha(V) and alpha(IIb) peptides were generated to determine if alpha(V) CT can modulate the activation of beta(3) integrins in comparison to alpha(IIb), and to identify the minimal bioactive sequences in alpha(V) CT. Using NMR structures and molecular models as guides, the initial peptides for study encompassed the alpha(II)beta homologous sequences of alpha(V) CT (alpha(V)(987-1006); V-1), its amino-terminus (alpha(V)(987-993); V-2), a turn motif (alpha(V)(993-1001); V-3), the carboxyl-terminus (alpha(V)(999-1006); V-4), and corresponding homologous alpha(IIb) peptides. Treatment of platelets and alpha(V)beta(3)-expressing cells with the peptides revealed that IIb-1 inhibited alpha(IIb)beta(3) activation and V-1 inhibited alpha(V)beta(3) activation, but not vice versa. The inhibitory capacity of these peptides was mapped to the central turn-motif region which was encompassed by V-3, but only partially by IIb-3. V-2 and IIb-2 activated both beta(3) integrins, while V-4 and IIb-4 were inactive. The use of truncation and mutant peptides confirmed the importance of the turn motif for inhibitory activity and identified the side-chain of alpha(V)(Q1001) as a critical inhibitory residue. The difference in the integrin inhibitory capacity of alpha(V) and alpha(IIb) peptides and their capacity to influence the assembly of kinases with integrin CTs, reveals a possible divergence in the regulatory control of the two beta(3) integrins.

Identification of residues of functional importance within the central turn motifs present in the cytoplasmic tails of integrin α IIb and α V subunits

Introduction Previous studies demonstrated that cell-permeable α IIb cytoplasmic peptides can modulate the activation of α IIbβ 3. An integrin activation motif was mapped to its membrane proximal region and a double proline mutant peptide and receptor indicated that its central turn motif had inhibitory capacity. However, the residues critical for inhibition of α IIbβ 3 activation were not identified. Using central turn peptides derived from α IIb and α V, residues critical for suppression of integrin activation were identified and the importance of these residues in protein–protein interactions was assessed. Materials and methods Cell-permeable peptides were used to determine the capacity of the central turn peptides to suppress α IIbβ 3 and α Vβ 3 activation. Far Western analysis was used to characterize the capacity of the peptides to interact with CIB1 and surface plasmon resonance was used to characterize the binding of an antibody to the cytoplasmic tails of α IIb and α V. Results and conclusions The central turn peptide from α V, α V(993–1001), has full inhibitory capacity while that derived from α IIb requires additional residues located adjacent to α IIb(995–1003). Within these two sequences there is a switch in the position of an asparaginine and leucine residue for a valine and glutamine (α IIb, RNRPPLEED; α V, RVRPPQEEQ). This switch had a dramatic effect on their inhibitory capacity and on protein–protein interactions. The two arginine and glutamic residues, juxtapositioned at identical locations in both subunits, appeared to be important in specifying the orientation by which proteins can dock to this region in α IIb and α V.

β-Strand Flipping and Slipping Triggered by Turn Replacement Reveal the Opportunistic Nature of β-Strand Pairing

We investigated how the register between adjacent beta-strands is specified using a series of mutants of the single-layer beta-sheet (SLB) in Borrelia OspA. The single-layer architecture of this system eliminates structural restraints imposed by a hydrophobic core, enabling us to address this question. A critical turn (turn 9/10) in the SLB was replaced with a segment with an intentional structural mismatch. Its crystal structure revealed a one-residue insertion into the central beta-strand (strand 9) of the SLB. This insertion triggered a surprisingly large-scale structural rearrangement: (i) the central strand (strand 9) was shifted by one residue, causing the strand to flip with respect to the adjacent beta-strands and thus completely disrupting the native side-chain contacts; (ii) the three-residue turn located on the opposite end of the beta-strand (turn 8/9) was pushed into its preceding beta-strand (strand 8); (iii) the register between strands 8 and 9 was shifted by three residues. Replacing the original sequence for turn 8/9 with a stronger turn motif restored the original strand register but still with a flipped beta-strand 9. The stability differences of these distinct structures were surprisingly small, consistent with an energy landscape where multiple low-energy states with different beta-sheet configurations exist. The observed conformations can be rationalized in terms of maximizing the number of backbone H-bonds. These results suggest that adjacent beta-strands "stick" through the use of factors that are not highly sequence specific and that beta-strands could slide back and forth relatively easily in the absence of external elements such as turns and tertiary packing.

Structural and functional characterization of the porcine proline–rich antifungal peptide SP‐B isolated from salivary gland granules

A 1905-Da cationic proline-rich peptide, named SP-B, was recently isolated by our group as the main component of salivary gland granules, and its primary sequence fully characterized by means of automated Edman sequencing and LC-MS/MS tools. In the present study SP-B is shown to possess antifungal activity when challenged with strains of Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, while only negligible antibacterial activity was detected. Furthermore, SP-B was found to be non-cytotoxic when tested on fibroblast cell lines. To obtain information regarding its structure affinity, capillary electrophoresis (CE), circular dichroism (CD) and attenuated total reflection (ATR)-FT/IR experiments were performed. CE revealed a pH dependence of the hydrodynamic radial dimensions both in aqueous and 2,2,2-trifluoroethanol solutions. CD and ATR-FT/IR measurements confirmed the structure-pH relationship, revealing a secondary structure composed of mixed proportions of polyproline-II, unordered and turn motifs, the last being more evident in the zwitterionic form of the peptide. From these findings SP-B peptide could be classified as a new member of the proline-rich antimicrobial peptide family.

Identification of Acidic Amino Acid Residues in the Protein Kinase Cα V5 Domain That Contribute to Its Insensitivity to Diacylglycerol

The protein kinase C (PKC) isoforms are maintained in an inactive and closed conformation by intramolecular interactions. Upon activation these are disrupted by activators, binding proteins and cellular membrane. We have seen that autophosphorylation of two sites in the C-terminal V5 domain is crucial to keep PKC alpha insensitive to the activator diacylglycerol, which presumably is caused by a masking of the diacylglycerol-binding C1a domain. Here we demonstrate that the diacylglycerol sensitivity of the PKC beta isoforms also is suppressed by autophosphorylation of the V5 sites. To analyze conformational differences, a fusion protein ECFP-PKC alpha-EYFP was expressed in cells and the FRET signal was analyzed. The analogous mutant with autophosphorylation sites exchanged for alanine gave rise to a substantially lower FRET signal than wild-type PKC alpha indicating a conformational difference elicited by the mutations. Expression of the isolated PKC alpha V5 domain led to increased diacylglycerol sensitivity of PKC alpha. We identified acidic residues in the V5 domain that, when mutated to alanines or lysines, rendered PKC alpha sensitive to diacylglycerol. Furthermore, mutation to glutamate of four lysines in a lysine-rich cluster in the C2 domain gave a similar effect. Simultaneous reversal of the charges of the acidic residues in the V5 and the lysines in the C2 domain gave rise to a PKC alpha that was insensitive to diacylglycerol. We propose that these structures participate in an intramolecular interaction that maintains PKC alpha in a closed conformation. The disruption of this interaction leads to an unmasking of the C1a domain and thereby increased diacylglycerol sensitivity of PKC alpha.