The deubiquitinating enzyme USP24 is a regulator of the UV damage response.

Regulation of p53 by ubiquitination and deubiquitination is important for its function. In this study, we demonstrate that USP24 deubiquitinates p53 in human cells. Functional USP24 is required for p53 stabilization, and p53 destabilization in USP24-depleted cells can be corrected by the forced expression of USP24. We show that USP24 depletion renders cells resistant to apoptosis after UV irradiation, consistent with the requirement of USP24 for p53 stabilization and PUMA activation in vivo. Additionally, purified USP24 protein is able to cleave ubiquitinated p53 in vitro. Importantly, cells with USP24 depletion exhibited significantly elevated mutation rates at the endogenous HPRT locus, implying an important role for USP24 in maintaining genome stability. Our data reveal that the USP24 deubiquitinase regulates the DNA damage response by directly targeting the p53 tumor suppressor.

Growth of a bacterium that apparently uses arsenic instead of phosphorus is a consequence of massive ribosome breakdown.

A recent study (Wolfe-Simon, F., Switzer Blum, J., Kulp, T. R., Gordon, G. W., Hoeft, S. E., Pett-Ridge, J., Stolz, J. F., Webb, S. M., Weber, P. K., Davies, P. C., Anbar, A. D., and Oremland, R. S. (2011) Science 332, 1163-1166) described the isolation of a special bacterial strain, GFAJ-1, that could grow in medium containing arsenate, but lacking phosphate, and that supposedly could substitute arsenic for phosphorus in its biological macromolecules. Here, we provide an alternative explanation for these observations and show that they can be reproduced in a laboratory strain of Escherichia coli. We find that arsenate induces massive ribosome degradation, which provides a source of phosphate. A small number of arsenate-tolerant cells arise during the long lag period prior to initiation of growth in +As/-P medium, and it is this population that undergoes the very slow, limited growth observed for both E. coli and GFAJ-1. These results provide a simple explanation for the reported growth of GFAJ-1 in arsenate without invoking replacement of phosphorus by arsenic in biological macromolecules.

Hydrophobic motif phosphorylation is not required for activation loop phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1).

p70 ribosomal protein S6 kinase 1 (S6K1) is regulated by multiple phosphorylation events. Three of these sites are highly conserved among AGC kinases (cAMP dependent Protein Kinase, cGMP dependent Protein Kinase, and Protein Kinase C subfamily): the activation loop in the kinase domain, and two C-terminal sites, the turn motif and the hydrophobic motif. The common dogma has been that phosphorylation of the hydrophobic motif primes S6K1 for the phosphorylation at the activation loop by phosphoinositide-dependent protein kinase 1 (PDK1). Here, we show that the turn motif is, in fact, phosphorylated first, the activation loop second, and the hydrophobic motif is third. Specifically, biochemical analyses of a construct of S6K1 lacking the C-terminal autoinhibitory domain as well as full-length S6K1, reveals that S6K1 is constitutively phosphorylated at the turn motif when expressed in insect cells and becomes phosphorylated in vitro by purified PDK1 at the activation loop. Only the species phosphorylated at the activation loop by PDK1 gets phosphorylated at the hydrophobic motif by mammalian target of rapamycin (mTOR) in vitro. These data are consistent with a previous model in which constitutive phosphorylation of the turn motif provides the key priming step in the phosphorylation of S6K1. The data provide evidence for regulation of S6K1, where hydrophobic motif phosphorylation is not required for PDK1 to phosphorylate S6K1 at the activation loop, but instead activation loop phosphorylation of S6K1 is required for mTOR to phosphorylate the hydrophobic motif of S6K1.

The conserved mitochondrial twin Cx9C protein Cmc2 Is a Cmc1 homologue essential for cytochrome c oxidase biogenesis.

Mitochondrial copper metabolism and delivery to cytochrome c oxidase and mitochondrially localized CuZn-superoxide dismutase (Sod1) requires a growing number of intermembrane space proteins containing a twin Cx(9)C motif. Among them, Cmc1 was recently identified by our group. Here we describe another conserved mitochondrial metallochaperone-like protein, Cmc2, a close homologue of Cmc1, whose function affects both cytochrome c oxidase and Sod1. In the yeast Saccharomyces cerevisiae, Cmc2 localizes to the mitochondrial inner membrane facing the intermembrane space. In the absence of Cmc2, cytochrome c oxidase activity measured spectrophotometrically and cellular respiration measured polarographically are undetectable. Additionally, mutant cmc2 cells display 2-fold increased mitochondrial Sod1 activity, whereas CMC2 overexpression results in Sod1 activity decreased to 60% of wild-type levels. CMC1 overexpression does not rescue the respiratory defect of cmc2 mutants or vice versa. However, Cmc2 physically interacts with Cmc1 and the absence of Cmc2 induces a 5-fold increase in Cmc1 accumulation in the mitochondrial membranes. Cmc2 function is conserved from yeast to humans. Human CMC2 localizes to the mitochondria and CMC2 expression knockdown produces cytochrome c oxidase deficiency in Caenorhabditis elegans. We conclude that Cmc1 and Cmc2 have cooperative but nonoverlapping functions in cytochrome c oxidase biogenesis.

Biophysical characterization reveals structural disorder in the developmental transcriptional regulator LBH.

Limb-bud and heart (LBH) is a key transcriptional regulator in vertebrates with pivotal roles in embryonic development and human disease. Herein, using a diverse array of biophysical techniques, we report the first structural characterization of LBH pertinent to its biological function. Our data reveal that LBH is structurally disordered with no discernable secondary or tertiary structure and exudes rod-like properties in solution. Consistent with these observations, we also demonstrate that LBH is conformationally flexible and thus may be capable of adapting distinct conformations under specific physiological contexts. We propose that LBH is a member of the intrinsically disordered protein (IDP) family, and that conformational plasticity may play a significant role in modulating LBH-dependent transcriptional processes.

Mechanism of PDK1-catalyzed Thr-229 phosphorylation of the S6K1 protein kinase.

PDK1 (phosphoinositide-dependent protein kinase-1) catalyzes phosphorylation of Thr-229 in the T-loop of S6K1 alpha II (the 70-kDa 40 S ribosomal protein S6 kinase-1 alpha II isoform), and Thr-229 phosphorylation is synergistic with C-terminal Thr-389 phosphorylation to activate S6K1 alpha II regulatory functions in protein translation preinitiation complexes. Unlike its common AGC kinase subfamily member S6K1 alpha II, PDK1 does not contain the synergistic C-terminal phosphorylation site, and it has been proposed that phosphorylated Thr-389 in S6K1 alpha II may initially serve to trans-activate PDK1-catalyzed Thr-229 phosphorylation. Herein, we report direct binding and kinetic studies that showed PDK1 to exhibit nearly equal binding affinities and steady-state kinetic turnover numbers toward native (K(d)(S6K1) = 1.2 microm and k(cat) = 1.1 s(-1)) and the phosphomimicking T389E mutant S6K1 alpha II (K(d)(S6K1) = 1.5 microm and k(cat) = 1.2 s(-1)), although approximately 2-fold enhanced specificity was displayed for the T389E mutant (k(cat)/K(m)(S6K1) = 0.08 microm(-1) s(-1) compared with 0.04 microm(-1) s(-1)). Considering that transient kinetic binding studies showed all nucleotide and S6K1 alpha II substrates and products to rapidly associate with PDK1 (k(on) = 1-6 mum(-1) s(-1)), it was concluded that positioning a negative charge at residue Thr-389 reduced approximately 2-fold the occurrence of nonproductive binding events that precede formation of a reactive ternary complex for Thr-229 phosphorylation. In addition, steady-state kinetic data were most simply accommodated by an Ordered Bi Bi mechanism with competitive substrate inhibition, where (i) the initially formed PDK1-ATP complex phosphorylates the nucleotide-free form of the S6K1 alpha II kinase and (ii) initial binding of S6K1 alpha II precludes ATP binding to PDK1.

Kinetic mechanism of fully activated S6K1 protein kinase.

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (Thr-229) and hydrophobic motif (Thr-389). Previously, we described production of the fully activated catalytic kinase domain construct, His(6)-S6K1alphaII(DeltaAID)-T389E. Now, we report its kinetic mechanism for catalyzing phosphorylation of a model peptide substrate (Tide, RRRLSSLRA). First, two-substrate steady-state kinetics and product inhibition patterns indicated a Steady-State Ordered Bi Bi mechanism, whereby initial high affinity binding of ATP (K(d)(ATP)=5-6 microM) was followed by low affinity binding of Tide (K(d)(Tide)=180 microM), and values of K(m)(ATP)=5-6 microM and K(m)(Tide)=4-5 microM were expressed in the active ternary complex. Global curve-fitting analysis of ATP, Tide, and ADP titrations of pre-steady-state burst kinetics yielded microscopic rate constants for substrate binding, rapid chemical phosphorylation, and rate-limiting product release. Catalytic trapping experiments confirmed rate-limiting steps involving release of ADP. Pre-steady-state kinetic and catalytic trapping experiments showed osmotic pressure to increase the rate of ADP release; and direct binding experiments showed osmotic pressure to correspondingly weaken the affinity of the enzyme for both ADP and ATP, indicating a less hydrated conformational form of the free enzyme.

Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1).

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in (1)H-(15)N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D(2)ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D(2)ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.

Baculovirus-mediated expression, purification, and characterization of a fully activated catalytic kinase domain construct of the 70 kDa 40S ribosomal protein S6 kinase-1 alphaII isoform (S6K1alphaII).

S6K1alphaII is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) regions of its catalytic kinase domain [S6K1alphaII(DeltaAID); deletion of C-terminal autoinhibitory domain residues 399-502]. With regard to mimicking the synergistic effect of full dual site phosphorylation, baculovirus-mediated expression and affinity purification of the His(6)-S6K1alphaII(DeltaAID)-T229E,T389E double mutant from Sf9 insect cells yielded enzyme with compromised activity. Higher activity preparations were generated using the Sf9 purified His(6)-S6K1alphaII(DeltaAID)-T389E single mutant isoform, which was in vitro phosphorylated by the upstream T229 kinase, PDK1 ( approximately 75 nmol/min/mg). Most significantly, we report that the His(6)-S6K1alphaII(DeltaAID)-T389E construct was generated in its most highly active form (250 nmol/min/mg) by baculovirus-mediated expression and purification from Sf9 insect cells that were coinfected with recombinant baculovirus expressing the catalytic kinase domain of PDK1 [His(6)-PDK1(DeltaPH)]. Approximately equal amounts of fully activated His(6)-S6K1alphaII(DeltaAID)-T389E (5+/-1 mg) and His(6)-PDK1(DeltaPH) (8+/-2 mg) were His(6) affinity co-purified 60 h after initial coinfection of 200 mL of Sf9 insect cells (2x10(6) cells/mL), which were resolved by MonoQ anion exchange chromatography. ESI-TOF mass spectrometry, MonoQ anion exchange chromatography, and kinetic assays showed His(6)-PDK1(DeltaPH) to phosphorylate T229 to approximately 100% after co-expression in Sf9 insect cells as compared to approximately 50% under in vitro conditions, raising interest to mechanistic components not fully achieved in the in vitro reaction. Generation of fully activated S6K1 will facilitate more rigorous analysis of its structure and mechanism.

Reconstitution of modular PDK1 functions on trans-splicing of the regulatory PH and catalytic kinase domains.

The serine-threonine protein kinases PDK1 and PKB each contain a pleckstrin homology (PH) domain that binds the membrane-bound phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] second messenger and is required for PDK1-catalyzed phosphorylation and activation of PKB. While X-ray structures have been reported for the individual regulatory PH and catalytic kinase domain constructs of both PDK1 and PKB, diffraction quality crystals of full length constructs have yet to be obtained, likely due to conformational heterogeneity. In developing alternative approaches to understanding the potential role of conformational dynamics in regulating PKB phosphorylation by PDK1, an efficient in vitro method for protein trans-splicing was developed, which utilizes the N- and C-terminal split inteins of the gene dnaE from Nostoc punctiforme [(N)NpuDnaE] and Synechocystis sp. strain PCC6803 [(C)SspDnaE], respectively. For conjugating the regulatory PH domain to the catalytic kinase domain of PDK1, the recombinant trans-splicing fusion constructs KINASE(AEY)-(N)NpuDnaE-His6 and GST-His6-(C)SspDnaE-(CMN)PH were designed, PCR assembled, overexpressed, and affinity purified. The cross-reacting (N)NpuDnaE and (C)SspDnaE inteins generated full length spliced-PDK1 with kobs = (2.8 +/- 0.3) x 10(-5) s(-1) and with < or =5% of any competing trans-cleavage reactions. Spliced-PDK1 was efficiently purified to > or =95% homogeneity from the reaction mixture by subsequent His6 affinity and ion exchange chromatography steps. In vitro kinase assays and phosphopeptide mapping studies confirmed that spliced-PDK1 retained the ability to colocalize and selectively phosphorylate Thr-309 of PKBbeta in a PI(3,4,5)P3-dependent manner. The high-level production and reconstitution of functional spliced-PDK1 establishes the feasibility of incorporating domain-specific biophysical probes for spectroscopic studies of regulatory PH domain mediated catalytic specificity.

Fluorescence resonance energy transfer in quantum dot-protein kinase assemblies.

In search of viable strategies to identify selective inhibitors of protein kinases, we have designed a binding assay to probe the interactions of human phosphoinositide-dependent protein kinase-1 (PDK1) with potential ligands. Our protocol is based on fluorescence resonance energy transfer (FRET) between semiconductor quantum dots (QDs) and organic dyes. Specifically, we have expressed and purified the catalytic kinase domain of PDK1 with an N-terminal histidine tag [His(6)-PDK1(DeltaPH)]. We have conjugated this construct to CdSe-ZnS core-shell QDs coated with dihydrolipoic acid (DHLA) and tested the response of the resulting assembly to a molecular dyad incorporating an ATP ligand and a BODIPY chromophore. The supramolecular association of the BODIPY-ATP dyad with the His(6)-PDK1(DeltaPH)-QD assembly encourages the transfer of energy from the QDs to the BODIPY dyes upon excitation. The addition of ATP results in the displacement of BODIPY-ATP from the binding domain of the His(6)-PDK1(DeltaPH) conjugated to the nanoparticles. The competitive binding, however, does not prevent the energy transfer process. A control experiment with QDs, lacking the His(6)-PDK1(DeltaPH), indicates that the BODIPY-ATP dyad adsorbs nonspecifically on the surface of the nanoparticles, promoting the transfer of energy from the CdSe core to the adsorbed BODIPY dyes. Thus, the implementation of FRET-based assays to probe the binding domain of PDK1 with luminescent QDs requires the identification of energy acceptors unable to interact nonspecifically with the surface of the nanoparticles.

Role of the PH domain in regulating in vitro autophosphorylation events required for reconstitution of PDK1 catalytic activity.

In addition to its catalytic domain, phosphoinsositide-dependent protein kinase-1 (PDK1) contains a C-terminal pleckstrin homology (PH) domain, which binds the membrane-bound phosphatidylinositol (3,4,5)-triphosphate [PI(3,4,5)P3] second messenger. Here, we report in vitro kinetic, phosphopeptide mapping, and oligomerization studies that address the role of the PH domain in regulating specific autophosphorylation events, which are required for PDK1 catalytic activation. First, 'inactive' unphosphorylated forms of N-terminal His6 tagged full length (His6-PDK1) and catalytic domain constructs [His6-PDK1(Delta PH)] were generated by treatment with Lambda protein phosphatase (lambda PP). Reconstitution of lambda PP-treated His6-PDK1(Delta PH) catalytic activity required activation loop Ser-241 phosphorylation, which occurred only upon trans-addition of 'active' PDK1 with an apparent bimolecular rate constant of (app)k1(S241) = 374+/-29 M(-1) s(-1). In contrast, full length lambda PP-treated His6-PDK1 catalyzed Ser-241 cis-autophosphorylation with an apparent first-order rate constant of (app)k1(S241) = (5.0+/-1.5) x 10(-4) s(-1) but remained 'inactive'. Reconstitution of lambda PP-treated His(6)-PDK1 catalytic activity occurred only when autophosphorylated in the presence of PI(3,4,5)P3 containing vesicles. PI(3,4,5)P3 binding to the PH domain activated apparent first-order Ser-241 autophosphorylation by 20-fold [(app)k1(S241) = (1.1+/-0.1) x 10(-2) s(-1)] and also promoted biphasic Thr-513 trans-autophosphorylation [(app)k2(T513) = (4.9+/-1.1) x 10(2) M(-1) s(-1) and(app)k3(T513) = (1.5+/-0.2) x 10(3) M(-1) s(-1)]. The results of mutagenesis studies suggest that Thr-513 phosphorylation may cause dissociation of autoinhibitory contacts formed between the contiguous regulatory PH and catalytic kinase domains.

Steady-state kinetic mechanism of PDK1.

PDK1 catalyzes phosphorylation of Thr in the conserved activation loop region of a number of its downstream AGC kinase family members. In addition to the consensus sequence at the site of phosphorylation, a number of PDK1 substrates contain a PIF sequence (PDK1-interacting fragment), which binds and activates the kinase domain of PDK1 (PDK1(deltaPH)). To gain further insight to PIF-dependent catalysis, steady-state kinetic and inhibition studies were performed for His6-PDK1(deltaPH)-catalyzed phosphorylation of PDK1-Tide (Tide), which contains an extended "PIF" sequence C-terminal to the consensus sequence for PDK1 phosphorylation. In two-substrate kinetics, a large degree of negative binding synergism was observed to occur on formation of the active ternary complex (alphaKd(ATP) = 40 microM and alphaKd(Tide) = 80 microM) from individual transitory binary complexes (Kd(ATP) = 0.6 microM and Kd(Tide) = 1 microM). On varying ATP concentrations, the ADP product and the (T/E)-PDK1-Tide product analog (p'Tide) behaved as competitive and noncompetitive inhibitors, respectively; on varying Tide concentrations, ADP and p'Tide behaved as noncompetitive and competitive inhibitors, respectively. Also, negative binding synergism was associated with formation of dead-end inhibited ternary complexes. Time progress curves in pre-steady-state studies under "saturating" or kcat conditions showed (i) no burst or lag phenomena, (ii) no change in reaction velocity when adenosine 5'-O-(thiotriphosphate) was used as a phosphate donor, and (iii) no change in reaction velocity on increasing relative microviscosity (0 < or = eta/eta0 < or = 3). Taken together, PDK1-catalyzed trans-phosphorylation of PDK1-Tide approximates a Rapid Equilibrium Random Bi Bi system, where motions in the central ternary complex are largely rate-determining.

Discovering new drug-targeting sites on flexible multidomain protein kinases: Combining segmental isotopic and site-directed spin labeling for nuclear magnetic resonance detection of interfacial clefts.

A novel structure-based approach to study the structure and dynamics of flexible multi-domain monomeric protein kinases, which otherwise do not yield diffraction quality crystals, is described. A combination of segmental 15N-isotopic labeling of a regulatory domain with site-directed paramagnetic nitroxide spin labeling of the kinase domain is employed. Nuclear magnetic resonance studies of the enhancement of amide proton relaxation rates of the 15N-isotopically labeled regulatory domain caused by insertion of the paramagnetic nitroxide spin label on the kinase domain provide long-range distance restraints for determination of both the average positional structure and the relative flexibility exhibited between the two contiguous domains. Clefts and crevices detected around the dynamic domain-domain interface provide new targeting sites for tethered-based extension of current small-molecule lead compounds to produce more potent and selective pharmaceutical agents.

Improved yields for baculovirus-mediated expression of human His(6)-PDK1 and His(6)-PKBbeta/Akt2 and characterization of phospho-specific isoforms for design of inhibitors that stabilize inactive conformations.

PDK1 and PKB/Akt have a pleckstrin homology (PH) domain at the C-terminus and N-terminus, respectively, which stabilizes an unphosphorylated, autoinhibited conformation. Binding of the PH domain to a phospholipid second messenger causes relief of autoinhibition, which results in kinase phosphorylation and activation. Baculovirus-mediated expression in Sf9 insect cells of both His(6)-PDK1 and His(6)-PKBbeta/Akt2 were optimized, which significantly improved the yields (5-fold) of the affinity purified enzymes over previously reported values. Isoelectric focusing (IEF) and Western analyses indicated that the apparent V(max)=192+/-13 U/mg and K(m) (PDK-Tide)=55+/-10 microM of purified His(6)-PDK1 results from a mixture of at least three different phospho-specific isoforms (pI values of 6.8, 6.5, and 6.4). A purely unphosphorylated isoform of His(6)-PDK1 (pI=6.8) was generated by treatment with lambda protein phosphatase (lambdaPP), which decreased V(max) to 2.4+/-0.4 U/mg and increased K(m) (PDK-Tide) to 217+/-61 microM. Isoelectric focusing and Western analyses indicated that the apparent V(max)=0.21+/-0.03 U/mg and K(m) (Crosstide)=87+/-30 microM of purified His(6)-PKBbeta/Akt2 results from a mixture of the enzyme monophosphorylated either at Ser-474 ( approximately 90%) or at Thr-309 ( approximately 10%). A purely unphosphorylated isoform of His(6)-PKBbeta/Akt2 (pI=6.4) was generated by treatment with lambdaPP, which decreased V(max) approximately 2-fold. The optimization of high-level production and detailed characterization of purified and lambdaPP-treated His(6)-PDK1 and His(6)-PKBbeta/Akt2 will facilitate detailed structural and kinetic studies aimed at understanding the mechanism of second messenger-induced activation.

Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences.

A novel thermodynamically-balanced inside-out (TBIO) method of primer design was developed and compared with a thermodynamically-balanced conventional (TBC) method of primer design for PCR-based gene synthesis of codon-optimized gene sequences for the human protein kinase B-2 (PKB2; 1494 bp), p70 ribosomal S6 subunit protein kinase-1 (S6K1; 1622 bp) and phosphoinositide-dependent protein kinase-1 (PDK1; 1712 bp). Each of the 60mer TBIO primers coded for identical nucleotide regions that the 60mer TBC primers covered, except that half of the TBIO primers were reverse complement sequences. In addition, the TBIO and TBC primers contained identical regions of temperature- optimized primer overlaps. The TBC method was optimized to generate sequential overlapping fragments (approximately 0.4-0.5 kb) for each of the gene sequences, and simultaneous and sequential combinations of overlapping fragments were tested for their ability to be assembled under an array of PCR conditions. However, no fully synthesized gene sequences could be obtained by this approach. In contrast, the TBIO method generated an initial central fragment (approximately 0.4-0.5 kb), which could be gel purified and used for further inside-out bidirectional elongation by additional increments of 0.4-0.5 kb. By using the newly developed TBIO method of PCR-based gene synthesis, error-free synthetic genes for the human protein kinases PKB2, S6K1 and PDK1 were obtained with little or no corrective mutagenesis.

PDK1 and PKB/Akt: ideal targets for development of new strategies to structure-based drug design.

Growth factor binding events to receptor tyrosine kinases result in activation of phosphatidylinositol 3-kinase (PI3K), and activated PI3K generates the membrane-bound second messengers phosphatidylinositol 3,4-diphosphate [PI(3,4)P2] and PI(3,4,5)P3, which mediate membrane translocation of the phosphoinositide-dependent kinase-1 (PDK1) and protein kinase B (PKB, also known as Akt). In addition to the kinase domain, PDK1 and PKB contain a pleckstrin homology (PH) domain that binds to the second messenger, resulting in the phosphorylation and activation of PKB by PDK1. Recent evidence indicates that constitutive activation of PKB contributes to cancer progression by promoting proliferation and increased cell survival. The indicating of PDK1 and PKB as primary targets for discovery of anticancer drugs, together with the observations that both PDK1 and PKB contain small-molecule regulatory binding sites that may be in proximity to the kinase active site, make PDK1 and PKB ideal targets for the development of new strategies to structure-based drug design. While X-ray structures have been reported for the kinase domains of PDK1 and PKB, no suitable crystals have been obtained for either PDK1 or PKB with their PH domains intact. In this regard, a novel structure-based strategy is proposed, which utilizes segmental isotopic labeling of the PH domain in combination with site-directed spin labeling of the kinase active site. Then, long-range distance restraints between the 15N-labeled backbone amide groups of the PH domain and the unpaired electron of the active site spin label can be determined from magnetic resonance studies of the enhancement effect that the paramagnetic spin label has on the nuclear relaxation rates of the amide protons. The determination of the structure and position of the PH domain with respect to the known X-ray structure of the kinase active site could be useful in the rational design of potent and selective inhibitors of PDK1 and PKB by 'linking' the free energies of binding of substrate (ATP) analogs with analogs of the inositol polar head group of the phospholipid second messenger. The combined use of X-ray crystallography, segmental isotopic and spin labeling, and magnetic resonance studies can be further extended to the study of other dynamic multidomain proteins and targets for structure-based drug design.

Structural basis of perturbed pKa values of catalytic groups in enzyme active sites.

In protein and RNA macromolecules, only a limited number of different side-chain chemical groups are available to function as catalysts. The myriad of enzyme-catalyzed reactions results from the ability of most of these groups to function either as nucleophilic, electrophilic, or general acid-base catalysts, and the key to their adapted chemical function lies in their states of protonation. Ionization is determined by the intrinsic pKa of the group and the microenvironment created around the group by the protein or RNA structure, which perturbs its intrinsic pKa to its functional or apparent pKa. These pKa shifts result from interactions of the catalytic group with other fully or partially charged groups as well as the polarity or dielectric of the medium that surrounds it. The electrostatic interactions between ionizable groups found on the surface of macromolecules are weak and cause only slight pKa perturbations (<2 units). The sum of many of these weak electrostatic interactions helps contribute to the stability of native or folded macromolecules and their ligand complexes. However, the pKa values of catalytic groups that are found in the active sites of numerous enzymes are significantly more perturbed (>2 units) and are the subject of this review. The magnitudes of these pKa perturbations are analyzed with respect to the structural details of the active-site microenvironment and the energetics of the reactions that they catalyze.