Daan M. F. van Aalten

High-Resolution Structure of the Pleckstrin Homology Domain of Protein Kinase B/Akt Bound to Phosphatidylinositol (3,4,5)-Trisphosphate

The products of PI 3-kinase activation, PtdIns(3,4,5)P3 and its immediate breakdown product PtdIns(3,4)P2, trigger physiological processes, by interacting with proteins possessing pleckstrin homology (PH) domains. One of the best characterized PtdIns(3,4,5)P3/PtdIns(3,4)P2 effector proteins is protein kinase B (PKB), also known as Akt. PKB possesses a PH domain located at its N terminus, and this domain binds specifically to PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with similar affinity. Following activation of PI 3-kinase, PKB is recruited to the plasma membrane by virtue of its interaction with PtdIns(3,4,5)P3/PtdIns(3,4)P2. PKB is then activated by the 3-phosphoinositide-dependent pro-tein kinase-1 (PDK1), which like PKB, possesses a PtdIns(3,4,5)P3/PtdIns(3,4)P2 binding PH domain. Here, we describe the high-resolution crystal structure of the isolated PH domain of PKB(alpha) in complex with the head group of PtdIns(3,4,5)P3. The head group has a significantly different orientation and location compared to other Ins(1,3,4,5)P4 binding PH domains. Mutagenesis of the basic residues that form ionic interactions with the D3 and D4 phosphate groups reduces or abolishes the ability of PKB to interact with PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The D5 phosphate faces the solvent and forms no significant interactions with any residue on the PH domain, and this explains why PKB interacts with similar affinity with both PtdIns(3,4,5)P3 and PtdIns(3,4)P2.

Structure of the LKB1-STRAD-MO25 complex reveals an allosteric mechanism of kinase activation

Solving Pseudokinases Mutations of the protein kinase LKB1 are associated with cancer in humans. Many kinases are activated by phosphorylation, but LKB1 is activated by STRADα, a pseudokinase that is similar to protein kinases and binds ATP, but does not phosphorylate substrates. By solving the crystal structure of an activating complex containing LKB, Zeqiraj et al. (p. 1707, published online 5 November) show that STRADα works with another protein, MO25α, to hold LKB1 in an active conformation. The results may help explain the evolutionary origin of pseudokinases, the biological roles of other pseudokinases, and the mechanisms of disease-causing mutations in LKB1. A “pseudokinase” activates the LKB1 tumor suppressor protein without catalyzing phosphorylation. The LKB1 tumor suppressor is a protein kinase that controls the activity of adenosine monophosphate–activated protein kinase (AMPK). LKB1 activity is regulated by the pseudokinase STRADα and the scaffolding protein MO25α through an unknown, phosphorylation-independent, mechanism. We describe the structure of the core heterotrimeric LKB1-STRADα-MO25α complex, revealing an unusual allosteric mechanism of LKB1 activation. STRADα adopts a closed conformation typical of active protein kinases and binds LKB1 as a pseudosubstrate. STRADα and MO25α promote the active conformation of LKB1, which is stabilized by MO25α interacting with the LKB1 activation loop. This previously undescribed mechanism of kinase activation may be relevant to understanding the evolution of other pseudokinases. The structure also reveals how mutations found in Peutz-Jeghers syndrome and in various sporadic cancers impair LKB1 function.

N-myristoyltransferase inhibitors as new leads to treat sleeping sickness.

African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for ∼30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target—T. brucei N-myristoyltransferase—leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.

Binding of Phosphatidylinositol 3,4,5-Trisphosphate to the Pleckstrin Homology Domain of Protein Kinase B Induces a Conformational Change

Protein kinase B (PKB/Akt) is a key regulator of cell growth, proliferation and metabolism. It possesses an N-terminal pleckstrin homology (PH) domain that interacts with equal affinity with the second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2, generated through insulin and growth factor-mediated activation of phosphoinositide 3-kinase (PI3K). The binding of PKB to PtdIns(3,4,5)P3/PtdIns(3,4)P2 recruits PKB from the cytosol to the plasma membrane and is also thought to induce a conformational change that converts PKB into a substrate that can be activated by the phosphoinositide-dependent kinase 1 (PDK1). In this study we describe two high-resolution crystal structures of the PH domain of PKBalpha in a noncomplexed form and compare this to a new atomic resolution (0.98 A, where 1 A=0.1 nm) structure of the PH domain of PKBalpha complexed to Ins(1,3,4,5)P4, the head group of PtdIns(3,4,5)P3. Remarkably, in contrast to all other PH domains crystallized so far, our data suggest that binding of Ins(1,3,4,5)P4 to the PH domain of PKB, induces a large conformational change. This is characterized by marked changes in certain residues making up the phosphoinositide-binding site, formation of a short a-helix in variable loop 2, and a movement of variable loop 3 away from the lipid-binding site. Solution studies with CD also provided evidence of conformational changes taking place upon binding of Ins(1,3,4,5)P4 to the PH domain of PKB. Our data provides the first structural insight into the mechanism by which the interaction of PKB with PtdIns(3,4,5)P3/PtdIns(3,4)P2 induces conformational changes that could enable PKB to be activated by PDK1.

High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site

3‐phosphoinositide dependent protein kinase‐1 (PDK1) plays a key role in regulating signalling pathways by activating AGC kinases such as PKB/Akt and S6K. Here we describe the 2.0 Å crystal structure of the PDK1 kinase domain in complex with ATP. The structure defines the hydrophobic pocket termed the ‘PIF‐pocket’, which plays a key role in mediating the interaction and phosphorylation of certain substrates such as S6K1. Phosphorylation of S6K1 at its C‐terminal PIF‐pocket‐interacting motif promotes the binding of S6K1 with PDK1. In the PDK1 structure, this pocket is occupied by a crystallographic contact with another molecule of PDK1. Interestingly, close to the PIF‐pocket in PDK1, there is an ordered sulfate ion, interacting tightly with four surrounding side chains. The roles of these residues were investigated through a combination of site‐directed mutagenesis and kinetic studies, the results of which confirm that this region of PDK1 represents a phosphate‐dependent docking site. We discuss the possibility that an analogous phosphate‐binding regulatory motif may participate in the activation of other AGC kinases. Furthermore, the structure of PDK1 provides a scaffold for the design of specific PDK1 inhibitors.

Structural Insights Into the Regulation of Pdk1 by Phosphoinositides and Inositol Phosphates

3‐phosphoinositide‐dependent protein kinase‐1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C‐terminal pleckstrin homology (PH) domain that interacts with PtdIns(3,4,5)P3/PtdIns(3,4)P2 and with lower affinity to PtdIns(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4. The structures reveal a ‘budded’ PH domain fold, possessing an N‐terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand‐binding site. Mutagenesis and lipid‐binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,3,4,5,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full‐length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,3,4,5,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6‐kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.

Structural insights into the mechanism and inhibition of eukaryotic O-GlcNAc hydrolysis

O‐linked N‐acetylglucosamine (O‐GlcNAc) modification of specific serines/threonines on intracellular proteins in higher eukaryotes has been shown to directly regulate important processes such as the cell cycle, insulin sensitivity and transcription. The structure, molecular mechanisms of catalysis, protein substrate recognition/specificity of the eukaryotic O‐GlcNAc transferase and hydrolase are largely unknown. Here we describe the crystal structure, enzymology and in vitro activity on human substrates of Clostridium perfringens NagJ, a close homologue of human O‐GlcNAcase (OGA), representing the first family 84 glycoside hydrolase structure. The structure reveals a deep active site pocket highly conserved with the human enzyme, compatible with binding of O‐GlcNAcylated peptides. Together with mutagenesis data, the structure supports a variant of the substrate‐assisted catalytic mechanism, involving two aspartic acids and an unusually positioned tyrosine. Insights into recognition of substrate come from a complex with the transition state mimic O‐(2‐acetamido‐2‐deoxy‐D‐glucopyranosylidene)amino‐N‐phenylcarbamate (Ki=5.4 nM). Strikingly, the enzyme is inhibited by the pseudosubstrate peptide Ala‐Cys(‐S‐GlcNAc)‐Ala, and has OGA activity against O‐GlcNAcylated human proteins, suggesting that the enzyme is a suitable model for further studies into the function of human OGA.

The Structural Basis of Acyl Coenzyme A-Dependent Regulation of the Transcription Factor Fadr

FadR is an acyl‐CoA‐responsive transcription factor, regulating fatty acid biosynthetic and degradation genes in Escherichia coli. The apo‐protein binds DNA as a homodimer, an interaction that is disrupted by binding of acyl‐CoA. The recently described structure of apo‐FadR shows a DNA binding domain coupled to an acyl‐CoA binding domain with a novel fold, but does not explain how binding of the acyl‐CoA effector molecule >30 Å away from the DNA binding site affects transcriptional regulation. Here, we describe the structures of the FadR‐operator and FadR‐myristoyl‐CoA binary complexes. The FadR‐DNA complex reveals a novel winged helix‐turn‐helix protein‐DNA interaction, involving sequence‐specific contacts from the wing to the minor groove. Binding of acyl‐CoA results in dramatic conformational changes throughout the protein, with backbone shifts up to 4.5 Å. The net effect is a rearrangement of the DNA binding domains in the dimer, resulting in a change of 7.2 Å in separation of the DNA recognition helices and the loss of DNA binding, revealing the molecular basis of acyl‐CoA‐responsive regulation.

Structure and metal-dependent mechanism of peptidoglycan deacetylase, a streptococcal virulence factor

Streptococcus pneumoniae peptidoglycan GlcNAc deacetylase (SpPgdA) protects the Gram-positive bacterial cell wall from host lysozymes by deacetylating peptidoglycan GlcNAc residues. Deletion of the pgda gene has been shown to result in hypersensitivity to lysozyme and reduction of infectivity in a mouse model. SpPgdA is a member of the family 4 carbohydrate esterases, for which little structural information exists, and no catalytic mechanism has yet been defined. Here we describe the native crystal structure and product complexes of SpPgdA biochemical characterization and mutagenesis. The structural data show that SpPgdA is an elongated three-domain protein in the crystal. The structure, in combination with mutagenesis, shows that SpPgdA is a metalloenzyme using a His-His-Asp zinc-binding triad with a nearby aspartic acid and histidine acting as the catalytic base and acid, respectively, somewhat similar to other zinc deacetylases such as LpxC. The enzyme is able to accept GlcNAc3 as a substrate (Km = 3.8 mM, kcat = 0.55 s-1), with the N-acetyl of the middle sugar being removed by the enzyme. The data described here show that SpPgdA and the other family 4 carbohydrate esterases are metalloenzymes and present a step toward identification of mechanism-based inhibitors for this important class of enzymes.

Analysis of the LKB1-STRAD-MO25 complex

Mutations in the LKB1 tumour suppressor threonine kinase cause the inherited Peutz-Jeghers cancer syndrome and are also observed in some sporadic cancers. Recent work indicates that LKB1 exerts effects on metabolism, polarity and proliferation by phosphorylating and activating protein kinases belonging to the AMPK subfamily. In vivo, LKB1 forms a complex with STRAD, an inactive pseudokinase, and MO25, an armadillo repeat scaffolding-like protein. Binding of LKB1 to STRAD-MO25 activates LKB1 and re-localises it from the nucleus to the cytoplasm. To learn more about the inherent properties of the LKB1-STRAD-MO25 complex, we first investigated the activity of 34 point mutants of LKB1 found in human cancers and their ability to interact with STRAD and MO25. Interestingly, 12 of these mutants failed to interact with STRAD-MO25. Performing mutagenesis analysis, we defined two binding sites located on opposite surfaces of MO25α, which are required for the assembly of MO25α into a complex with STRADα and LKB1. In addition, we demonstrate that LKB1 does not require phosphorylation of its own T-loop to be activated by STRADα-MO25α, and discuss the possibility that this unusual mechanism of regulation arises from LKB1 functioning as an upstream kinase. Finally, we establish that STRADα, despite being catalytically inactive, is still capable of binding ATP with high affinity, but that this is not required for activation of LKB1. Taken together, our findings reinforce the functional importance of the binding of LKB1 to STRAD, and provide a greater understanding of the mechanism by which LKB1 is regulated and activated through its interaction with STRAD and MO25.