Martin Klima

The crystal structure of the phosphatidylinositol 4‐kinase IIα

Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4‐kinase type IIα (PI4K IIα), in complex with ATP solved by X‐ray crystallography at 2.8 Å resolution. The structure revealed a non‐typical kinase fold that could be divided into N‐ and C‐lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C‐lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane.

Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane

TRAIL, a ligand of the TNFα family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.

Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein.

Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.

The high-resolution crystal structure of phosphatidylinositol 4-kinase IIβ and the crystal structure of phosphatidylinositol 4-kinase IIα containing a nucleoside analogue provide a structural basis for isoform-specific inhibitor design.

Phosphatidylinositol 4-phosphate (PI4P) is the most abundant monophosphoinositide in eukaryotic cells. Humans have four phosphatidylinositol 4-kinases (PI4Ks) that synthesize PI4P, among which are PI4K IIβ and PI4K IIα. In this study, two crystal structures are presented: the structure of human PI4K IIβ and the structure of PI4K IIα containing a nucleoside analogue. The former, a complex with ATP, is the first high-resolution (1.9 Å) structure of a PI4K. These structures reveal new details such as high conformational heterogeneity of the lateral hydrophobic pocket of the C-lobe and together provide a structural basis for isoform-specific inhibitor design.

Inhibition of vacuolar ATPase attenuates the TRAIL-induced activation of caspase-8 and modulates the trafficking of TRAIL receptosomes.

Tumour necrosis factor (TNF) related apoptosis inducing ligand (TRAIL), a membrane‐bound ligand from the TNF family, has attracted significant attention due to its rather specific and effective ability to induce apoptotic death in various types of cancer cells via binding to and activating its pro‐apoptotic death receptors. However, a significant number of primary cancer cells often develop resistance to TRAIL treatment, and the signalling platform behind this phenomenon is not fully understood. Upon blocking endosomal acidification by the vacuolar ATPase (V‐ATPase) inhibitors bafilomycin A1 (BafA1) or concanamycin A, we observed a significantly reduced initial sensitivity of several, mainly colorectal, tumour cell lines to TRAIL‐induced apoptosis. In cells pretreated with these inhibitors, the TRAIL‐induced processing of caspase‐8 and the aggregation and trafficking of the TRAIL receptor complexes were temporarily attenuated. Nuclear factor κB or mitogen activated protein/stress kinase signalling from the activated TRAIL receptors remained unchanged, and neither possible lysosomal permeabilization nor acid sphingomyelinase was involved in this process. The cell surface expression of TRAIL receptors and their TRAIL‐induced internalization were not affected by V‐ATPase inhibitors. The inhibitory effect of BafA1, however, was blunted by knockdown of the caspase‐8 inhibitor cFLIP. Altogether, the data obtained provide the first evidence that endosomal acidification could represent an important regulatory node in the proximal part of TRAIL‐induced pro‐apoptotic signalling.

T-cell activation triggers death receptor-6 expression in a NF-κB and NF-AT dependent manner

Death receptor-6 (DR6) apparently participates in the regulation of T-cell activation and/or activity as its genetic disruption results in enhanced CD4+ T-cell expansion, the production of Th2 cytokines, and interestingly also the compromised migration of CD4+ T cells to sites of inflammation. However, the mechanism of regulation of DR6 expression in cells of the immune system is not fully understood. In this communication we show that DR6 is not expressed in resting T cells from human peripheral blood or murine lymph nodes but that its expression is significantly upregulated in CD3 crosslinking- or PMA/ionomycin-activated T lymphocytes. DR6 expression is transiently increased in both activated human CD4+ and CD8+ T cells and it is apparently dependent on the activation of NF-κB and NF-AT signaling pathways. In contrast to primary peripheral blood T cells, the widely used model lymphoblastic leukemia T-cell line Jurkat is DR6-positive and unexpectedly, TCR-mediated stimulation of Jurkat cells strongly downregulates DR6 expression via suppression of its transcription.

Norbornane-based nucleoside and nucleotide analogues locked in North conformation

We report on the synthesis of novel conformationally locked nucleoside and nucleotide derivatives, which are structurally closely related to clinically used antivirals such as didanosine and abacavir. As a suitable conformationally rigid substitute of the sugar/pseudosugar ring allowing a permanent stabilization of the nucleoside in North conformation we employed bicyclo[2.2.1]heptane (norbornane) substituted in the bridgehead position with a hydroxymethyl group and in the C-3 position with a nucleobase. Prepared nucleoside derivatives were also converted into appropriate phosphoramidate prodrugs (ProTides) in order to increase delivery of the compounds in the cells. All target compounds were evaluated in a broad antiviral and cytostatic assay panel.

Crystal structures of a yeast 14-3-3 protein from Lachancea thermotolerans in the unliganded form and bound to a human lipid kinase PI4KB-derived peptide reveal high evolutionary conservation.

14-3-3 proteins bind phosphorylated binding partners to regulate several of their properties, including enzymatic activity, stability and subcellular localization. Here, two crystal structures are presented: the crystal structures of the 14-3-3 protein (also known as Bmh1) from the yeast Lachancea thermotolerans in the unliganded form and bound to a phosphopeptide derived from human PI4KB (phosphatidylinositol 4-kinase B). The structures demonstrate the high evolutionary conservation of ligand recognition by 14-3-3 proteins. The structural analysis suggests that ligand recognition by 14-3-3 proteins evolved very early in the evolution of eukaryotes and remained conserved, underlying the importance of 14-3-3 proteins in physiology.

Kobuviral Non-structural 3A Proteins Act as Molecular Harnesses to Hijack the Host ACBD3 Protein.

Picornaviruses are small positive-sense single-stranded RNA viruses that include many important human pathogens. Within the host cell, they replicate at specific replication sites called replication organelles. To create this membrane platform, they hijack several host factors including the acyl-CoA-binding domain-containing protein-3 (ACBD3). Here, we present a structural characterization of the molecular complexes formed by the non-structural 3A proteins from two species of the Kobuvirus genus of the Picornaviridae family and the 3A-binding domain of the host ACBD3 protein. Specifically, we present a series of crystal structures as well as a molecular dynamics simulation of the 3A:ACBD3 complex at the membrane, which reveals that the viral 3A proteins act as molecular harnesses to enslave the ACBD3 protein leading to its stabilization at target membranes. Our data provide a structural rationale for understanding how these viral-host protein complexes assemble at the atomic level and identify new potential targets for antiviral therapies.

Structural analysis of phosphatidylinositol 4-kinase IIIβ (PI4KB) – 14-3-3 protein complex reveals internal flexibility and explains 14-3-3 mediated protection from degradation in vitro

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is responsible for the synthesis of the Golgi and trans-Golgi network (TGN) pool of phosphatidylinositol 4-phospahte (PI4P). PI4P is the defining lipid hallmark of Golgi and TGN and also serves as a signaling lipid and as a precursor for higher phosphoinositides. In addition, PI4KB is hijacked by many single stranded plus RNA (+RNA) viruses to generate PI4P-rich membranes that serve as viral replication organelles. Given the importance of this enzyme in cells, it has to be regulated. 14-3-3 proteins bind PI4KB upon its phosphorylation by protein kinase D, however, the structural basis of PI4KB recognition by 14-3-3 proteins is unknown. Here, we characterized the PI4KB:14-3-3 protein complex biophysically and structurally. We discovered that the PI4KB:14-3-3 protein complex is tight and is formed with 2:2 stoichiometry. Surprisingly, the enzymatic activity of PI4KB is not directly modulated by 14-3-3 proteins. However, 14-3-3 proteins protect PI4KB from proteolytic degradation in vitro. Our structural analysis revealed that the PI4KB:14-3-3 protein complex is flexible but mostly within the disordered regions connecting the 14-3-3 binding site of the PI4KB with the rest of the PI4KB enzyme. It also predicted no direct modulation of PI4KB enzymatic activity by 14-3-3 proteins and that 14-3-3 binding will not interfere with PI4KB recruitment to the membrane by the ACBD3 protein. In addition, the structural analysis explains the observed protection from degradation; it revealed that several disordered regions of PI4KB become protected from proteolytical degradation upon 14-3-3 binding. All the structural predictions were subsequently biochemically validated.