<i>Escherichia coli</i> Diacylglycerol Kinase Is an α-Helical Polytopic Membrane Protein and Can Spontaneously Insert into Preformed Lipid Vesicles

The function of membrane proteins is inextricably linked to the proper packing and assembly of their independently helical transmembrane (TM) segments. Here we examined whether an externally added TM peptide analogue could specifically inhibit the function of the membrane protein from which it is derived by competing for native TM helix packing sites, thereby producing a non-functional peptide-protein complex. This hypothesis was tested using Lys-tagged peptides synthesized with sequences corresponding to the three TM segments of the homotrimeric Escherichia coli diacylglycerol kinase (DGK). The peptide corresponding to wild-type DGK TM-2 inhibited the protein's enzymatic activity in a dose-dependent manner through formation of an inactive pseudo-complex, whereas peptides derived from TM-1 and TM-3 were benign toward DGK structure/function. Also, substitution of a conserved residue (Glu-69) within the TM-2 peptide abolished these effects, demonstrating the strict sequence requirements for TM-2-mediated association. This strategy, coupled with the practical advantages of the water solubility of Lys-tagged TM peptides, may constitute an attractive approach for the design of therapeutic membrane protein modulators even in the absence of a high resolution structure.The folding and oligomerization of integral membrane proteins can be divided into two energetically distinct steps (1). In this "two-stage" model, transmembrane (TM) 1 segments first fold into stable ␣-helices in the lipid bilayer. Upon establishment of the proper secondary structure elements, the nativelike spatial arrangement of side chains facilitates high affinity helix-helix association (2-5) mediated primarily by van der Waals packing and inter-helical H-bonding. The specificity of these interactions is crucial for proper membrane protein folding, as emphasized by the strict sequence requirements of residues at the helical interfaces of self-associating TM segments from such proteins as glycophorin A (6, 7), the influenza A M2 proton channel (8), phospholamban (9), synatobrevin (10), and the major coat protein from M13 bacteriophage (3,11,12).Here, we hypothesize that synthetic peptides composed of the TM segment sequences from a membrane protein target can specifically prevent proper protein folding by competing for native helix-helix packing sites. Conceptually analogous studies performed on the single-spanning dimeric glycophorin A (13) and two multi-spanning G-protein coupled receptors (14, 15) illustrated the ability of synthetic peptides to disrupt transmembrane helix-helix interactions. Furthermore, we sought to inquire whether appropriately designed TM segment peptides derived from a multi-spanning membrane protein could not only interact with the intact protein but also inhibit its function.The practical use of TM peptides in this context has been limited by the challenges associated with purifying and characterizing these inherently hydrophobic species. However, previous work in our laboratory has established the fact that flanking TM segm...

Section: Resultsmentioning

confidence: 99%

A Transmembrane Segment Mimic Derived from Escherichia coli Diacylglycerol Kinase Inhibits Protein Activity

Partridge

Melnyk

Yang

et al. 2003

“…DAGK of E. coli (dgk) is a small protein of 121 amino acids that, due to its hydrophobic character, can insert spontaneously in active form into membranes (59). For expression of this protein in chloroplasts we transformed tobacco with a chimeric construct encoding a fusion of the bacterial DAGK with an N-terminal leader peptide derived from the small subunit of ribulosebisphosphate carboxylase/oxygenase.…”

Section: Transgenic Plants Are Smaller Show Dagk Activity In the Envmentioning

confidence: 99%

Channeling of Eukaryotic Diacylglycerol into the Biosynthesis of Plastidial Phosphatidylglycerol

Fritz

Lokstein

Hackenberg

et al. 2007

Plastidial glycolipids contain diacylglycerol (DAG) moieties, which are either synthesized in the plastids (prokaryotic lipids) or originate in the extraplastidial compartment (eukaryotic lipids) necessitating their transfer into plastids. In contrast, the only phospholipid in plastids, phosphatidylglycerol (PG), contains exclusively prokaryotic DAG backbones. PG contributes in several ways to the functions of chloroplasts, but it is not known to what extent its prokaryotic nature is required to fulfill these tasks. As a first step toward answering this question, we produced transgenic tobacco plants that contain eukaryotic PG in thylakoids. This was achieved by targeting a bacterial DAG kinase into chloroplasts in which the heterologous enzyme was also incorporated into the envelope fraction. From lipid analysis we conclude that the DAG kinase phosphorylated eukaryotic DAG forming phosphatidic acid, which was converted into PG. This resulted in PG with 2-3 times more eukaryotic than prokaryotic DAG backbones. In the newly formed PG the unique ⌬3-trans-double bond, normally confined to 3-transhexadecenoic acid, was also found in sn-2-bound cis-unsaturated C18 fatty acids. In addition, a lipidomics technique allowed the characterization of phosphatidic acid, which is assumed to be derived from eukaryotic DAG precursors in the chloroplasts of the transgenic plants. The differences in lipid composition had only minor effects on measured functions of the photosynthetic apparatus, whereas the most obvious phenotype was a significant reduction in growth.

“…Early experiments showed that DGK activity occupies a central position in phospholipid synthesis; this enzyme attracted additional interest when it was shown to participate in intracellular signaling (1). DGK activity is found in organisms from bacteria to mammals, although the protein identified as a DGK in bacteria is an integral membrane protein (2) and has little structural homology with the DGK characterized in multicellular organisms (3). Ten DGK isoforms have been identified in mammals and grouped into five subtypes based on the presence of various domains in their primary sequences, which define distinct regulatory motifs.…”

mentioning

confidence: 99%

Role of the Diacylglycerol Kinase α-Conserved Domains in Membrane Targeting in Intact T Cells

Merino¹,

Sanjuán²,

Moraga

et al. 2007

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGK␣ is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGK␣ domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr 335. This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.The transient generation of lipids at the cell membrane by the concerted action of lipases, kinases, and phosphatases is a very effective mechanism for the localization and activation of signaling molecules. The majority of lipid-modifying enzymes are cytosolic proteins that must in turn translocate to the membrane to modify their substrates. Characterization of the mechanism that governs membrane translocation of these proteins is, thus, essential to assess precisely their role in the regulation of cell responses.The diacylglycerol kinases (DGK) 5 are a family of enzymes that phosphorylate diacylglycerol (DAG) to produce phosphatidic acid (PA); this modulates the levels of these two lipids, both of which have recognized second messenger functions. Early experiments showed that DGK activity occupies a central position in phospholipid synthesis; this enzyme attracted additional interest when it was shown to participate in intracellular signaling (1). DGK activity is found in organisms from bacteria to mammals, although the protein identified as a DGK in bacteria is an integral membrane protein (2) and has little structural homology with the DGK characterized in multicellular organisms (3). Ten DGK isoforms have been identified in mammals and grouped into five subtypes based on the presence of various domains in their primary sequences, which define distinct regulatory motifs.Translocation from one subcellular compartment to another appears to be a general theme in the regulation of DGK proteins. Many of the early data regarding DGK activity regulation refer to changes in DGK activity or localization based on enzymatic studies without specifying the isoform concerned (4, 5). Characterization of the DGK isoforms has broadened the concept that these proteins reg...