A transmembrane segment mimic derived from Escherichia coli diacylglycerol kinase inhibits protein activity

Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
Journal of Biological Chemistry (Impact Factor: 4.6). 07/2003; 278(24):22056-60. DOI: 10.1074/jbc.M210685200
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: A systematic study of the crystallization of an α-helical, integral membrane enzyme, diacylglycerol kinase, DgkA, using the lipidic cubic mesophase or in meso method is described. These trials have resulted in the production of blocky, rhombohedron-shaped crystals of diffraction quality currently in use for structure determination. Dramatic improvements in crystal quality were obtained when the identity of the lipid used to form the mesophase bilayer into which the protein was reconstituted as a prelude to crystallogenesis was varied. These monoacylglycerol lipids incorporated fatty acyl chains ranging from 14 to 18 carbon atoms long with cis olefinic bonds located toward the middle of the chain. Best crystals were obtained with a lipid that had an acyl chain 15 carbon atoms long with the double bond between carbons 7 and 8. It is speculated that the effectiveness of this lipid derives from hydrophobic mismatch between the target integral membrane protein and the bilayer of the host mesophase. Low temperature (4 °C) worked in concert with the short chain lipid to provide high quality crystals. Recommended screening strategies for crystallizing membrane proteins that include host lipid type and low temperature are made on the basis of this and related in meso crystallization trials.
    Crystal Growth & Design 07/2013; 13(7):2846-2857. DOI:10.1021/cg400254v · 4.56 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: While interactions of single-span transmembrane helices have been studied to a significant extent in the past years, the folding of polytopic α-helical transmembrane proteins as well as their oligomerization, are far less analyzed and understood. The goal of the few thus far performed thermodynamic studies, in which unfolding of polytopic TM proteins was described, was to achieve a mild, potentially reversible unfolding process, to finally derive thermodynamic parameters for the reverse folding pathway. In the first part of this review, we summarize the studies analyzing the thermodynamic stability and folding pathways of polytopic transmembrane proteins. Based on these studies, we deduce some common principles, guiding transmembrane protein unfolding and folding, important for the design of future folding/unfolding studies. Furthermore, the discussed observations can conceptually guide an experimental search for proper in vitro transmembrane protein refolding conditions. In many of the resolved membrane protein structures, individual monomers interact to form higher ordered oligomers. In most cases, oligomerization of those monomeric units appears to be intimately linked to the protein function, and folding of the individual protomers might even occur only after interaction. In the second part of this review, we discuss folding pathways of oligomeric α-helical transmembrane proteins as well as causes and consequences of α-helical transmembrane protein oligomerization.
    Archives of Biochemistry and Biophysics 12/2014; 564. DOI:10.1016/ · 3.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Drug-resistant bacteria use several families of membrane-embedded transporters to remove antibiotics from the cell. One such family is the small multidrug resistance proteins (SMRs) that, because of their relatively small size (ca. 110 residues with four transmembrane [TM] helices), must form (at least) dimers to efflux drugs. Here, we use a Lys-tagged synthetic peptide with exactly the same sequence as TM4 of the full-length SMR Hsmr from Halobacterium salinarum [TM4 sequence: AcA(Sar)(3)-VAGVVGLALIVAGVVVLNVAS-KKK (Sar = N-methylglycine)] to compete with and disrupt the native TM4-TM4 interactions believed to constitute the locus of Hsmr dimerization. Using a cellular efflux assay of the fluorescent SMR substrate ethidium bromide, we determined that bacterial cells containing Hsmr are able to remove cellular ethidium via first-order exponential decay with a rate constant (k) of 10.1 × 10(-3) ± 0.7 × 10(-3) s(-1). Upon treatment of the cells with the TM4 peptide, we observed a saturable ~60% decrease in the efflux rate constant to 3.7 × 10(-3) ± 0.2 × 10(-3) s(-1). In corresponding experiments with control peptides, including scrambled sequences and a sequence with d-chirality, a decrease in ethidium efflux either was not observed or was marginal, likely from nonspecific effects. The designed peptides did not evoke bacterial lysis, indicating that they act via the α-helicity and membrane insertion propensities of the native TM4 helix. Our overall results suggest that this approach could conceivably be used to design hydrophobic peptides for disruption of key TM-TM interactions of membrane proteins and represent a valuable route to the discovery of new therapeutics.
    Antimicrobial Agents and Chemotherapy 04/2012; 56(7):3911-6. DOI:10.1128/AAC.00158-12 · 4.45 Impact Factor