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

Anthony W. Partridge {ddagger} §, Roman A. Melnyk {ddagger} §, Dawn Yang ¶, James U. Bowie ¶ and Charles M. Deber {ddagger} ||

From the {ddagger} Division of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8 and the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, Department of Chemistry and Biochemistry, UCLA-Department of Energy Laboratory of Structural Biology and Molecular Medicine, Los Angeles, California 90095-1570

Received for publication, October 18, 2002 , and in revised form, March 13, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
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 {alpha}-helices in the lipid bilayer. Upon establishment of the proper secondary structure elements, the native-like spatial arrangement of side chains facilitates high affinity helix-helix association (25) 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 segments with a defined number of lysine residues renders them water soluble without affecting their ability to fold into {alpha}-helices in micelles and participate in native-like helix-helix contacts (5). In this work, we have synthesized the three putative TM helices from Escherichia coli diacylglycerol kinase (DGK) (16), a ~13-kDa membrane protein that directly phosphorylates diacylglycerol (DAG) by Mg·ATP (17) and exists as a homotrimer in its functional state (18). This protein is an attractive system because it is a relatively small, well characterized protein whose enzymatic activity is dependent on homotrimerization proposed to be mediated by TM-TM interactions (19). We demonstrate here that a Lys-tagged peptide corresponding to a TM segment important for proper DGK folding/assembly both interacts with and inhibits enzyme function when added to the folded full-length protein.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
TM Finder—Transmembrane sequences of DGK chosen for synthesis were identified using the web-based program TM Finder (www.bioinformatics-canada.org/TM/) (20) with the following parameters: N- and C-terminal windows were set at 3; the core length was set at 10; the gap was set at 3; and the segment length was set at 10.

Peptide and Protein Preparation and Purification—Peptides were synthesized and purified as described previously (5). DGK proteins were prepared and purified as described previously (21); pure enzyme was stored at–80 °C in a buffer containing 50 mM sodium phosphate (pH 8.0), 0.3 M NaCl, 0.25 mM imidazole, and 0.5% (w/v) decyl maltoside.

Circular Dichroism Spectroscopy—Circular dichroism spectra were recorded using a Jasco J-720 circular dichroism spectrometer. Samples were measured at peptide concentrations between 20 and 50 µM and were dissolved in a buffer containing 50 mM SDS, 10 mM Tris, and 10 mM NaCl, pH 7.2. Measurements were taken using a quartz cuvette with a 0.1-mm path-length. Spectral scans were performed from 250–190 nm with a step-resolution of 0.2 nm, a speed of 20 nm/min, and a bandwidth of 1.0 nm.

SDS-PAGE Gel Shift Assay—Peptide samples were subjected to SDS-polyacrylamide gel electrophoresis using 10–20% Tricine precast gels (Novex, San Diego, CA). Mixing of peptide and protein (~2 µg of each) was performed in detergent and heated at 85 °C for 2 min prior to electrophoretic separation.

Enzyme Activity Assays—5 µl of peptide stock solutions in 500 mM dithiothreitol were added to 45 µl of 0.14 µM wild-type DGK in 50 mM sodium phosphate, pH 7.4, 300 mM sodium chloride, and 0.5% decyl maltoside. After incubation for various times at room temperature, 10-µl aliquots were assayed as described by Lau et al. in 1999 (25). Briefly, DGK activity was measured using a colorimetric method in which the ADP generated by the DGK-catalyzed reaction is coupled to the oxidation of NADH using phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase. For the dose-dependent studies, protein was incubated with peptide for 2 h.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Peptide Design—Peptide mimics of the three putative transmembrane domains from the DGK protein (TM-1, TM-2, and TM-3) were designed and synthesized. The sequence boundaries in these peptides were chosen by considering previous experimental work (16, 22) in conjunction with the output from TM Finder, a program that identifies TM sequences based on the dual requirements of hydrophobicity and membrane helical propensity (20). To ensure that the entire TM region was included, we incorporated a number of putative juxtamembranous residues that likely reside at the membrane-water interface. The experimentally determined topology of the full-length DGK protein (16) and the sequences of the Lys-tagged peptides corresponding to the protein TM segments used in this study are presented in Fig. 1. Consistent with previous applications of this hydrophilic tagging approach, we found that DGK TM peptides designed in this manner had the beneficial attributes of water solubility and ease of purification (3, 5, 12, 23, 24). To eliminate the possibility that the homophilic nature of the TM-3 construct observed (see below) was due to disulfide bonding, its native Cys residue was changed to an Ala residue. We noted that all peptides herein displayed the ability to spontaneously insert into SDS and n-octyl-{beta}-D-glucopyranoside ({beta}-OG) detergent micelles, where they adopt a high degree of helicity as measured by circular dichroism spectroscopy (data not shown).



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FIG. 1.
a, topological model of the diacylglycerol kinase protein as determined by Smith et al. (16). The protein consists of two putative surface bound amphipathic helices (green) and three transmembrane (TM) helices (gold). Encircled numbers indicate the amino acid residues that were chosen as the sequence boundaries for the synthesized peptides. b, sequences of synthetic Lys-tagged DGK TM peptides. Numbers of flanking Lys residues for each peptide are calculated from sequence hydrophobicity as the minimum required for aqueous solubility and do not affect efficacy of helix-helix tertiary contacts.

 

Oligomerization of Wild-type and Cys-less DGK Proteins in SDS Detergent Micelles—Cross-linking studies and biochemical evidence have demonstrated that the DGK protein exists in its functional form as a homotrimer under non-denaturing conditions (18, 25). Here we observed that briefly heating DGK samples to 80 °C in SDS before electrophoretic separation (see "Experimental Procedures") resulted in a mixture of monomers-dimers-trimers, as judged by Rf analysis (not shown), exclusively for both wild-type and Cys-less DGK proteins, indicating that disulfide bonds were not responsible for the observed oligomerization as demonstrated previously (26). The increased interactions observed between DGK monomers under these conditions is likely due to the increased opportunity for DGK species to interact and equilibrate (Fig. 2a; lanes 1 and 2, respectively). It has been noted that, without sample heating, monomeric DGK is the predominant species observed in SDS (18, 26).



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FIG. 2.
a, oligomerization of intact DGK protein as determined by SDS-PAGE analysis. Protein stock was added to an SDS-containing sample buffer and heated briefly prior to electrophoretic separation on 10–20% Tricine gels. Wild-type DGK (lane 1) and Cys-less DGK (lane 2) each ran as a mixture of three species consistent with monomers, dimers, and trimers as determined by Rf analysis. Oligomeric states are indicated by black circles to the left of the gel; values obtained from molecular weight markers are given to the right. b, interactions between wild-type DGK and DGK-TM-peptides. Protein and peptides were mixed and heated briefly prior to electrophoresis (see "Experimental Procedures" for details). Free DGK protein is shown in lane 1. Rf analysis of the Lys-tagged DGK TM peptides run in the absence of protein indicated that TM-1 (lane 2) was monomeric, whereas TM-2 (lane 4) and TM-3 (lane 6) were homodimeric. When mixed with wild-type DGK protein, TM-2 peptide interacts with the protein as evidenced by the appearance of a novel band (lane 5). Protein/peptide mixtures involving either TM-1 (lane 3) or TM-3 (lane 7) did not show any such interactions, as the wild-type DGK protein in these lanes ran in an equivalent manner to the control lane of protein alone (compare lane 1).

 

TM Peptide Interactions with Wild-type DGK Protein—To ascertain which, if any, of the Lys-tagged TM peptides interact with the full-length DGK protein, a gel shift assay was employed wherein protein was incubated in the absence and presence of the each of the three TM peptides in detergent micelles prior to electrophoresis. When the individual TM peptide mimics were run on SDS-PAGE in the absence of the DGK protein, TM-1 ran at a molecular weight consistent with a monomer, whereas both TM-2 and TM-3 seemed to migrate as homodimers according to Rf analysis (Fig. 2b, lanes 2, 4, and 6, respectively). Note that, in our experience, Lys-tagged TM peptides migrate true to the molecular weight markers in contrast to their untagged counterparts. The dimeric state of TM-2 is supported by the presence of an additional monomer band observed in some experiments (e.g. Fig. 4a, lane 2). Furthermore, TM-2 migrates slower than TM-1 despite its lower molecular weight and decreased overall positive charge. Finally, a longer version of TM-2 peptide (22% higher molecular weight) migrated unambiguously as a dimeric species on SDS-PAGE.



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FIG. 4.
Glu-69 in DGK TM-2 is required for binding and inhibition of DGK. a, gel shift-assay. DGK alone runs as a mixture of monomer, dimer, and trimer (lane 1). DGK and wild-type TM-2 interact as is indicated by a gel-shift (lane 3). The E69A TM-2 peptide does not interact with DGK, as no gel shift is evident (lane 5) compared with peptide alone (lane 4). b, circular dichroism spectra of wild-type TM-2 and TM-2-E69A (dashed curve) peptides in 50 mM SDS, 10 mM Tris, and 10 mM NaCl, pH 7.2. Ellipticity ({theta}) is given in degrees x cm2 per decimol. Spectra are essentially superimposable, confirming that E69A does not disrupt the TM helix. c, percent inhibition of DGK activity in the presence of wild-type TM-2 and E69A TM-2 peptides relative to "no peptide" after 185 min of pre-incubation.

 

In the presence of DGK protein, TM-2 consistently produced a shift in migration for both protein and peptide. Specifically, this interaction between the TM-2 peptide and DGK protein was evidenced by the following: (i) a decrease in TM-2 peptide dimer band intensity in the presence of protein (Fig. 2b, lane 4 versus lane 5); (ii) a decrease in intensity of the DGK protein monomer band (Fig 2b, lane 1 versus lane 5); and (iii) the appearance of a novel band between the protein monomer and dimer positions (Fig. 2b, lane 5). The position of the upper extent of the novel peptide/protein band was consistent with that of a peptide dimer interacting with a protein monomer. However, because of the diffuse nature of this band, we cannot exclude the possibility of the presence of a protein monomer/peptide monomer complex. In parallel experiments, neither TM-1 or TM-3 peptides altered the migration of DGK, nor did their migration change in the presence of protein (Fig. 2, lanes 2, 3, 6, and 7), indicating that the interaction observed between the TM-2 peptide and the full-length protein was specific.

Inhibition of DGK Activity by TM Peptides—To determine the effect of Lys-tagged peptides on DGK enzymatic activity, mixtures of protein and peptide in decyl maltoside were assayed for their ability to inhibit DGK activity (see "Experimental Procedures"). To ensure appropriate mixing between peptide and protein, peptides were incubated with DGK for various times prior to the measurement of DGK activity expressed as Vmax. In the presence of TM-1 and TM-3 peptides, no significant reduction in DGK activity was observed up to 135 min of pre-incubation (Fig. 3a). However, in the presence of TM-2, DGK activity decreased ~50% between 5 and 40 min of preincubation, after which it remained constant during longer incubations. The equilibrium inhibition of DGK activity by TM peptides is shown in Fig. 3b. In conjunction with SDS-PAGE results, these findings implicate TM-2 as a critical determinant for DGK folding and function.



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FIG. 3.
Effect of TM peptides on wild-type DGK enzyme activity in decyl maltoside micelles. a, the Vmax of wild-type DGK (0.14 µM) was measured using saturating substrate concentrations in the absence and presence of each TM peptide (100 µM). Peptides and protein were pre-incubated for various times prior to the addition of substrates to allow for equilibration of the mixture (see "Experimental Procedures"). Activity assays at each time point were performed in triplicate for each peptide and averaged. B, percent inhibition of DGK activity in the presence of TM peptides relative to "no peptide" after 185 min of pre-incubation.

 

Conserved Residue Glu-69 Is Necessary for Protein Binding and Inhibition—Sequence alignment of all known DGK homologues from bacterial species revealed that, within the TM-2 helix, only the Glu-69 and Asn-72 residues were absolutely conserved (data not shown). For our studies, the Glu-69 position was chosen for investigation because previous reports suggested that this residue might play an important structural role, whereas the Asn-72 residue was shown to be part of the active site (25). To gain insights into the manner in which TM-2 peptide binds to and inhibits the DGK protein, we synthesized and purified a TM-2 peptide containing a Glu-to-Ala mutation at position 69 (sequence KKK-50VDAITRVLLISSVMLVMIVE69ILNSAI75-KKK). We found that the TM-2-E69A peptide did not bind to or inhibit the DGK protein (Fig. 4, a and c). Notably, however, the E69A mutation did not alter the dimeric nature of TM-2, indicating that an H-bond involving Glu-69 is not a requirement for the observed SDS-resistant wild-type TM-2 peptide dimer (Fig. 4a). Additionally, the E69A mutation did not produce any change in the secondary structure of the TM-2 peptide as ascertained by circular dichroism spectroscopy (Fig. 4b).

Dose-dependent Inhibition of DGK—To investigate whether the inhibition by TM-2 peptide displayed dose dependence, we titrated increasing amounts (0.1–300 µM) of TM-2 peptide and measured DGK activity (Fig. 5). The results show that wild-type TM-2 peptide gave rise to a dose-dependent decrease in DGK activity, whereas the E69A control peptide did not.



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FIG. 5.
Dose-dependent inhibition of DGK by wild-type TM-2 peptide. DGK activity was measured in the presence of samples of wild-type ({blacktriangleup}) and E69A ({diamondsuit}) TM-2 peptides over the concentration range 0.1–300 µM. DGK concentration, 0.14 µM. Prior to an assay of enzyme activity, incubations of peptide/protein mixtures were carried out in 50 mM sodium phosphate, pH 7.4, 300 mM sodium chloride, and 0.5% decyl maltoside.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Despite the absence of a high-resolution structure for DGK, previous cross-linking data identified TM-2 as a potential oligomeric determinant of the protein homotrimer (19). A goal of our work was to use designed TM peptides to provide further insight into the role of TM-2 vis-à-vis that of TM-1 and TM-3 segments in DGK folding and oligomerization.

When the individual TM peptides were run on SDS-PAGE, TM-1 ran as a monomer, whereas both TM-2 and TM-3 migrated as homodimers (Fig. 2). The monomeric state of TM-1 is in agreement with the previous demonstration that this helix acts as a passive TM anchor and can be replaced with nonspecific sequences (27). The observed self-association of the TM-2 peptide is in agreement with the notion that helix 2 is involved in the homo-oligomerization of the full-length protein. The fact that this helix forms a homodimer in SDS rather than a homotrimer may indicate some added complexity in the pathway toward DGK oligomerization; for instance, homotrimerization of intact DGK may require additional structural units distal to TM-2. The TM-3 peptide similarly formed a homodimer in SDS detergent micelles despite the fact that TM-3 peptide did not bind to or inhibit DGK, suggesting that TM-3 self-association likely does not occur in vivo or that it is involved with processes other than DGK trimerization.

Data from SDS-PAGE analysis (Fig. 2b) and DGK inhibition assays (Figs. 3, a and b, and 5) demonstrate that TM-2, but not TM-1 or TM-3, interacts specifically with the protein in a dose-dependent fashion. The further observation that the TM-2 E69A peptide did not bind to or inhibit the DGK protein (Fig. 4, ac) reinforces the structural significance of Glu-69 while also providing insight into the mechanism of TM-2 binding. The necessity for a glutamic acid residue at position 69 suggests that the peptide/protein interaction is mediated, at least in part, by an interhelical H-bond, either to Glu-69 or another distal residue. This H-bond in the functional DGK protein would provide a rigid electrostatic link that may serve to stabilize the oligomeric state of DGK in vivo.

Previous work demonstrated that DGK contains three active sites per trimer, wherein each of these active sites is formed from two half-sites contributed by different monomers (25). In this framework, free monomers lack a complete active site and are thus enzymatically inactive. As a mechanism for TM-2 inactivation of DGK, we may therefore propose a scenario involving the formation of peptidedimer-proteinmonomer pseudo-trimeric complexes (Fig. 6) as arises in mixing experiments in SDS detergent (Fig. 2b, lane 5), where we noted the appearance of a novel band corresponding to a molecular weight that would be expected for a peptide-protein complex with a 2:1 stoichiometry. Additionally, as indicated in Fig. 6, populations of 1:1 protein-peptide complexes could also lead to inhibitory species. Complex formation is further validated by the concomitant reduction in both DGK monomer and free TM-2 band intensities (compare Fig. 2b, lanes 1 and 4 with lane 5) when TM-2 is mixed with protein. Such an interaction effectively reduces the monomeric DGK species present in the monomer-dimer-trimer reaction scheme, thereby impeding the equilibrium toward DGK trimers and hence resulting in the observed reduction of enzymatic activity (Fig. 3). The absence of any interaction between peptide with the DGK dimeric population likely stems from the fact that free TM-2 peptide already forms an SDS-resistant dimer. Similarly, the lack of any significant reduction of the dimeric or trimeric populations of DGK in the presence of TM-2 may reflect slow equilibration of proteins in SDS detergent micelles. Because TM-2 appears to associate only with the DGK monomer in SDS-PAGE experiments, the ability of this peptide to inhibit DGK enzyme activity suggests that either DGK is a mixture of species including monomer in decyl maltoside micelles and/or that the self-association of DGK trimers in the native state is of sufficiently low affinity as to be susceptible to competition/dissociation in the presence of added TM-2 peptide.



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FIG. 6.
Proposed mechanism for TM-2 inactivation of DGK protein. The trimeric protein species (bottom left) with three complete active sites is shown in equilibrium with dimeric and monomeric DGK protein species with one and zero active site(s), respectively. Binding of the TM-2 peptide dimer to the monomeric DGK species would result in the off-pathway formation of inactive pseudo-trimer complex (bottom right), thereby shifting the overall protein equilibrium away from the fully functional DGK trimer toward less or non-functional lower order oligomers. Numbers in the circles refer to TM-1, TM-2, and TM-3 helices.

 

As an alternative mechanism for DGK inhibition, the results obtained here cannot explicitly exclude the possibility that the TM-2 peptide may directly bind to and alter intramolecular rather than intermolecular packing of the DGK helices. For example, the TM-2 peptides may associate with either TM-1 or TM-3 and alter the arrangement of the helices within the same molecule, thereby disrupting the architecture of the DGK protein. However, the affinities for such intramolecular associations appear to be weak, as we found no significant heterodimerization between each of the TM peptides when mixed together in all possible combinations on SDS-PAGE (data not shown).


    CONCLUSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
A single TM helical peptide corresponding to TM-2 of the homotrimeric membrane protein diacylglycerol kinase has been demonstrated to possess sufficient specificity to bind to the protein and to inhibit its enzymatic function. As inhibitors, TM-based peptides have the advantages of synthetic accessibility and the autonomous folding nature and binding specificity of their membrane-inserted helices. The potential of TM peptides as therapeutics is augmented if these species are synthesized as Lys-tagged constructs, as water-solubility expands the scope of routes for drug delivery. Such modulation of membrane protein activity by TM peptides is a potentially effective paradigm for the treatment of pathogenic infections and certain forms of human disease.


    FOOTNOTES
 
* This work was supported in part by grants from the Canadian Institutes of Health Research (CIHR) (to C. M. D.), the Natural Sciences and Engineering Research Council of Canada (NSERC) (to C. M. D.), and the National Institutes of Health (to J. U. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work and hold Canadian Institutes of Health Research Doctoral Awards. Back

|| To whom correspondence should be addressed: Research Institute, Structural Biology & Biochemistry, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5924; Fax: 416-813-5005; E-mail: deber{at}sickkids.ca.

1 The abbreviations used are: TM, transmembrane, DGK, diacylglycerol kinase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
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