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

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 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 (1Popot J.L. Engelman D.M. Biochemistry. 1990; 29: 4031-4037Crossref PubMed Scopus (821) Google Scholar). In this ”two-stage“ model, transmembrane (TM) 1The abbreviations used are: TM, transmembrane, DGK, diacylglycerol kinase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.1The abbreviations used are: TM, transmembrane, DGK, diacylglycerol kinase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. segments first fold into stable α-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 (2Popot J.L. Gerchman S.E. Engelman D.M. J. Mol. Biol. 1987; 198: 655-676Crossref PubMed Scopus (244) Google Scholar, 3Wang C. Deber C.M. J. Biol. Chem. 2000; 275: 16155-16159Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 4Ding F.X. Xie H. Arshava B. Becker J.M. Naider F. Biochemistry. 2001; 40: 8945-8954Crossref PubMed Scopus (39) Google Scholar, 5Melnyk R.A. Partridge A.W. Deber C.M. Biochemistry. 2001; 40: 11106-11113Crossref PubMed Scopus (84) Google Scholar) 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 (6Lemmon M.A. Flanagan J.M. Hunt J.F. Adair B.D. Bormann B.J. Dempsey C.E. Engelman D.M. J. Biol. Chem. 1992; 267: 7683-7689Abstract Full Text PDF PubMed Google Scholar, 7Mingarro I. Elofsson A. von Heijne G. J. Mol. Biol. 1997; 272: 633-641Crossref PubMed Scopus (36) Google Scholar), the influenza A M2 proton channel (8Bauer C.M. Pinto L.H. Cross T.A. Lamb R.A. Virology. 1999; 254: 196-209Crossref PubMed Scopus (61) Google Scholar), phospholamban (9Arkin I.T. Adams P.D. MacKenzie K.R. Lemmon M.A. Brunger A.T. Engelman D.M. EMBO J. 1994; 13: 4757-4764Crossref PubMed Scopus (174) Google Scholar), synatobrevin (10Fleming K.G. Engelman D.M. Proteins. 2001; 45: 313-317Crossref PubMed Scopus (49) Google Scholar), and the major coat protein from M13 bacteriophage (3Wang C. Deber C.M. J. Biol. Chem. 2000; 275: 16155-16159Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 11Deber C.M. Khan A.R. Li Z. Joensson C. Glibowicka M. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11648-11652Crossref PubMed Scopus (89) Google Scholar, 12Melnyk R.A. Partridge A.W. Deber C.M. J. Mol. Biol. 2002; 315: 63-72Crossref PubMed Scopus (61) Google Scholar). 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 (13Gerber D. Shai Y. J. Biol. Chem. 2001; 276: 31229-31232Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and two multi-spanning G-protein coupled receptors (14Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar, 15Tarasova N.I. Rice W.G. Michejda C.J. J. Biol. Chem. 1999; 274: 34911-34915Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) 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 α-helices in micelles and participate in native-like helix-helix contacts (5Melnyk R.A. Partridge A.W. Deber C.M. Biochemistry. 2001; 40: 11106-11113Crossref PubMed Scopus (84) Google Scholar). In this work, we have synthesized the three putative TM helices from Escherichia coli diacylglycerol kinase (DGK) (16Smith R.L. O'Toole J.F. Maguire M.E. Sanders II, C.R. J. Bacteriol. 1994; 176: 5459-5465Crossref PubMed Google Scholar), a ∼13-kDa membrane protein that directly phosphorylates diacylglycerol (DAG) by Mg·ATP (17Badola P. Sanders II, C.R. J. Biol. Chem. 1997; 272: 24176-24182Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and exists as a homotrimer in its functional state (18Vinogradova O. Badola P. Czerski L. Sonnichsen F.D. Sanders II, C.R. Biophys. J. 1997; 72: 2688-2701Abstract Full Text PDF PubMed Scopus (63) Google Scholar). 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 (19Nagy J.K. Lau F.W. Bowie J.U. Sanders C.R. Biochemistry. 2000; 39: 4154-4164Crossref PubMed Scopus (43) Google Scholar). 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. TM Finder—Transmembrane sequences of DGK chosen for synthesis were identified using the web-based program TM Finder (www.bioinformatics-canada.org/TM/) (20Deber C.M. Wang C. Liu L.P. Prior A.S. Agrawal S. Muskat B.L. Cuticchia A.J. Protein Sci. 2001; 10: 212-219Crossref PubMed Scopus (111) Google Scholar) 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 (5Melnyk R.A. Partridge A.W. Deber C.M. Biochemistry. 2001; 40: 11106-11113Crossref PubMed Scopus (84) Google Scholar). DGK proteins were prepared and purified as described previously (21Lau F.W. Bowie J.U. Biochemistry. 1997; 36: 5884-5892Crossref PubMed Scopus (226) Google Scholar); 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 (25Lau F.W. Chen X. Bowie J.U. Biochemistry. 1999; 38: 5521-5527Crossref PubMed Scopus (50) Google Scholar). 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. 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 (16Smith R.L. O'Toole J.F. Maguire M.E. Sanders II, C.R. J. Bacteriol. 1994; 176: 5459-5465Crossref PubMed Google Scholar, 22Sanders II, C.R. Czerski L. Vinogradova O. Badola P. Song D. Smith S.O. Biochemistry. 1996; 35: 8610-8618Crossref PubMed Scopus (60) Google Scholar) 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 (20Deber C.M. Wang C. Liu L.P. Prior A.S. Agrawal S. Muskat B.L. Cuticchia A.J. Protein Sci. 2001; 10: 212-219Crossref PubMed Scopus (111) Google Scholar). 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 (16Smith R.L. O'Toole J.F. Maguire M.E. Sanders II, C.R. J. Bacteriol. 1994; 176: 5459-5465Crossref PubMed Google Scholar) 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 (3Wang C. Deber C.M. J. Biol. Chem. 2000; 275: 16155-16159Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 5Melnyk R.A. Partridge A.W. Deber C.M. Biochemistry. 2001; 40: 11106-11113Crossref PubMed Scopus (84) Google Scholar, 12Melnyk R.A. Partridge A.W. Deber C.M. J. Mol. Biol. 2002; 315: 63-72Crossref PubMed Scopus (61) Google Scholar, 23Liu L.P. Deber C.M. Biochemistry. 1997; 36: 5476-5482Crossref PubMed Scopus (104) Google Scholar, 24Partridge A.W. Melnyk R.A. Deber C.M. Biochemistry. 2002; 41: 3647-3653Crossref PubMed Scopus (54) Google Scholar). 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-β-d-glucopyranoside (β-OG) detergent micelles, where they adopt a high degree of helicity as measured by circular dichroism spectroscopy (data not shown). 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 (18Vinogradova O. Badola P. Czerski L. Sonnichsen F.D. Sanders II, C.R. Biophys. J. 1997; 72: 2688-2701Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 25Lau F.W. Chen X. Bowie J.U. Biochemistry. 1999; 38: 5521-5527Crossref PubMed Scopus (50) Google Scholar). 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 (26Lau F.W. Nauli S. Zhou Y. Bowie J.U. J. Mol. Biol. 1999; 290: 559-564Crossref PubMed Scopus (53) Google Scholar). 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 (18Vinogradova O. Badola P. Czerski L. Sonnichsen F.D. Sanders II, C.R. Biophys. J. 1997; 72: 2688-2701Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 26Lau F.W. Nauli S. Zhou Y. Bowie J.U. J. Mol. Biol. 1999; 290: 559-564Crossref PubMed Scopus (53) Google Scholar). 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. 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 longer The of DGK activity by TM peptides is in Fig. In conjunction with SDS-PAGE these TM-2 as a for DGK folding and function. for Protein and of all DGK from species that, within the TM-2 only the and residues were conserved (data not shown). For our studies, the position was chosen for because previous that this residue an important whereas the residue was to be of the (25Lau F.W. Chen X. Bowie J.U. Biochemistry. 1999; 38: 5521-5527Crossref PubMed Scopus (50) Google Scholar). To into the manner in which TM-2 peptide to and inhibits the DGK protein, we synthesized and purified a TM-2 peptide containing a at position We found that the peptide did not to or inhibit the DGK protein (Fig. 4, a and the did not the dimeric nature of TM-2, indicating that an is not a for the observed wild-type TM-2 peptide dimer (Fig. the did not change in the secondary structure of the TM-2 peptide as by circular dichroism spectroscopy (Fig. Inhibition of whether the by TM-2 peptide displayed we of TM-2 peptide and measured DGK activity (Fig. 5). The that wild-type TM-2 peptide to a dose-dependent decrease in DGK activity, whereas the peptide did the absence of a structure for DGK, previous identified TM-2 as a of the protein homotrimer (19Nagy J.K. Lau F.W. Bowie J.U. Sanders C.R. Biochemistry. 2000; 39: 4154-4164Crossref PubMed Scopus (43) Google Scholar). A of our work was to use designed TM peptides to into the of TM-2 that of TM-1 and TM-3 segments in DGK folding and When the individual TM peptides were run on 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 with the previous that this helix as a TM and can be with sequences Y. J. Bowie J.U. Biol. 1997; PubMed Scopus Google Scholar). The observed of the TM-2 peptide is in with the that helix 2 is in the of the full-length protein. The fact that this helix a in SDS than a homotrimer may some added in the toward DGK for homotrimerization of intact DGK may additional to The TM-3 peptide a in SDS detergent micelles despite the fact that TM-3 peptide did not to or inhibit DGK, that TM-3 likely not in or that it is with than DGK from SDS-PAGE analysis (Fig. and DGK 3, a and and demonstrate that TM-2, but not TM-1 or interacts specifically with the protein in a dose-dependent The that the TM-2 peptide did not to or inhibit the DGK protein (Fig. 4, the of also into the of TM-2 The for a residue at position that the peptide/protein interaction is at in by an to or residue. This in the functional DGK protein a that may to the state of DGK in work demonstrated that DGK three wherein each of these is from two by monomers (25Lau F.W. Chen X. Bowie J.U. Biochemistry. 1999; 38: 5521-5527Crossref PubMed Scopus (50) Google Scholar). In this monomers a and are a for TM-2 of DGK, we may a the formation of (Fig. as in mixing experiments in SDS detergent (Fig. 2b, lane where we noted the appearance of a novel band corresponding to a molecular weight that be for a peptide-protein with a as in Fig. 6, of could also to species. formation is by the reduction in both DGK monomer and TM-2 band Fig. 2b, lanes 1 and 4 with lane when TM-2 is with protein. an interaction the monomeric DGK species in the reaction thereby the toward DGK and in the observed reduction of enzymatic activity (Fig. The absence of interaction between peptide with the DGK dimeric likely from the fact that TM-2 peptide an the of significant reduction of the dimeric or of DGK in the presence of TM-2 may of proteins in SDS detergent TM-2 to only with the DGK monomer in SDS-PAGE experiments, the ability of this peptide to inhibit DGK enzyme activity that DGK is a mixture of species monomer in decyl maltoside micelles that the of DGK in the native state is of affinity as to be to in the presence of added TM-2 peptide. an for DGK the here cannot exclude the possibility that the TM-2 peptide may directly to and than packing of the DGK For the TM-2 peptides may with TM-1 or TM-3 and the arrangement of the helices within the thereby the of the DGK protein. However, the for such to be as we found no significant between each of the TM peptides when in all on SDS-PAGE (data not shown). A TM helical peptide corresponding to TM-2 of the homotrimeric membrane protein diacylglycerol kinase has been demonstrated to specificity to to the protein and to inhibit its enzymatic function. peptides have the advantages of synthetic and the folding nature and specificity of their The of TM peptides as is if these species are synthesized as Lys-tagged as the of for of membrane protein activity by TM peptides is a for the of and of

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