NMR study of the preferred membrane orientation of polyisoprenols (dolichol) and the impact of their complex with polyisoprenyl recognition sequence peptides on membrane structure

Guo-Ping Zhou2 and Frederic A. Troy, II1,3

2 The Center for Hemostasis, Thrombosis and Vascular Biology, Beth Israel Deaconess Medical Center Harvard Medical School, Boston, MA 02115, and 3 Department of Biochemistry and Molecular Medicine, University of California School of Medicine, Davis, CA 95616


1 To whom correspondence should be addressed; e-mail: fatroy{at}ucdavis.edu

Received on August 23, 2004; revised on November 2, 2004; accepted on November 17, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Earlier NMR studies showed that the polyisoprenols (PIs) dolichol (C95), dolichylphosphate (C95-P) and undecaprenylphosphate (C55-P) could alter membrane structure by inducing in the lamellar phospholipid (PL) bilayer a nonlamellar or hexagonal (HexII) structure. The destabilizing effect of C95 and C95-P on host fatty acyl chains was supported by small angle X-ray diffraction and freeze-fracture electron microscopy. Our present 1H- and 31P-NMR studies show that the addition of a polyisoprenol recognition sequence (PIRS) peptide to nonlamellar membranes induced by the PIs can reverse the hexagonal structure phase back to a lamellar structure. This finding shows that the PI:PIRS docking complex can modulate the polymorphic phase transitions in PL membranes, a finding that may help us better understand how glycosyl carrier-linked sugar chains may traverse membranes. Using an energy-minimized molecular modeling approach, we also determined that the long axis of C95 in phosphatidylcholine (PC) membranes is oriented ~ parallel to the interface of the lipid bilayer, and that the head and tail groups are positioned near the bilayer interior. In contrast, the phosphate head group of C95-P is anchored at the PC bilayer, and the angle between the long axis of C95-P and the bilayer interface is about 758, giving rise to a preferred conformation more perpendicular to the plane of the bilayer. Molecular modeling calculations further revealed that up to five PIRS peptides can bind cooperatively to a single PI molecule, and this tethered structure has the potential to form a membrane channel. If such a channel were to exist in biological membranes, it could be of functional importance in glycoconjugate translocation, a finding that has not been previously reported.

Key words: glycosyl carrier lipid (dolichol) structure / glycosyl translocation / nuclear magnetic resonance / polyisoprenyl recognition sequence peptides / polyisoprenol membrane orientation


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
The molecular details of how glycosyl carrier lipid-linked sugar chains are translocated across biological membranes remains an important yet unsolved problem in membrane glycobiology. This unresolved problem transcends issues of fundamental importance in both prokaryotic and eukaryotic biology. Synthesis of all major bacterial cell envelope structures, for example, requires the participation of sugars linked to undecaprenylphosphate (C55-P), whereas synthesis of all N-linked glycoproteins and some O-linked and glycosyl phosphatidylinositol anchors in eukaryotes require that the sugars be linked to dolichylphosphate (C95-P). The complexity of figuring out translocation pathways is exemplified by the fact that a number of elegant biochemical and molecular genetic approaches over the past ~ 25 years have not been able to determine how the sugar moieties are moved from one side of the membrane to the other.

To gain insight into this problem, our initial idea was to isolate a simpler aspect of the problem that could be analyzed and understood by nuclear magnetic resonance (NMR). These earlier studies focused on the molecular motions and conformations of the polyisoprenols (PIs) in model phospholipid (PL) membranes. The results of these extensive electron paramagnetic resonance (EPR), and 1H-, 2H- and 31P-NMR studies were recently summarized (Zhou and Troy, 2003Go). Lacking in these studies, however, was an effort to determine if there was a direct physical interaction between the PIs and any glycosyltransferase. Following the discovery of a postulated dolichol recognition sequence (DRS), or the more generic PI recognition sequences (PIRS) in three yeast glycosyltransferases, Dpm1, Alg1, and Alg7 (Albright et al., 1989Go; Datta and Lehrman, 1993Go; Kelleher et al., 1992Go; Scocca and Krag, 1990Go; Troy, 1992Go; Zhu and Lehrman, 1990Go; Zimmerman and Robbins, 1993Go), some studies provided new insight into this problem that suggested a biophysical approach to study the possible role that these sequence motifs and the PIs may play in biosynthetic and translocation processes. This was an alternate strategy to the molecular genetic approaches that had led to ambiguity in experimental findings on the functional importance of DRS/PIRS in membrane-mediated glycosylation and translocation reactions. Site-directed mutagenesis studies showed, for example, that such conserved residues were essential for hamster UDP-N-acetylglucosamine:dolichol-P N-acetylglucosamine-1-P transferase activity (Dan et al., 1996Go). In contrast, deletion analysis of the hydrophobic DRS coding region in the yeast Dol-P-Man synthase gene showed that the consensus sequence was not essential for protein glycosylation (Zimmerman and Robbins, 1993Go). These conflicting results thus emphasized the potential limitation of deletion and mutational approaches to determine unambiguously the functional importance of a DRS/PIRS binding domain and highlighted the need for direct structural information.

The results of our recent 2D 1H-NMR nuclear Overhauser enhancement spectroscopy (NOESY) studies have confirmed a highly specific interaction between the PIs and the PIRS peptide in NeuE, a protein implicated in the membrane-directed synthesis of polysialic acid (polySia) in neuroinvasive Escherichia coli K1 (Zhou and Troy, 2003Go). Key contact amino acids were also identified in PIRS peptides from Dpm1 and Alg7, two eukaryotic glycosyltransferases involved in N-linked glycosylation. It thus appears that these recognition sequences are the active domain of PI-binding proteins that constitute a highly specific binding motif for interacting with the PIs. These studies also showed that the conformation and motional properties of both the PIs and PIRS peptides changed after docking (Zhou and Troy, 2003Go). Furthermore, our discovery of a PIRS in KpsM, a protein with no known catalytic function but implicated only in the translocation of polySia chain across the inner membrane in E. coli K1 (Pavelka et al., 1991Go; Pigeon and Silver, 1994Go), led to the proposal that the unusual length and poly-cis geometry of these PIs may provide a flexible matrix for the spatial organization of multienzymes to coordinate biosynthesis and translocation (Troy, 1992Go). Support for this hypothesis has now come from our 2D 1H-NMR NOESY and energy minimization studies. 3D structures of the molecular complexes between the PIs and PIRS peptides has revealed that a single PI molecule can bind more than one PIRS peptide. These findings thus support the hypothesis that the PIs may serve as a structural scaffold to organize and tether in functional domains PIRS-containing proteins within multiglycosyltransferase complexes that participate in both biosynthetic and translocation processes (Zhou and Troy, 2003Go). Thus, it is not only protein–protein interactions between glycosyltransferases within the endoplasmic reticulum that may be important for synthesis and translocation but protein–PI interactions as well.

Based on these findings, we initiated the present 1H- and 31P-NMR spectroscopic studies to determine how the PIRS:PI interactions may influence the conformational and motional properties of PL membranes and the preferred orientation of the PIs in model membranes. The objectives of the present studies were fourfold: (1) to determine by 1H- and 31P NMR how PIs and PIRS peptides interactions may influence the organization of the PL fatty acyl chains in model PL membranes; (2) to determine by 31P-NMR if the PIRS peptides can reverse the ability of the PIs to induce nonlamellar states in PL membrane; (3) to determine by energy minimized molecular modeling the preferred orientation of C95 and C95-P in model membranes, as a first approximation in understanding how PIs may modulate membrane PL polymorphism; and (4) to determine the number of PIRS peptides that can bind to a single PI molecule and the 3D structure of the binding complex. The results from our spectroscopic studies show that the addition of a PIRS peptide to nonlamellar membranes induced by the PIs can reverse the nonlamellar hexagonal phase back to a lamellar structure. Our molecular modeling calculations have shown that as many as five PIRS peptides can bind to a single PI molecule. These findings lead to the hypothesis that formation of a hexagonal structure induced by the PIs may have the potential of forming a membrane channel that could facilitate glycoconjugate translocation processes. This is an alternate hypothesis to the possible existence of hypothetical flippases to accomplish movement of hydrophilic sugar chains across hydrophobic membranes.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Rationale for studying C95 and C95-P and different PIRS peptides
Although most of the experiments in this study were carried out using the PIs, C95 and C-95-P, and the PIRS peptide NeuE, studies using C55-P and the PIRS peptides Dpm1 and Alg7 were also performed. This was done to compare different parameters, including the phosphorylated and nonphosphorylated PIs and their chain length, and to determine if the prokaryotic (NeuE) and eukaryotic (Dpm1 and Alg7) PIRS peptides behaved similarly. The rationale for using the different PIs was also to determine experimentally their molecular motions and preferred orientation in the membrane, and if the different species had the same or different effect on perturbing membrane structure. Similarly, the use of different PIRS peptides allowed us to determine if each species could modulate or reverse the nonlamellar conformation induced by the PI back to a bilayer structure. This hypothesis was predicted from the structural similarities among the class of the PIs and the PIRS peptides, as reported previously (Zhou and Troy, 2003Go), but it has not been tested experimentally. The basis of our present studies follows from these earlier findings that predicted that the close structural similarities between the 3D structure of C55-P and C95-P and NeuE and DPM1 could allow other PIRS peptides to be used to study the specificity of their interactions with the PIs (Zhou and Troy, 2003Go, p. 59). The findings reported herein support this supposition. Finally, a major portion of the poly-cis-polyisoprenols (PIs) in both prokaryotic and eukaryotic membranes are often present as the "inactive" or free alcohol (or fatty acid ester). Relatively little is known about the conformation or function of these abundant, neutral PIs. Our studies thus provide new information on the organization and motions of both the free and phosphorylated forms of the PIs within the membrane (Table I), findings that have not been previously described.


View this table:
[in this window]
[in a new window]
 
Table I. Effect of the PIRS peptide:C95 binding complex on the motion of the host phospholipid head groups in PE:PC (2:1) membranes

 

Chemical shift differences in 1H-NMR spectra demonstrate that C95 and C95-P interact directly with host phospholipids to alter membrane structure
To determine if C95 and C95-P could interact directly with host PLs and if so could alter membrane structure, control 1D 1H-NMR studies were carried out to measure the chemical shifts of the methyl (CH3), methylene (CH2), and -N(CH3)3 protons in 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC) vesicles alone. These chemical shifts were recorded to be 0.88, 1.28, and 3.24 ppm, respectively (Figure 1A[a]).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. 500 MHz 1D 1H-NMR spectra reveals differences in chemical shift of protons in DOPC vesicles containing C95, C95-P, and the Dpm1:PI binding complex. (A): (a) DOPC vesicles alone; (b) DOPC vesicles containing 10 mol % C95; (c) DOPC vesicles containing 10 mol % of the PIRS peptide, Dpm1; (d) DOPC vesicles containing 10 mol % each of Dpm1 and C95. (B): (a) DOPC vesicles alone; (b) DOPC vesicles containing 10 mol % C95P; (c) DOPC vesicles containing 10 mol % each of Dpm1 and C95-P. Small unilamellar vesicles were prepared from aqueous dispersions of DOPC by sonication as described under Materials and methods. All spectra were recorded in D2O at 25°C. 1H shifts were measured relative to the methyl proton resonance of internal DSS (0 ppm).

 

To examine the extent of membrane perturbation induced by C95, 10 mol % of the PI was incorporated into the same vesicles, resulting in a chemical shift of the same protons to 0.82, 1.24, and 3.21 ppm, respectively (Figure 1A[b]). These results were consistent with a hydrophobic interaction occurring between C95 and the CH3 and CH2 protons in the fatty acyl chains of DOPC, likely reflecting a change in membrane structure. Similarly, after incorporating 10 mol % C95-P into the vesicles, the chemical shift of the same host protons changed to 0.83, 1.24, and 3.19 ppm, respectively (Figure 1B[b]). Changes in the chemical shift induced by C95-P in the -N(CH3)3 protons was slightly greater (3.25 versus 3.19) than those induced by C95 (3.25 versus 3.21). These results thus confirmed a direct interaction between C95 and C95-P and host PLs, suggesting that incorporation of either C95 or C95-P into DOPC vesicles perturbed the structure of the host membrane lipids. The effect was somewhat more pronounced with C95-P than C95. Importantly, these 1H-NMR finding extend results obtained using 31P-NMR (Duijn et al., 1987Go; Knudsen and Troy, 1989Go; Valtersson et al., 1985Go). They are also in accord with and extend earlier 2H-NMR studies showing that the head and tail groups of C95 had nearly identical T1 times in host membranes (de Ropp et al., 1987Go). Furthermore, our molecular structure of C95 (Zhou and Troy, 2003Go) is also consistent with these NMR results and shows that the head and tail groups of the PI are both oriented near the ester carbonyl groups of the PL, which is in close proximity to the aqueous bilayer interface.

1H-NMR studies establish that PIRS peptides alone do not alter membrane structure
To determine if a PIRS peptide itself could alter membrane bilayer structure, 10 mol % Dpm1 was incorporated into DOPC vesicles. 1H-NMR spectra showed the chemical shifts of the CH3, CH2, and -N(CH3)3 protons were shifted only slightly (0.88, 1.27, and 3.23 ppm, respectively), relative to the same protons in DOPC vesicles (Figure 1B[c]).In contrast to the chemical shift induced by C95 and C95-P, these values were nearly identical to that of DOPC membranes alone. A similar finding was obtained with two other PIRS-containing peptides, NeuE and Alg7 (data not shown). Relative to the change in membrane structure induced by the PIs, these results suggested a much weaker interaction occurs between the relatively hydrophobic PIRS peptides and the fatty acyl chains in DOPC vesicles. On this basis, we conclude that the PIRS peptides alone have little perturbing effect on membrane PL structure.

1H-NMR spectroscopy shows that the C95- or C95-P:Dpm1 binding complex can reverse the nonlamellar structure induced by the PIs back to a lamellar conformation
When 10 mol % of C95 and Dpm1 were incorporated together into DOPC vesicles, forming a PI:PIRS binding complex (Zhou and Troy, 2003Go), the chemical shift in the CH3, CH2, and -N(CH3)3 protons was determined to be 0.90, 1.29, and 3.27 ppm, respectively (Figure 1A[d]). These values were nearly identical to the corresponding chemical shifts in DOPC vesicles alone (Figure 1A[a]), and in contrast to the larger chemical shifts induced by C95 or C95-P (Figure 1A[b] and 1B[b]), respectively. These results suggested that the nonlamellar conformation induced by C95 was reversed back to a lamellar bilayer structure by the C95:Dpm1 peptide binding complex. Similarly, the C95-P:Dpm1 peptide complex could also reverse the nonlamellar membrane structure induced by C95-P back to a bilayer configuration (Figure 1B[c]). It thus appears that PIRS peptides, when in complex with C95 or C95-P, can modulate the change in membrane structure induced by the PIs. The ability of PIs to destabilize the membrane and for the PI:PIRS peptide binding complex to transiently modulate this disordering could be an important factor in understanding how glycoconjugates might traverse biological membranes, as developed next.

31P-NMR studies confirms that the PI-induced nonlamellar phase in PL membranes can be reversed by the PI:PIRS peptide binding complex
31P-NMR spectroscopy records the time averaged powder pattern behavior of PL head group orientations and has been used to characterize and determine different organizational states of PL in membranes (Cevc and Marsh, 1987Go; Chan et al., 1981Go; Cullis and de Kruijff, 1979Go; Devaux et al., 1986Go; Duijn, 1987Go; Kohler and Klein, 1977Go; Valtersson et al., 1985Go). Our previous studies used phosphatidylcholine (PC) or phosphatidylethanolamine (PE):PC (2:1) vesicles to measure the 31P-NMR spectra of the PL head groups in the presence and absence of PIs (Knudsen and Troy, 1989Go; Troy, 1991Go). These studies showed that 5 mol % C55 or C95 or their phosphorylated derivatives C55-P and C95-P, could induce a nonlamellar organization of the PL in membranes, consistent with structural features interpreted by others to be a hexagonal (HexII) structure (Duijn, 1987Go; Cullis and de Kruijff, 1979Go; Gruner, 1992Go; Kohler and Klein, 1977Go; Valtersson et al., 1985Go).

In the present study, we carried out 31P-NMR experiments using 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE):DOPC (2:1) vesicles to extend these observations and, importantly, to determine what effect the PI:PIRS peptide complex might have on modulating or reversing the membrane lipid polymorphism induced by the PIs. As shown in Figure 2a, proton-decoupled31P-NMR showed a spectrum expected for model membranes where the PLs exhibit a typical bilayer configuration, characterized by a broad asymmetrical line shape resulting from the anisotropic motions of the PL head groups, as described in previous work.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2. 31P-NMR evidence that the nonlamellar phase in aqueous dispersions of DOPE:DOPC membranes induced by C95 can be reversed by the PIRS peptide, NeuE. Proton decoupled 31P-NMR spectra of DOPE:DOPC (2:1) vesicles containing C95 and the PIRS peptide, NeuE. (a) DOPE:DOPC (2:1) vesicles alone; (b) DOPE:DOPC (2:1) vesicles containing 5 mol %; (c) DOPE:DOPC (2:1) vesicles 5 mol % each of C95 and NeuE. Aqueous dispersions of the multilamellar vesicles were prepared as described under Materials and methods, and all spectra were recorded in D2O at 18°C. DSS was used as a chemical shift reference set at 0 ppm.

 

The overall low field shoulder and higher field signal with a chemical shift range of ~ 40 ppm is in agreement with that reported for the lamellar bilayer line shape profile. This bilayer spectrum is distinctly different, however, from the sharp 31P NMR spectrum seen after incorporating 5 mol % C95 into the vesicles (Figure 2b). As shown, the addition of the PI decreased the line width nearly twofold, and the asymmetry of the spectrum was reversed. These spectral features are the characteristic hallmarks of PLs in the nonlamellar (HexII) phase wherein an isotropic peak is shifted downfield by ~ 5 ppm, and the broad component (~ 20 ppm) is shifted upfield. Nearly identical spectra were recorded when 5 mol % C95-P was incorporated into the same PL vesicles (data not shown). As the 31P-NMR spectra shown in Figure 2b reflects the mirror image of the lamellar spectrum (Figure 2a), we conclude that C95 (and C95-P) can induce a nonlamellar phase organization of the PL molecules in the membrane, a result in accord with our 1H-NMR findings as described. (See the cited references for a full description of the 31P-NMR spectral characteristics of lamellar (BL) and nonlamellar (non-BL) membrane structure).

When 5 mol % of NeuE and C95 were incorporated together into DOPE:DOPC (2:1) vesicles, the line shape of the proton-decoupled 31P-NMR spectrum and the chemical shift of the peaks (Figure 2c) became nearly identical to the lamellar spectrum of DOPE:DOPC alone (Figure 2a). As shown in Figure 2c, the nonlamellar features of the 31P-NMR line shapes in 2b decreased, whereas the anisotropic features characteristic of the bilayer phase became more prominent. We interpret this finding to mean that the C95:NeuE binding complex can reverse the formation of the nonlamellar phase induced by C95,as shown in Figure 2b. We believe this is an important finding because it demonstrates that the PI:PIRS peptide binding complex can modulate the phase state of host PLs. A similar finding was observed with C95-P, or when the PIRS peptide from Alg7, a yeast glycosyltransferase, also predicted to bind C95-P (Albright et al., 1987) was incorporated into C95-containing PL membranes (data not shown). These results thus provide further evidence that a thermodynamically favorable equilibrium exists between lamellar and nonlamellar conformations and the PI:PIRS peptide binding complex can modulate or shift the equilibrium from the nonlamellar phase induced by the PI back to the lamellar conformation.

Molecular motions of PIs in model membranes
Table I shows the 31P-NMR-derived longitudinal or spin-lattice relaxation times (T1) for different proton resonances of the host PL head groups in the presence and absence of C95 and the PIRS peptides, NeuE, as studied in Fig. 2.

The host PL head groups showed increased T1 times when C95 was incorporated into PE:PC vesicles (560 ms) compared with the T1 values of the vesicles alone (385–420 ms). In the presence of the C95:NeuE peptide binding complex, however, the T1 time was shifted back to that of the PL bilayer (385–420 ms). Although the molecular motions that occur in membranes are very complex, as summarized by Wuthrich (1987)Go, T1 is sensitive to the slower motion (i.e., longer tc values) as observed for our PE:PC vesicles in Table I. The converse, that faster motions are associated with decreased correlation times, also generally holds. Consistent with this interpretation is the 31P line shape, which is known to depend directly on correlation times that reflect lipid motion (Chan et al., 1981Go and references therein; McLaughlin et al., 1977Go). Accordingly, we interpret our T1 results to suggest that the PL head groups had decreased correlation times and therefore faster motions in the presence of C95, as predicted for the organization of PL in the nonlamellar state. Taken together, these T1 results provide additional information on the rates of lipid motions that correlate with the motional and conformational states of the PL, as observed by 31P-NMR (Figure 2). These T1 findings thus provide further evidence that the PI:PIRS peptide binding complex can modulate the change in membrane structure induced by the PIs alone.

It is likely that the molecular motions, membrane organization, and 3D structures of both the free and phosphorylated PIs are relevant to their function and to a better understanding of the precursor–product relationship between C95 and C95-P. For example, our finding that C95 is oriented to one leaflet of the bilayer in contrast to the orientation of C95-P in both leaflets (see later discussion) could be important for a better understanding of the topology/function of dolichol kinase and/or Dol-P and Dol-P-P phosphatases. Thus models of the topology and how/where C95 is phosphorylated and Dol-P and Dol-P-P dephosphorylated should consider the limits imposed by the orientation of both C95 and C95-P in the membrane and their molecular motions.

Earlier, de Ropp et al. measured the T1 time of the acetyl ester head group and tail group for 2H-labeled PIs, using 2H-NMR (de Ropp and Troy, 1985Go; de Ropp et al., 1987Go). These studies showed that the head and tail groups of the neutral PIs had the same T1 and correlation times. More recently, we measured the T1 times of the poly-CH, poly-CH2, and poly-CH3 protons in the coil region of C95 and C95 -P by 1H-NMR (Zhou and Troy, 2003Go). These findings showed that the T1 value of the poly-CH protons in C95 (910 ms) was close to that of the CH3 protons adjacent to the head group (950 ms), whereas the T1 value of the poly-CH protons in C95-P (840 ms) was much shorter than that of the CH3 protons adjacent to the head group of C95-P (1190 ms) (Zhou and Troy, 2003Go, table 5). These studies thus confirmed that the three segments of C95, in contrast to C95-P, have motions similar to that expected for a location within the bilayer interior. Our molecular modeling of the preferred orientation of C95 in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) membranes to be described also showed that the head, tail, and the central coiled regions of C95 were localized on the same side of the bilayer and in close apposition with the bilayer interface (see later discussion). This orientation accounts for the different T1 values between these same three regions of C95-P, which are much greater than the T1 values and orientation of C95 (Zhou and Troy, 2003Go).

Based on 31P-NMR spectra of C95-P in digalactosyl-diglyceride, Valtersson et al. (1984) concluded that the charged phosphate group undergoes motions similar to those experienced by PL head groups in a bilayer. Duijn et al. (1987) presented a model in which the phosphate head group of an extended form of C95-P (~ 100 Å) was located at the lipid–water interface, a minor part of the isoprene chain ran parallel to the fatty acid chains, and most of the nonpolar isoprene units were located between the two monolayers. Our NMR and molecular modeling studies of the PIs have shown, however, that the energy-minimized structures do not exist in an extended conformation but rather are tripartite molecules, with their three domains arranged in a coiled, helical structure with lengths of 32 Å (C95-P), 33 Å (C95), and 22 Å (C55-P) (Zhou and Troy, 2003Go, figure 5). With this new structural information, we carried out studies to determine the preferred orientation of C95 and C95-P in PL membranes and the impact that their complex with the PIRS peptides have on membrane structure.

Thermodynamic reversibility of the C95-induced nonlamellar phase transition in DOPE/DOPC membranes
The 31P-NMR spectra in Figure 2c showed that the PIRS peptide, NeuE, was able to reverse the nonlamellar phase induced by C95 in multilamellar dispersions of DOPE/DOPC membranes. The rate of this reversibility was relatively fast, as the spectral change back to bilayer occurred under ~2 h at 18°C. This suggested that the NeuE:C95 binding complex stabilized the bilayer form of the lipids. This finding led us to ask what the spontaneous rate of reversibility of the C95-induced HexII phase back to bilayer would be in the absence of the PIRS peptide. As shown in Figure 3, the reversibility of the phospholipid polymorphism in DOPC/DOPE membranes containing 10 mol % C95 occurred at a significantly slower rate.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3. Thermodynamic reversibility of nonlamellar phase transition in DOPC/DOPE membranes. induced by C95. The nonlamellar phase in lipid dispersions of DOPC/DOPE (2:1) multilamellar vesicles was induced by incorporating 10 mol % of C95, as described in the text. 31P-NMR spectra were recorded on the same sample after 2, 3, 4, 5, and 6 days, as shown in panels a–e, respectively. All spectra were recorded in D2O at 18°C, and DSS was used as a chemical shift reference set at 0 ppm.

 

Figure 3a shows spectral features of a nonlamellar phase, characterized by the relatively sharp signal at the resonance position of the PLs undergoing isotropic motion (~4 ppm), and the reversed asymmetry of the spectrum. This phase reverted back to a near bilayer spectrum after six days (Figure 3e). Intermediate spectra consistent with a combination of both lamellar and nonlamellar phases during this transition are shown in parts b–d. We interpret the reversibility of the nonbilayer to bilayer structure as further evidence that this phase transition is a thermodynamically favorable process. Stabilization of the membrane BL structure by the NeuE:C95 binding complex (Figure 2c) thus shows that membrane PL polymorphism can be modulated by PIRS peptides. This could be important in our understanding of the molecular details of the role that the PIs play in the transbilayer movement of PI-linked sugars across biological membranes.

Determination of the preferred conformation of the PIs and the PI:PIRS peptide complex in phospholipid membranes
Based on EPR studies, a model in which C95 was sandwiched between the two faces of a PL bilayer was proposed (McCloskey and Troy, 1980aGo,bGo). Subsequent 2H-NMR (de Ropp and Troy, 1984Go, 1985Go) and 31P-NMR studies (Knudsen and Troy, 1989Go; Valtersson et al., 1985Go) supported this model and further revealed that both ends of acetyl esterified PIs were localized in the bilayer interior. This led to the conclusion that the shorter chain esters of C10, C15, and C45 did not adopt a conventional "head group at interface" orientation. More detailed information, however, on what is the 3D conformation of the PIs, their preferred orientation in PL membranes, and how this orientation might effect PI interactions with host PL or transmembrane proteins, has not been previously addressed in any system. A helpful beginning to understanding these questions was the recent elucidation of the 3D conformation of the PIs and their binding complexes with PIRS peptides (Zhou and Troy, 2003Go).

A molecular model of dolicholbased on small-angle X-ray scattering and molecular mechanics data was first constructed by Murgolo et al. (1989)Go. The dimensions of C95 were reported to be 53.07 Å (length) x 30.94 Å (width), and the molecule was proposed to consist of three geometrical regions, a central coiled segment and two flanking regions. Based on our recent NMR and energy minimization studies with respect to all atoms using the assisted modeling with energy refinements (AMBER) force field (Chou et al., 2000Go; Weiner et al., 1984Go), we determined a different 3D structures for C95 and also derived structures for C95-P and C55-P (Zhou and Troy, 2003Go). The force constants for the phosphate and pyrophosphate groups in AMBER are now well defined, which was not the case for the earlier modeling studies. In our molecular modeling, the dimensions of C95 were determined to be 31.87 Å (length) x 15.41 Å (width). These findings also revealed that the 3D conformation of C95, C95-P, and C55-P were nearly identical tripartite molecules with their three domains arranged in a coiled, helical structure (Zhou and Troy, 2003Go, figure 5). In our calculations, the lowest steric energy conformations of C95-P and C55-P also gave rise to three geometrical regions, consisting of a central coiled segment and two flanking arms, representing the head and tail domains, respectively. Our models for the PIs further revealed that the coil and tail regions of C95-P and C55-P were more compact relative to that of C95.

In the present study, the preferred orientation of C95 and C95-P in a PL membrane, a bilayer of DMPC was first constructed using energy minimization calculations, as previously described (Zhou and Troy, 2003Go). Our 3D models for both C95 and C95-P were built based on our 2D 1H-NMR NOESY results and refined by energy minimization using the AMBER force field. These structures were then inserted in different orientations in the DMPC bilayer to determine the orientation that resulted in a minimum docking energy for each PI. An important structural feature to emerge from the study of C95 (Figure 4A) was that the long axis of C95 was oriented more parallel to the interface of the bilayer, in contrast to the more perpendicular orientation of C95-P that would be required to span a bilayer (Figure 4B), as described next.



View larger version (96K):
[in this window]
[in a new window]
 
Fig. 4. Preferred orientation of C95 and C95-P in DMPC membranes. Energy-minimized 3D structure of C95 and C95-P in a DMPC bilayer (A and B, respectively) Each model was rotated through 0° (a), 90° (b), and 180° (c). C95 and C95-P are shown in yellow. The oxygen atom of each PI is shown in orange. The DMPC bilayer is shown in blue, and the fatty acyl chains showing the most significant conformational change after insertion of the PI are shown in green. The phosphorus atoms of DMPC are shown in red. Water molecules are shown in pink (oxygen) and white (hydrogen). Energy minimization and molecular modeling calculations were carried out as described under Materials and methods.

 

The C95:DMPC model (Figure 4A) shows the structure as it is rotated through 0°, 90°, and 180°, respectively. Viewing these different angles allows the relative orientation of C95 to be observed in the membrane. From these different perspectives it can be seen that the oxygen atom (orange) at the polar head group of C95 is positioned near the carbonyl group of DMPC. The conformation of the fatty acyl chains also changed on intercalation of C95 into the PC bilayer. The fatty acyl chains showing the most prominent alterations are colored green. Because C95 remains oriented in the upper leaflet of the bilayer, the fatty acyl chains that undergo the most significant conformational change are localized in the middle of the upper monolayer and in close proximity to the coiled region of C95. This orientation indicates that C95 interacts with the PL mainly through hydrophobic interactions between the coil region of the PI and the fatty acyl chains of the PL, a finding supported by the NMR results already described.

Similarly, we sought to determine the preferred orientation of C95-P in a DMPC bilayer. An energy-minimized, space-filling model of C95-P in the DMPC bilayer is shown in Figure 4B. In contrast to the nearly horizontal orientation of C95, the angle between the long axis of C95-P and the interface of the bilayer was about 75°, the phosphorus atom of C95-P being anchored near the aqueous interface. A comparison of the energy minimized models of C95 (Figure 4A) with C95-P (Figure 4B) showed that C95-P was more disruptive of membrane structure because it perturbed a greater number of fatty acyl chains (green) than C95. Again, the fatty acyl chains of the PL are seen to interact with the central coiled segment of C95-P. Similar to C95, C95-P is also sandwiched in the intrabilayer region. Unlike C95, however, C95-P was tilted about 75° in the plane of the bilayer, as noted. A second distinguishing feature was that C95-P extends beyond the bilayer midplane, being partially oriented in both leaflets of the host bilayer (Figure 4B). This is in contrast to the orientation of C95, which is localized principally to only one leaflet at the interface of the bilayer interior (Figure 4A). Based on these findings, we conclude that the preferred orientation of C95 is primarily horizontal to the plane of the bilayer, whereas C95-P is preferentially arranged more perpendicular to the plane of the bilayer. The C95-P orientation is favored by the more hydrophilic phosphate head group on these amphipathic molecules that seek to maximize their interaction with the aqueous interface. In both cases, however, C95 and C95-P molecules are in close apposition with the acyl chains of the host Pls.

To determine the effect of a PI:PIRS binding complex on the structure of DMPC bilayers, a NeuE:C95-P docking structure was inserted into the membrane using the energy-minimization methods that were used to model C95 and C95-P (Zhou and Troy, 2003Go). Steroview structures of this binding complex in DMPC membranes revealed, as in Figure 4, that the head group of C95-P was at the lamellar interface and that a significant portion of the docking structure was located near the middle of the bilayer. They also showed that both C95-P and NeuE interacted with the fatty acyl chains of DMPC (data not shown). These results thus support our NMR findings suggesting that these hydrophobic interactions likely induce the PI-mediated conformational change in membrane structure and the PI:PIRS peptide binding complex reverses the nonlamellar phase back to the bilayer conformation. To determine how this NeuE:C95-P docking structure derived from the NMR results might change after being intercalated into a DMPC bilayer, we modeled the complex after the bilayer was removed, thus revealing an energetically favorable docking structure (Figure 5).



View larger version (47K):
[in this window]
[in a new window]
 
Fig. 5. Energy-minimized, space-filling model of the NeuE:(C95-P) binding structure after removal of the DMPC bilayer. The same energy minimized binding structure shown in Figure 4B was modeled after removal of DMPC bilayer. The structure is shown after rotation through 0° (a), 90° (b), and 180° (c). C95-P is shown in yellow. The three contact amino acids in NeuE that dock to C95-P are shown in turquoise (Ile3 and Ile7) and pink (Leu6). The remaining NeuE residues are shown in green. The phosphorus atom in the head groups of C95-P is shown in red. The 3D structure was built by energy minimization with respect to all atoms using the AMBER force field, as described under Materials and methods.

 

As shown in this structure, the key contact amino acids previously described in NeuE that constituted a binding motif for interacting with the PIs (Ile3, Leu6, and Ile7), still remaineddocked to the central coiled region of C95-P. These energ-minimization finding thus confirm the specificity of the PIRS residues in binding to C95-P, results originally determined by our 2D NMR studies (Zhou and Troy, 2003Go, table 9).

The effect of PI orientation in membranes on the number and cooperative binding of PIRS peptides
Binding of theNeuE peptide to C95 or C95-P induced a different conformation in the peptide resulting from the fact that three protons in the contact amino acids in the peptide are bound to C95, whereas four protons in NeuE mediate binding to C95-P (Zhou and Troy, 2003Go). Binding of NeuE to C95 or C95-P also induced a subtle conformational change in the PI. This change facilitates the binding of subsequent NeuE peptides to the opposite side of the PI. Thus each PI molecule is capable of binding several NeuE molecules. As shown in Table II, an unexpected finding was that the free energy of PIRS binding to the PI became significantly less for each subsequent NeuE bound.


View this table:
[in this window]
[in a new window]
 
Table II. Binding of NeuE to C55-P is cooperative: increase in the free energy of binding of multiple NeuE peptides to a single C55-P molecule

 

The free energy of binding ranged from –9 Kcal/mol for binding of the first NeuE to C55-P to –32 Kcal/mol for binding of the fifth NeuE peptide, which is the maximum number of peptides that can bind a single PI molecule. These results show that binding of the first PI molecule initiated cooperative binding of subsequent PIRS peptides, a finding that may be of biological relevance in the formation of a membrane pore or channel, as will be discussed.

A second important finding to emerge from these studies was that C95 and C95-P have different preferred orientations in host PL membranes, as already described. These differences may account for the different number of PIRS peptides that can bind to a PI. For example, because of the more horizontal orientation of C95 in the interface region between the two leaflets (Figure 4A), the distance between one side of C95 and the bilayer interface is about 3.5 Å. Because the diameter of a PIRS peptide (~16 Å; Zhou and Troy, 2003Go) is greater than this distance, it is possible for a C95 molecule intercalated in a horizontal orientation to bind only three NeuE peptides. In contrast, energy minimization and molecular modeling calculations showed that a C95-P (or C55-P) that is oriented more perpendicular to the plane of the bilayer can bind as many as five NeuE peptides (Table II), because this orientation eliminates the size constraints noted for C95 (Figure 5).

As shown in this figure, protons on Ile3, Leu6, and Ile7 in each NeuE peptide are the key protons that mediate binding to the CH2 and CH3 protons of the PI coil region. In contrast, NeuE peptides are bound to C95 by the protons of amino acid residues Leu1 and Leu6 (Zhou and Troy, 2003Go). The different binding properties of C95 and C95-P between PIRS peptides could potentially influence the catalytic activities of different glycosyltransferases within a multiglycosyltransferase complex. Specifically, the conformational change induced in such a glycosyltransferase on binding to the active, phosphorylated PI within such a complex may be a determinant in regulating enzyme activity. Attenuation in the level of enzyme activity could occur when the productive complex dissociates. Our studies suggest that these catalytic effects may be dependent on specific binding between their transmembrane PIRS sequences and C95-P or C55-P.

Possible involvement of PI:PIRS binding complex in channel formation
As shown in Figure 6A, a single C55-P molecule can tether up to five PIRS peptides to form a potential channel in the membrane. Such a channel might facilitate the transport of N-linked oligosaccharides or other PI-linked sugars, including polysialic acid, across biological membranes. How might it be possible for such a channel to form? We have reported that the 9th or 10th amino acid residue within the PIRS domain of many glycosyltransferases involved in N-linked glycosylation is proline (Zhou and Troy, 2003Go), which leads to a break in the helices of proteins (Piela et al., 1987Go). Other studies have indicated a bias toward proline residues within the transmembrane-spanning domains of proteins involved in transport functions (Brandl and Deber, 1986Go; Deber et al., 1986Go). Although the significance of these findings is not fully understood, the cyclic side chain of proline prevents it from forming a hydrogen bond with the amino acid residue in the preceding turn of the {alpha}-helix. This may favor the formation of structures in which a hydrogen bond is provided by a specific interaction with a residue from another membrane protein or lipid. This could facilitate polar interactions between several different or identical PIRS-containing proteins that are tethered to the same PI molecule. The result of these interactions may induce the conformational change in both the PI and PIRS peptides that we have reported (Zhou and Troy, 2003Go), and the subsequent release of the PI from the PI:PIRS complex, with the concomitant formation of a channel. An energy-minimized model of such a PI-induced channel consisting of five PIRS-containing peptides is shown in Figure 6B.



View larger version (68K):
[in this window]
[in a new window]
 
Fig. 6. Energy-minimized 3D model of five NeuE peptides binding to a single C55-P molecule. (A) Front (a) and vertical view (b) of an energy-minimized binding structure consisting of the (NeuE)5:C55-P docking structure. The binding complex is modeled after removal of DMPC bilayer. C55-P is shown in yellow. The three contact amino acids in each of the five NeuE peptides that bind C95-P are shown in pink (Ile3 and Ile7) and turquoise (Leu6). The remaining NeuE residues are shown in green. The phosphorus atom in C55-P is shown in red. (B) An energy-minimized 3D structure of the (NeuE)5:C55-P binding complex shown in A after removal of C55-P. The front and vertical views of the model are shown in (a) and (b), respectively. The NeuE peptides are color coded as described in A. The 3D structure was built by energy minimization with respect to all atoms using the AMBER force field, as described under Materials and methods.

 

Based on our experimental data and the molecular modeling calculations, the insertion of C95 or C95-P into host PL membranes induces small changes in the thickness of bilayer and the distance between neighboring phosphorus atoms (Zhou and Troy, 2003Go).These changes reflect a conformational change in the head groups of the PC membranes. Studies carried out by Janas et al. (1994)Go indicated that C55-P decreased DOPC membrane conductance and ionic permeability. This means that the translocation of the highly polar and anionic charged polysialic acid across the inner membrane in E. coli K1, for example, cannot depend solely on C55-P, results in agreement with our findings that a tethered complex of a PI with PIRS may be important. Although the effect on membrane permeability by PIs is not well established, our NMR studies show that docking between the NeuE peptide and C95 in PE:PC (2:1) vesicles can inhibit the rapid motion of the PIs and thereby change the nonlamellar phase induced by C95 or C95-P back to a lamellar structure. This implies that the reversible bilayer to nonbilayer change may be controlled by whether a PI molecule is free within the membrane and therefore capable of inducing the nonlamellar configuration or bound to a PIRS-containing protein, in which case it can change membrane structure back to a bilayer.

Based on previous studies, we proposed that PIs may function as a flexible matrix or scaffolding to organize and tether proteins of multienzyme complexes to coordinate both biosynthetic and translocation processes (Troy, 1992Go; Zhou and Troy, 2003Go). In the present study, we extend this experimentally testable model by showing that inverted hexagonal (HexII) membranes have the potential to form hydrophilic pores in PL membranes, and that the PIs can induce such lipid polymorphism. An attractive feature of such a model is that it may lessen the energy barrier for the vectorial translocation of hydrophilic sugar chains across or through biological membranes. Thus lipid polymorphism in membrane structure between the thermodynamically favorable bilayer-nonlayer structure is a model in contrast to the flip-flop model proposed by Waechter and colleagues. In this model, flippases are hypothesized to catalyze the movement of C95-P-linked sugars across hydrophobic membranes (Schenk et al., 2001Go). Though such hypothetical enzymes might reduce the energy barrier for sugar translocation by an unknown mechanism, the flippase model does not account for the considerable energy that would be required to drive sugar chains through lipid bilayers (Lennarz, 1987Go). A detailed molecular description of how transmembrane transport of glycoconjugates occurs remains unclear in any biological system.

The general aim of this study was to investigate the interaction of C95, C95-P, and their docking complex with PIRS peptides with host fatty acyl chains in model PL membranes. To this end we used a combination of 1H-NMR and 31P-NMR spectroscopy and molecular modeling calculations to probe these important biological systems. A key finding to emerge is that addition of C95 or C95-P to PL bilayers promotes formation of nonlamellar phase states. Interestingly, formation of PI:PIRS peptide complexes stabilized the lamellar phase. The molecular modeling aspects of these spectroscopic studies also revealed differences in the preferred orientation of C95 and C95-P in the membrane. Finally, these studies suggest the existence of a potential channel with functional importance for glycoconjugate translocation.

Our conclusions are based on several key findings. First, the 1H-NMR chemical shifts of the host fatty acyl chain CH3, CH2, and the head group -N(CH3)3 protons in DMPC vesicles changed relative to that of DOPC alone. Second, C95 induced a 31P-NMR line width change in PE:PC (2:1) membranes that was characteristic of a nonlamellar structure. Third,the phosphorus atoms of the PL in C95 doped PE:PC vesicles revealed longer spin-lattice relaxation times (T1) than either the same vesicles alone or vesicles containing the NeuE:C95 or Alg7:C95 binding complex. The longer T1 times in the presence of 5 mol % C95 reflected the motion of the PL phosphate head groups in a nonlamellar conformation. Thus both the 1H-NMR and 31P-NMR results revealed that C95 and C95-P altered membrane structure by perturbing the fatty acyl chains and the head groups of the PL. In contrast, the 31P-NMR spectra of PL in PE:PC vesicles containing a PI:PIRS peptide binding complex (C95:Dpm1) showed line widths more characteristic of a bilayer structure. These same membranes also showed slower motions of the PL head groups than those of the same vesicles containing only C95. Furthermore, the 1H-NMR results revealed that the chemical shifts of the CH3, CH2, and -N(CH3)3 protons in DOPC vesicles containing C95 and the PIRS peptide, Dpm1,were nearly the same as DOPC alone.Similar chemical shifts for the CH3, CH2 and -N(CH3)3 protons in DOPC and DOPC vesicles containing either the Dpm1 or the NeuE:C95-P binding complex were observed (data not shown).

We interpret these results to mean that PL membranes containing a PI:PIRS peptide binding complex can reverse the C95-induced perturbation of membrane structure, thereby stabilizing the conformation of the PL in the bilayer structure. 31P-NMR-derived T1 studies were used to further verify these findings. As shown in Table I, the shortest T1 time for the phosphorus head group was detected when the PIRS peptides, NeuE (or Alg7) bound to C95. Taken together, these findings indicate that (1) the 1H-NMR results are consistent with those obtained by 31P-NMR for studying the effects of the PIs and PI:PIRS binding complexes on membrane structure, and (2) that the PI:PIRS docking structure can modulate or change the PI-induced nonlamellar structure back to a lamellar conformation. Neither conclusion has been reported previously. The spontaneous and reversible transformation of the nonlamellar phase transition induced by the PIs and modulated by the PIRS peptides are consistent with the thermodynamically favorable phase transitions that may be necessary to accompany the translocation of glycoconjugates across biological membranes.

But how might this work? Gruner (1992) has noted that biological membranes consist of large amounts of PL prone to form HexII and that these PL endow bilayers with "mesomorphic plasticity." The consequence of this plasticity leads to the disruption of the lipid–water interface and therefore to the nonbilayer alteration in membrane structure. We suggest that the lipid polymorphism resulting from the nonlamellar lipid phase induced by the PIs may provide a hydrophilic pore or channel to facilitate the translocation of lipid-linked hydrophilic sugar chains through membranes. The idea is that the PI may function to tether several PIRS-containing proteins, which, because of a positive binding cooperativity between the proteins, may form a transitory hydrophilic channel. This cooperativity results from both electrostatic and van der Waals interactions between the proteins, as previously described (Zhou and Troy, 2003Go). Such a channel could thus facilitate the passage of sugar chains linked to the PI from one side of the membrane to the other, without having to directly traverse a hydrophobic bilayer. The energetics of the latter is a difficult problem to reconcile, given the large energy requirement that such a translocation would entail, as previously discussed (Lennarz, 1987Go; Zhou and Troy, 2003Go).

The results of our orientation studies show that C95-P is oriented nearly perpendicular to the plane of the bilayer, suggesting that there is repulsion between the phosphate head groups of C95-P, and an electrostatic attractive interaction between the negatively charged phosphorus group of C95-P and the positively charged -N(CH3)3 group of DMPC. These interactions might be expected to perturb bilayer structure. This perturbation alone, however, does not induce a bilayer to nonbilayer change. This is possibly so because of the significant hydrophobic interactions between the other regions of C95-P, in particular the central coil region and the fatty acyl chains of DMPC. As we have shown (Figure 4B), the intercalation of C95-P into the membrane significantly perturbs the fatty acyl chains in both leaflets of the DMPC bilayer. Thus the combination of these interactions likely results in the greater C95-P-induced conformational change in the structure of PC membranes. It is anticipated that this information may help us better understand the possible molecular mechanisms that may be involved in PI-mediated biosynthetic and translocation processes that ferry glycoconjugates across membranes. The details of this important aspect of glycobiology is not understood in any biological system.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Preparation and structure of the PIRS peptides
The three 13-amino-acid peptides that encompass the PIRS domain in NeuE, Dpm1, and Alg7 (Zhou and Troy, 2003Go) were synthesized by the California Peptide Research (Napa, CA) using standard t-BOC chemistry. The resin containing the peptides was dried in high vacuum, and the peptide cleaved from the resin by 90% HF at 0°C. The released peptides were purified by reverse-phase high-performanc liquid chromatography, and were judged to be at least 80% pure. The extremely hydrophobic peptides were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) as a stock solution and stored at –20°C. The sequence of the three peptides were as follows:

Deuterated DOPC, DMPC, and DOPE were purchased from Avanti Polar Lipids (Alabaster, AL). C95, C95-P, and C55-P of > 98% purity were purchased from Larodan Fine Chemical Laboratory (Malmö, Sweden). Analytical thin-layer chromatography (TLC) was carried out on precoated silica gel F254 plates (250 mm x 10 mm x 20 cm) purchased from Merck (Darmstadt, Germany). The following solvent systems were used: (A) CHCl3-CH3OH-H2O (65:25:4 v/v); (B) CHCl3-CH3OH-NH4OH (14.8 M)-H2O (70:30:4:1); (C) CH3Cl3-MeOH (5:1); (D) CH3Cl3-MeOH-H2O (10:10:3). All PIs gave single spots on TLC in several solvent systems. C95, C95-P, C55-P, and the different PLs were dissolved in deuterated chloroform (99.8 atom % D, CDCl3, Aldrich, St. Louis, MO) and stored as stock solutions at –20°C.

Preparation of model PL vesicles
For the 31P-NMR studies, aqueous dispersion of large-diameter multilamellar vesicles of DOPC and DOPE/DOPC (2:1) were prepared as described previously (McCloskey and Troy, 1980aGo,bGo). Briefly, the solvent from appropriate aliquots of a stock solution of the lipids was removed under a stream of N2 gas. Traces of solvent were removed under high vacuum overnight. The lipids were resuspended in 0.5 ml D2O containing 50 mM Tris, 0.1 M NaCl, pH 7.3, and vortexed for 10 min to achieve complete suspension of the large (~ 1–2500 Å) multilamellar vesicles (MLVs). For the high-resolution 1H-NMR study (Fig. 1), an XL2020 Sonicator (Heat Systems-Ultrasonics, USA) with microprobe was used to sonicate the MLV suspension on ice for 15 min under an inert atmosphere of argon. This produced the smaller diameter (~ 280–300 Å) unilamellar vesicles (SUVs). After sonication, the resulting clear dispersion was centrifuged at 13,000 x g for 30 min and the vesicles in the supernatant were transferred to a 5-mm NMR tube.

Preparation of PL vesicles containing PIs and PIs plus PIRS peptide
Incorporation of the PIs and PIRS peptides into dispersions of the PL vesicles was carried out as follows.DOPC or DOPE/DOPC (2:1) was mixed with: (1) 10 mol % of the PIRS peptides (either Dpm1/DOPC, NeuE/DOPE/DOPC [2:1]) or Alg7/DOPE/DOPC [2:1]) such that the molar ratio of peptide to the PL was 1:8; (2) 10 mol % of PI (either C95/DOPC, C55-P/DOPC, or C95/DOPC/DOPE [2:1]) was mixed such that the ratio of the PIs to the PL in the vesicles was 1:8; and (3) 10 mol % of the PIRS peptide and PIs (C95/Dpm1/DOPC or C95/Alg7/DOPC/DOPE [2:1]) was mixed to give the desired molar ratios as described in the text for each experiment. The bulk of the solvent was removed under a stream N2 gas. The samples were then lyophilized overnight (15 h). One-half milliliter D2O was added and the samples were incubated at room temperature for 2 h and vortexed for ~ 15 min to achieve dispersion of the larger multilamellar vesicles. For the 1H-NMR experiment (Figure 1), aqueous dispersion of the DOPC vesicles containing 10 mol % C95 (Figure 1A[b]), 10 mol % Dpm1 (Figure 1A[c]), and 10 mol % each of C95 and Dpm1 (Figure 1A[d]) or 10 mol % C95-P (Figure 1B[b]) and 10 mol % each of C95-P and Dpm1 (Figure 1B[c]) were prepared by sonication, as described. After sonication, the samples were centrifuged at 13,000 x g for 30 min, and SUVs in the supernatant were transferred to a 5-mm NMR tube.

NMR measurements
All 1H-NMR measurements were carried out on the Nicolet NT-500 instrument (General Electric) operating at 500 MHz. 1H shifts were measured relative to the methyl proton resonance of internal sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSS). The 1D proton spectra were recorded with a sweep width of 8000 Hz and 16K data points. A total of 1024 scans were accumulated, and an acquisition time of 2.05 s was used for the peptide and the mixture of peptide and PIs in the host PL vesicles. A presaturated pulse sequence was used to inhibit the strong water resonance peak.

Proton-decoupled 31P-NMR spectra were acquired at 121.5 MHz on a GE Omega 300 MHz spectrometer. A 90° radiofrequency pulse with a 1–2 s interpulse time was used with a 40 KHz spectral sweep width. Sixteen thousand data points and 10,000 scans were collected for the spectra shown in Figure 2a and b and Figure 3, whereas 4096 scans were collected for the spectrum shown in Figure 2c.

Molecular modeling calculations to determine the preferred conformation of C95 and C95-P in DMPC membranes
Molecular modeling calculations for the PIRS peptide NeuE were carried out on a Silicon Graphic Krebs computer using DGII (NMRchitect, Biosym/MSI, San Diego, CA), as previously described (Zhou and Troy, 2003Go). In these studies we described the 3D structures of C95, C95-P, and C55-P, which were built by energy minimization using the AMBER force field (Chou et al., 1999, 2000). In AMBER, the force constants for the phosphate and pyrophosphate groups are well defined (Weiner et al., 1984Go). The bilayer models, consisting of DMPC molecules were constructed using the INSIGHTII program (Biosym/MSI, San Diego, CA). The energy minimization calculations of the PIs and PIRS-PI in DMPC model membranes were also carried out on a Silicon Graphic Krebs computer using DISCOVER of INSIGHTII with 2500 cycles of steepest descent followed by 2500 cycles of conjugate gradient minimization.Minimization moves the atoms in the molecular system to the nearest local minimum, which is not necessarily the global minimum. Minimization was carried out in two steps. First, an equation describing the energy of the system as a function of its coordinates was defined and evaluated for a given conformation. Second, the conformation was adjusted to the lowest value of the target function.


    Acknowledgements
 
We are grateful to Dr. K. C. Chou for help in molecular modeling of the 3D structures using AMBER and to Professors Tom Jue and John C. Voss for critical help and advice. This work was funded in part by National Institutes of Health Research Grants GM 55703 and AI09352 (F.A.T.).

This article is dedicated to Professor William J. Lennarz for his many seminal and landmark contributions to glycobiology and, in particular, to his studies to elucidate the molecular events underlying the dolichol-mediated pathway. His distinguished career as an outstanding scientist and mentor was recently recognized by his being honored as the recipient of the 2004 Karl Meyer Award from the Society of Glycobiology.


    Abbreviations
 
AMBER, assisted modeling with energy refinements; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine; DMSO, dimethyl sulfoxide; DOPC, 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine; DOPE,1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; DRS, dolichol recognition sequence; DSS, 4,4-dimethyl-4-silapentane-1-sulfonate; EPR, electron paramagnetic resonance; MLV, multiple lamellar vesicles; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser enhancement spectroscopy; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, polyisoprenol; PIRS, polyisoprenol recognition sequence; PL, phospholipid; SLV, single lamellar vesicles


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Albright, C.F., Orlean, P., and Robbins, P.W. (1989) A 13-amino acid peptide in three yeast glycosyltransferases may be involved in dolichol recognition. Proc. Natl Acad. Sci. USA, 86, 7366–7369.[Abstract]

Brandl, C.J. and Deber, C.M. (1986) Hypothesis about the function of membrane-buried proline residues in transport proteins. Proc. Natl Acad. Sci. USA, 83, 917–921.[Abstract]

Cevc, G. and Marsh, D. (1987) Phospholipid bilayers: physical principles and models. John Wiley & Sons, New York, pp. 408–425.

Chan, S.I., Bocian, D.F., and Petersen, N.O. (1981) Nuclear magnetic resonance studies of the phospholipids bilayer membrane. In: E. Grell, (Ed.), Membrane spectroscopy. Springer-Verlag, New York.

Chou, K.C., Tomasselli, A.G., and Heinrikson, R.L. (2000) A novel approach to predict active sites of enzyme molecules. FEBS Lett., 470, 249–256.[CrossRef][ISI][Medline]

Cullis, P.R. and de Kruijff, B. (1979) Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim. Biophys. Acta, 559, 399–420.[ISI][Medline]

Dan, N., Middleton, R.B., and Lehrman, M.A. (1996) Hamster UDP-N-acetylglucosamine:dolichol-PN-acetylglucosamine-1-P transferase has multiple transmembrane spans and a critical cytosolic loop. J. Biol. Chem., 271, 30717–30724.[Abstract/Free Full Text]

Datta, A.K. and Lehrman, M.A. (1993) Both potential dolichol recognition sequences of hamster GlcNAc-1-phosphate transferase are necessary for normal enzyme function. J. Biol. Chem., 268, 12663–12668.[Abstract/Free Full Text]

Deber, C.M., Brandl, C.J., Deber, R.B., Hsu, L.C., and Young, X.K. (1986) Amino acid composition of the membrane and aqueous domains of integral membrane proteins. Arch. Biochem. Biophys., 251, 68–76.[ISI][Medline]

de Ropp, J.S. and Troy, F.A. (1984) Chemical synthesis and 2H NMR investigations of polyisoprenols: dynamics in model membranes. Biochemistry, 23, 2691–2695.[ISI][Medline]

de Ropp, J.S. and Troy, F.A. (1985) 2H NMR investigation of the organization and dynamics of polyisoprenols in membranes. J. Biol. Chem., 260, 15669–15674.[Abstract/Free Full Text]

de Ropp, J.S., Knudsen, M.J., and Troy, F.A. (1987) 2H NMR investigation of the dynamics and conformation of polyisoprenols in model membranes. Chemica Scripta, 27, 101–108.[ISI]

Devaux, P.F., Hoatson, G.L., Favre, E., Fellmann, P., Farren, B., MacKay, A.L., and Bloom, M. (1986) Interaction of cytochrome c with mixed dimyristoylphosphatidylcholine-dimyristoylphosphotidylserine bilayers: a deuterium nuclear magnetic resonance study. Biochemistry, 25, 3804–3812.[CrossRef][ISI][Medline]

Duijn, G.V., Verkleij, A.J., de Kruijff, B., Valtersson, C., Dallner, G., and Chojnacki, T. (1987) Influence of dolichols on lipid polymorphism in model membranes and the consequences for phospholipid flip-flop and vesicle fusion. Chemica Scripta, 27, 95–100.[ISI]

Gruner, S.M. (1992) Nonlamellar lipid phases. In: P. Yeagel (Ed.), The structure of biological membranes. CRC Press, Ann Arbor, MI, pp. 211–250.

Janas, T., Chojnacki, T., Swiezewska, E., and Janas, T. (1994) The effect of undecaprenol on bilayer lipid membranes. Acta Biochim. Polon., 41, 351–358.[Medline]

Kelleher, D.J., Kreibich, G., and Gilmore, R. (1992) Oligosaccharyltransferase activity is associated with a protein complex composed of ribophorins I and II and a 48 kd protein. Cell, 69, 55–65.[ISI][Medline]

Knudsen, M.J. and Troy, F.A. (1989). Nuclear magnetic resonance studies of polyisoprenols in model membranes. J. Chem. Physics Lipids, 51, 205–212.[CrossRef]

Kohler, S.J. and Klein, M.P. (1977) Orientation and dynamics of phospholipid head groups in bilayers and membranes determined from phosphorus-31 nuclear magnetic resonance chemical shielding tensors. Biochemistry, 16, 519–526.[CrossRef][ISI][Medline]

Lennarz, W.J. (1987) Protein glycosylation in the endoplasmic reticulum: current topological issues. Biochemistry, 26, 7205–7210.[ISI][Medline]

McCloskey, M.A. and Troy, F.A. (1980a) Paramagnetic isoprenoid carrier lipids. 1. Chemical synthesis and incorporation into model membranes. Biochemistry, 19, 2056–2060.[ISI][Medline]

McCloskey, M.A. and Troy, F.A. (1980b) Paramagnetic isoprenoid carrier lipids. 2. Dispersion and dynamics in lipid membranes. Biochemistry, 19, 2061–2066.[ISI][Medline]

McLaughlin, A.C., Cullis, P.R., Hemming, M., Brown, F.F., and Brocklehurst, J. (1977) Magnet resonance studies of model and biological membranes. In: R.A Dwek, I.D. Campbell, R.E. Richards, and R.J.P. Williams (Ed.), NMR in biology. Academic Press, New York.

Murgolo, N.J., Patel, A., Stivala, S.S., and Wong, T.K. (1989) The conformation of dolichol. Biochemistry, 28, 253–260.[ISI][Medline]

Pavelka, M.J., Wright, L.F., and Silver, R.P. (1991) Identification of two genes, kpsM and kpsT, in region 3 of the polysialic acid gene cluster of Escherichia coli K1. J. Bacteriol., 173, 4603–4610.[ISI][Medline]

Piela, L., Nemethy, G., and Scheraga, H.A. (1987) Proline-induced constraints in alpha-helices. Biopolymers, 26, 1587–1600.[ISI][Medline]

Pigeon, R.P. and Silver, R.P. (1994) Topological and mutational analysis of KpsM, the hydrophobic component of the ABC-transporter involved in the export of polysialic acid in Escherichia coli K1. Mol. Microbiol., 14, 871–881.[ISI][Medline]

Schenk, B., Fernandez, F., and Waechter, C.J. (2001) The in(side) and out(side) of dolichyl phosphate biosynthesis and recycling in the endoplasmic reticulum. Glycobiology, 11(5), 61R–70R.[Abstract/Free Full Text]

Scocca, J.R. and Krag, S.S. (1990) Sequence of a cDNA that specifies the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase from Chinese hamster ovary cells. J. Biol. Chem., 265, 20261–20626.

Troy, F.A. (1991) NMR studies of polyisoprenyl derivatives on bilayer structure and the translocation of polysialic acid chains across the E. coli K1 inner membrane. Nineteenth Nobel Conference on Biosynthesis, Regulation and Products of the Mevalonate Pathway. Carolinska Medico Chirurgiska Institute Press, pp. 84–87.

Troy, F.A. (1992) Polysialylation: from bacteria to brains. Glycobiology, 2, 5–23.[ISI][Medline]

Valtersson, C., Van Duijn, G., Verkleij, A.J., Chojnacki, T., De Kruijff, B., and Dallner, G. (1985) The influence of dolichol, dolichol esters, and dolichyl phosphate on phospholipid polymorphism and fluidity in model membranes. J. Biol. Chem., 260, 2742–2751.[Abstract]

Weiner, S.J., Kollman, P.A., Case, D.A., Singh, C., Ghio, C., Alagona, G., Profeta, S., and Weiner, P. (1984) A new force field for molecular mechanical simulation of nucleic acids and proteins. J. Am. Chem. Soc., 106, 765–784.[ISI]

Wuthrich, K. (1987) NMR techniques for studies of protein-DNA interactions. In: E.B. Thompson and J. Papaconstantinou (Eds.), DNA: protein interaction and gene regulation. University of Texas Press, Austin, pp. 87–94.

Zhou, G.P. and Troy, F.A. (2003) Characterization by NMR and molecular modeling of the binding of polyisoprenols and polyisoprenyl recognition sequence peptides: 3D structure of the complexes reveals sites of specific interactions. Glycobiology, 13, 51–71[Abstract/Free Full Text]

Zhu, X. and Lehrman, M.A. (1990) Cloning, sequence, and expression of a cDNA encoding hamster UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase. J. Biol. Chem., 265, 14250–14255.[Abstract/Free Full Text]

Zimmerman, J.W. and Robbins, P.W. (1993) The hydrophobic domain of dolichyl-phosphate-mannose synthase is not essential for ezyme activity or growth in Saccharomyces cerevisiae. J. Biol. Chem., 268, 16746–16753.[Abstract/Free Full Text]





This Article
Abstract
FREE Full Text (PDF)
Correction to PDF
A correction has been published
All Versions of this Article:
15/4/347    most recent
cwi016v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Disclaimer
Request Permissions
Google Scholar
Articles by Zhou, G.-P.
Articles by Troy, F. A.
PubMed
PubMed Citation
Articles by Zhou, G.-P.
Articles by Troy, F. A., II