Department of Biological Chemistry, University of California Schoolof Medicine, One Shields Avenue, Davis, CA 95616-8635, USA
Received on April 16, 2002; revised on August 27, 2002; accepted on September 6, 2002
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Abstract |
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Key words: conformation-structure / glycosyl carrier lipid (dolichol) / glycosyl translocation / nuclear magnetic resonance / polyisoprenyl recognition sequence peptides
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Introduction |
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In prokaryotes, C55-P is the carrier of sugar residues involved in synthesis of cell wall peptidoglycans, lipopolysaccharides, common antigen, and capsular polysaccharides (Osborn, 1971; Troy, 1979
). The polysialic acid (polySia) capsule is a neurovirulent determinant in neurotropic E. coli K1 and Neisseria meningitidis as well as an example of a specific type of bacterial capsule whose synthesis involves C55-P as an intermediate carrier of Sia residues (Troy et al., 1975
; Masson and Holbein, 1983
).
The molecular details of how these structurally unique lipid cofactors function are unknown. It is hypothesized that the sugar residues linked to the lipid by a pyrophosphate bridge at the polar end of the isoprene molecules are translocated from one side of the membrane to the other, and the hydrocarbon chains presumably remain anchored in the nonpolar region of the membrane. This postulate, though widely reported in the literature, has not been well documented by direct experimental evidence (McCloskey and Troy, 1980a,b
). Although much is known regarding the biochemistry of PIs, including their synthesis and the transfer reactions they mediate, there is a dearth of biophysical information as to how they may ferry saccharide units across membranes (Lennarz, 1987
).
Based on the unusual length and poly-cis geometry of undecaprenyl and dolichyl derivatives (Figure 1), it was proposed earlier that these properties might endow the PIs with some unique physiochemical properties important for their biological function (McCloskey and Troy, 1980a). As a first step in understanding the role of the PIs in transmembrane glycoconjugate processes, previous studies focused on the organization and molecular motions of the PIs in model membranes and the effect these so-called superlipids had on membrane structure. These studies used a variety of biophysical approaches, including electron paramagnetic resonance (McCloskey and Troy, 1980a
,b
; Troy, 1991
), 1H- and 2H-nuclear magnetic resonance (NMR) (de Ropp and Troy, 1984
, 1985
; de Ropp et al., 1987
), 31P-NMR (Vigo et al., 1984
; Valtersson et al., 1985
; de Ropp et al., 1987
; Knudsen and Troy, 1989a
), differential scanning calorimetry, and fluorescence depolarization (Vigo et al., 1984
; Valtersson et al., 1985
). Several key findings emerged from studies using spin-labeled glycosyl carrier lipids, including information on the transbilayer diffusion rates and self-association of the PIs (McCloskey and Troy, 1980a
,b
). Information on the location and dynamics of the
-terminus of the polyprenols was subsequently provided by 2H-NMR studies of deuteruim-labeled PIs in host phospholipid vesicles (de Ropp and Troy, 1984
, 1985
). Importantly, these studies also revealed that C55 and C95 derivatives altered the membrane host packing matrix, thus potentially modulating membrane lipid polymorphism (de Ropp and Troy, 1984
, 1985
; Knudsen and Troy, 1989a
; Troy, 1991
). 31P-NMR studies confirmed that the longer-chain PIs induced the formation of nonbilayer or inverted hexagonal organization of phospholipid molecules in phosphatidylcholine (PC) and PC/phosphatidylethanolamine membrane vesicles (Valtersson et al., 1985
; de Ropp et al., 1987
; Knudsen and Troy, 1989a
; Troy, 1991
).
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Albright et al. (1989) first described a 13-amino-acid peptide consensus sequence in the presumed membrane-spanning domain of three yeast glycosyltransferases, Dpm1, Alg1, and Alg7, which was postulated to be a potential dolichol recognition sequence (DRS). A fourth yeast enzyme, Sec59 (a dolichol kinase), also contained a potential DRS (Heller et al., 1992
). These findings were extended to mammalian glycosylphosphotransferases (GlcNAc-1- phosphotransferases; GPTs), which catalyzed synthesis of GlcNAc-P-P-C95 (Scocca and Krag, 1990
; Zhu and Lehrman, 1990
; Lehrman, 1991
; Zhu et al., 1992
; Datta and Lehrman, 1993
). GPT was found to contain two potential DRSs, both of which were shown by mutational analysis to be required for enzyme function (Datta and Lehrman, 1993
). In contrast, deletion and mutational analysis of the DRS in yeast dolichylphosphomannose synthase showed that this domain was not essential for enzyme activity (Zimmerman and Robbins, 1993
). These conflicting results emphasized the potential limitation of deletion and mutational approaches to determine unambiguously the functional importance of the DRS and highlighted the need for direct structural information. Similar PI-binding sequences were later identified in other proteins of the C95 pathway in yeast and mammalian cells, including Alg2 (Jackson et al., 1993
) and ribophorin I and II (Kelleher et al., 1992
; Knauer and Lehle, 1999
). Ribophorin I and II are two of the nine nonidentical membrane protein subunits that make up the oligosaccharyltransferase (OST) complex (Kelleher et al., 1992
; Knauer and Lehle, 1999
; Kim et al., 2000
).
NeuE and KpsM, two proteins in the multienzyme polysialyltransferase complex in neuroinvasive E. coli K1, were also discovered to contain potential C95 recognition sequences in their membrane-spanning domains (Troy, 1992). Because these prokaryotic proteins would interact with the shorter chain PI, C55-P, we designated the PI binding domain by the more generic descriptor polyisoprenyl recognition sequence (PIRS) to denote that such sequences can interact with PIs other than C95. NeuE was initially postulated to be a sialyltransferase that may begin polySia chain synthesis by catalyzing the transfer of Sia from CMP-Sia to C55-P (Troy, 1992
). However, we were surprised to discover a potential PIRS in KpsM because this protein had no known biosynthetic function, having been implicated only in polySia chain translocation (Pavelka et al., 1991
; Troy, 1992
; Pigeon and Silver, 1994
). Table I compares the sequence of the 13-amino-acid PIRS peptides in the eukaryotic glycosyltransferases with the NeuE and KpsM proteins from E. coli K1.
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Our 2D NMR and molecular modeling results of the PI:PIRS complexes now provide the first biophysical evidence for a direct binding complex between specific contact amino acids in the PIRS peptides and the PIs. We have estimated the energetics of this binding and have determined that a single PI molecule can bind several PIRS peptides. Though the physiological significance of this interaction in vivo remains to be determined, it is anticipated that results from studies using model membranes may eventually help us better understand the importance of key PI:protein interactions in biological membranes, and thus their potential role in glycoconjugates synthesis/translocation processes. They might also lead to a better understanding of the molecular mechanisms of hypoglycosylation associated with some of the CDGs as well as the conformational disorders related to hyperglycosylation (Freeze, 2001a,b
).
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Results |
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The structures of the free NeuE peptide derived from the 2D NOE data, energy minimization, and simulated annealing calculations are shown in Figure 3. The top panel shows a stereoview of the backbone conformation after superimposing nine calculated NeuE structures. The lower panel shows a 3D energy minimized structural model of the peptide. The structures were determined using distance constraints and backbone dihedral angle constraints derived from 55 NOE cross-peaks and coupling constants. The average root mean squared deviation (RMSD) of these structures was 2.09 Å for all atoms. This conformation revealed that Leu1, Ile3, Leu6, and Ile7 were located on the same outer surface of the helical domain of the peptide. Superimposition of the terminal residues, 1 and 13, was not as well defined as with the other amino acids, as these residues were more flexible and thus showed fewer NOEs.
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1H-NMR resonance assignments for C95 and C95-P
Partial resonance assignments for C95 were initially reported in 1976 (Mankowski et al., 1976). Our present 1H-NMR studies include assignments for protons in C95-P and C55-P and extend the preliminary assignments made earlier for C95. No 1H-NMR assignments for C95-P and C55-P have been reported previously. Significantly, we found that the resonance assignments for C55-P, C95-P, and C95 in the membrane mimetic solvent dimethyl sulfoxide (DMSO) and PC vesicles were essentially identical. This finding confirms and extends other studies showing that hydrophobic peptides can adopt the same structure in DMSO as in the native protein, thus providing further evidence that no artifacts are induced in DMSO (Albert and Yeagle, 2000
; Yeagle et al., 2000a
). The chemical shifts observed in the 1D 1H NMR spectra for the eight different types of relevant protons in C95 and C95-P (Figure 1ah) are summarized in Table III.
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Our 2D 1H NMR studies showed that the NOESY spectra of C95 and C95-P were similar (Figure 4a,b). For example, three cross-peaks were observed in the spectrum of C95-P (Figure 4b), representing NOEs between (1) polyCH-polyCH2, (2) polyCH-polyCH3, and (3) polyCH2-polyCH3, respectively. The same cross-peaks were also observed in the NOESY spectrum of C95 (Figure 4a). The protons responsible for these cross-peaks were all in the unsaturated isoprene units. Because there were no cross-peaks that represented connectivity between the protons of the unsaturated and saturated -isoprene units, these data further revealed that the unsaturated polyisoprene units, which make up most of the PI backbone, are either in close contact or associated together. Based on these NMR findings, the 3D structures of C95, C95-P, and C55-P were built by energy minimization with respect to all atoms using AMBER force field (Weiner et al., 1984
) as described under Materials and methods. The force constants for the phosphate and pyrophosphate groups in AMBER are well defined. These findings revealed that the 3D conformation of the PIs were nearly identical tripartite molecules with their three domains arranged in a coiled, helical structure (Figure 5).
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The complete assignment of all resonance protons in the NeuE peptide in the presence and absence of C95 and C95-P is shown in Table II. The sequential connectivities observed for the NeuE peptide in the presence of C95 and C95-P are summarized in Figure 2b and c, respectively. This diagram shows the (i,i+1), (i,i+2), (i,i+3), (i,i+5), (i,i+7), and (i,i+8) contacts observed in the NOESY spectra, and the spinspin coupling constants, 3JHN, measured from the 1D 1H-NMR spectra. The temperature coefficient of NH protons (ppb/K) are also shown. In the presence of C95 or C95-P, the resonance and sequential assignments of NeuE were similar to those of the free peptide, except that changes in the chemical shift of some residues were observed, as will be described.
Structure of the NeuE peptide after binding to C95
The dNN(i,i+1) connectivities for residues 38 and 1013; the dN(i,i+1) connectivities for residues 14, 58, and 1013; and the medium NOE for d
N(i,i+3) connectivity between residues 2 and 5 that were observed for the free NeuE peptide (Figure 2a) were also observed when NeuE bound to C95 (Figure 2b). A comparison of the stereoview of the backbone conformation of the NeuE peptide before and after binding to C95 is shown in Figure 6a and b, respectively. As with the free peptide (Figure 6a), the NeuE:C95 docking structure (Figure 6b) is based on both the 2D NOE results and on energy minimization and simulated annealing. The stereoview conformations represent the superimposition of nine NeuE structures calculated using distance and backbone dihedral angle constraints, derived from 85 NOE cross-peaks and coupling constants. The average RMSD for the NeuE:C95 docking structure was 1.87 Å for all atoms.
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The backbone conformation of NeuE when bound to C95 (Figure 6b) also revealed that both Leu1 and Leu6 were again positioned on the same outer surface of the helical segment of the molecule, as was observed for the free NeuE peptide (Figure 6a). The superimposition fit for residues 113 in the bound state was better than for NeuE alone, because more NOEs were observed. As with the free NeuE peptide, the superimposed conformers of bound NeuE (Figure 6b) showed neither a characteristic -helix or ß-sheet structure. In contrast with the more extended structure seen in the free peptide (Figure 6a), residues 1013 in the bound structure were in a bent conformation (Figure 6b). This conformational change resulted in the detection of dßN connectivities between residues 5 and 13 (Figure 2b). Therefore, based on the sequential connectivities for NeuE in the presence and absence of C95 (Figure 2a,b) and by comparing the stereoview backbone conformations of free NeuE with the peptide in the bound NeuE:C95 structure (Figure 6a,b), we conclude that docking of NeuE to C95 induced a conformational change in the peptide, resulting in a more compact structure in the carboxyl terminal segment.
Structure of the NeuE peptide after binding to C95-P
Similar to the structure of free NeuE peptide and the structure of NeuE after binding to C95 (Figure 6), strong dN(i,i+1) connectivities in NeuE were observed in both segments of the peptide after binding to the phosphorylated PI, C95-P (Figure 2c). Segment 1 (residues 18), for example, contained more
-helix-like features, as indicated by a strong d
ß(i,i+3) connectivity between residues 2 and 5, a medium dNN(i,i+1) connectivity between residues 4 to 8, a medium d
N(i,i+3) connectivity between residues 3 and 6, and a medium d
N(i,i+1) connectivity between residues 3 and 5 (Figure 2c). Unexpectedly, we found different connection characteristics in both the N- and C-terminal segments of the peptide after binding to C95-P compared with C95. In the interaction between NeuE and C95-P, medium and strong d
ß(i,i+7) connectivities were observed between residues 6 and 13 and between residues 5 and 12 (Figure 2c). In contrast, in the NeuE:C95 complex only a medium dßN(i,i+8) connectivity between residues 5 and 13 was observed (Figure 2b). This suggested that the C-terminal segment of the peptide in the NeuE:C95-P complex was even more compact than in the NeuE:C95 structure. In support of this conclusion, no dNN(i,i+1) connectivity between residues 11 and 12 was observed after NeuE bound C95-P (Figure 2c), as there was in the free peptide (Figure 2a) or in the NeuE:C95 complex (Figure 2b).
A strong NOE was also observed between residues 9 and 10 for dN(i,i+1), in the NeuE:C95-P structure, as shown in Figure 2c. These sequential connectivity data thus verified that a slightly different conformational change occurred in the NeuE peptide after binding to the phosphorylated PI, C95-P (Figure 6c), compared with the nonphosphorylated alcohol, C95 (Figure 6b). Like the structure of the free NeuE peptide (Figure 6a) and the peptide after binding to C95 (Figure 6b), the conformation of NeuE bound to C95-P (Figure 6c) was determined by superimposing nine peptide structures, calculated by using distance and backbone dihedral angle constraints that were derived from 111 NOE cross-peaks and coupling constants. The average RMSD for the NeuE:C95-P structure was 1.80 Å for all atoms. These structures revealed that Ile3, Leu6, and Ile7 were located on the same outer surface of the helical segment of the peptide molecule (Figure 6c). The conformation of the N-terminal segment of the peptide (residues 28) in the NeuE:C95-P docked complex was better defined than the structure of free NeuE or the peptide in the NeuE:C95 complex. In the NeuE:C95-P structure, for example, the C-terminal backbone segment is bent toward the N-terminal segment, such that
ß connectivities between residues 5 and 12 and residues 6 and 13 were detected, as shown in Figure 2c. Energy minimized space-filling models of the NeuE peptide before and after binding to C95 and C95-P are shown in Figure 7ac, respectively.
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The specificity of interaction between PIs and PIRS peptides
A comparison of the 500 MHz 1H-NMR spectra of the NH proton resonances of the NeuE peptide before and after binding C95 or C95-P revealed that most resonances were shifted downfield after binding. A summary of all chemical shifts observed for the amino acid residues in NeuE after binding to C95 and C95-P is shown in Table II. These differences in chemical shift were the first suggestion of a direct interaction between NeuE and the PIs. This binding was further substantiated by chemical shift changes observed for the CH2 and CH3 protons in C55-P after binding NeuE peptide and in the 1H NMR spectra of C55-P in the presence and absence of the eukaryotic Dpm1 peptide in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) vesicles (Figure 8). Because of the near identity in these chemical shifts and in the close similarities between the 3D structures of C55-P and C95-P (Figure 5), as well as between NeuE and Dpm1, these findings suggest that the other PIRS peptides (Table I) could also be used to study the specificity of the interaction between these PIRS-containing peptides and the PIs.
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PI derivatives can bind more than one NeuE peptide
Energy minimization and simulated annealing studies showed that a single C95, C95-P, or C55-P molecule could bind more than one NeuE peptide. Tertiary structural models of these energetically favorable docking structures revealed that C95 and C95-P could each bind at least two NeuE peptides (Figure 12). For C95, the most favorable binding energy for the docking between the three proton pairs on NeuE and the PI was -10 kcal/mol (Table VII). All three of the amino acids involved in this binding are located on the opposite side of the C95 alcohol head group (right side of the PI in Figure 12). When the second NeuE peptide was bound to the opposite or head group side of the same PI (left side of the PI in Figure 12), the binding energy was slightly less favorable (7 kcal/mol). Similarly, as shown in Figure 12 (top right), one C95-P molecule could bind at least two NeuE peptides via four proton docking pairs, as summarized in Table IX. Again, the most favorable docking energy (9 kcal/mol) was for the NeuE peptide binding to the helical conformation on the opposite side of the phosphorylated head group (right side of the PI in Figure 12), and the binding energy for the second peptide docking to the head group side of the PI (left side of the PI in Figure 12) was -7 kcal/mol. It was possible to determine by energy minimization and molecular modeling a docking structure for NeuE and C55-P by using the NMR-derived conformation of NeuE, determined after binding to C95-P (Figures 6 and 7c) and the 3D structural similarity between C55-P and C95-P (Figure 5).
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On the basis of these findings, we conclude that the physical interaction between the NeuE and Dpm 1 peptides with PIs observed in the present study were specific and not due to nonspecific or irrelevant hydrophobic interactions. Studies to determine more precisely the contribution of each of these amino acids in the binding of PIRS peptides to PIs will require additional NMR experiments in which each of these key residues have been systematically replaced.
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Discussion |
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Limits of interpretation of experimental results
Our findings raise several relevant points that should be considered with respect to the limits of interpretation of the data. The following discussion addresses these limits and also covers the potential significance of our results in support of the hypothesis that the glycosyl carrier PI lipids may have a bifunctional role in glycosylation/translocation processes. The conformational changes induced on binding and a description of the molecular motions and energetics of binding are also presented.
First, although our biophysical approach was not designed to determine the potential physiological importance of the binding of PIRS peptides to PIs, the high degree of specificity of the interaction suggests a likely biological role. Accordingly, the lack of a demonstrated physiological role of PIprotein interactions should not diminish the potential significance of such a complex, as these interactions may be as important as the multiple proteinprotein interactions required for function of the OST complex (Kelleher et al., 1992; Spirig et al., 1997
; Knauer and Lehle, 1999
; Kim et al., 2000
). The biophysical strategy developed here should therefore be viewed as representing the beginning of studies to determine the role of PIPIRS interactions in glycosylation/translocation events. In this regard, it is noted that neither genetic (deletion or mutational analyses) nor biochemical approaches have been able to unambiguously determine the physiological importance of the PIRS peptides (Scocca and Krag, 1990
; Zhu and Lehrman, 1990
; Lehrman, 1991
; Zhu et al., 1992
; Datta and Lehrman, 1993
; Jackson et al., 1993
; Zimmerman and Robbins, 1993
; Rush et al., 1998
; Rush and Waechter, 1998
).
A second consideration is whether the same structural features present in a 13-amino-acid PIRS peptide exists when the peptide is contained within a full-length protein. This is likely to be true, based on a number of studies showing that small peptides of integral membrane proteins have the same secondary structure as that of the peptide contained within the full-length protein (Dyson et al., 1992; Kahn et al., 1992
; Blanco, 1994
; Adler et al., 1995
; Albert and Yeagle, 2000
; Katragadda et al., 2000
; Yeagle et al., 2000a
,b
). For example, one of the more thoroughly studied proteins is rhodopsin, where peptides of both the 6 and 7 transmembrane helical domains have the same 3D structure determined by NMR as the secondary structure of the peptides in the native protein (Kahn et al., 1992
; Albert and Yeagle, 2000
; Katragadda et al., 2000
; Yeagle et al., 2000a
). Similarly, studies on the four-helix bundle of myohemerythrin have also shown that small peptides of the protein retain the same secondary structure as the intact protein (Dyson et al., 1992
). Based on these findings we conclude that our 3D structure of the transmembrane domain of NeuE is likely similar to the same peptide segment in the intact NeuE protein.
A third consideration to note is that a number of studies have shown that the 1H-NMR-derived solution structure of hydrophobic transmembrane peptide helicies, for example, those of bacteriorhodopsin, are similar in DMSO as the corresponding peptide region in the crystal structure of the protein (reviewed in Yeagle et al., 2000a; Albert and Yeagle, 2000
; Katragadda et al., 2000
). This indicates that this membrane mimetic solvent can be used for high-resolution NMR studies of hydrophobic peptides.
Conformational features of PIRS peptides
Calculations of structural parameters from spectroscopic data using NMR theory can be limited, particularly for smaller peptides that may undergo conformational changes faster than can be observed on the NMR time scale (Jardetzky, 1981). Because of the flexibility in the terminal residues of the NeuE peptide, the structure shown in Figure 3 may time average several different conformations and thus approximate only one of several potential minimum energy structures. However, application of Chou and Fasman rules (Chou and Fasman, 1974
) predicted that the C-terminal segment of the peptide would be in an extended conformation, which is what we found experimentally for the nine calculated backbone conformations of the NeuE peptide. A more rigid C-terminal region was also found in the peptide after docking to C95 or C95-P, showing that binding induced a conformational change in the peptide (Figure 6). Thus the derived structure of the NeuE peptide, particularly after binding to the PI, is likely to be a reasonably accurate conformation.
The Pro9 residue in NeuE induced a distortion or bend after the N-terminal segment (residues 18) in the peptide (Figure 3). This is in accord with previous studies on the effect that Pro residues have on the conformation of polypeptide chains (Brandl and Deber, 1986; Deber et al., 1986
; Gennis, 1989
). Sequence analyses of the other PIRS peptides revealed that they too contained Pro in position 9 or 10, with the exception of Dpm1 and ribophorin I (Table I). All of the Pro-containing PIRS peptides had a similar conformational bend, thus forming two segments within the peptide backbone. A second characteristic common to most of the PIRS peptides is that the N-terminal segment usually contained Leu, Met, Ala, or Cys residues, amino acids that have a higher probability of forming
helices. The key Leu and Ile residues in the NeuE peptide that are involved in PI binding are all within this N-terminal segment, which, due to its distorted helix-like structure, is more rigid than the C-terminal segment. This structural perturbation within the peptide may aid and stabilize its interaction with the PIs. Based on the structural similarities between NeuE and the other PIRS peptides, we predict that the PI binding domain for these peptides will likely be located in their N-terminal segment.
Most of the C-terminal segment (residues 913) of the PIRS peptides contained Phe, Tyr, or Asp, residues, which have a higher probability of forming ß-sheet or ß-turn structures (Gennis, 1989). Thus, this segment of NeuE has a predicted higher probability of forming an extended ß-like structure, as do the other PIRS-peptides shown in Table I. Notably, each C-terminal segment also contained Pro, Asn, or Tyr, which have been implicated as the most likely functional residues in transport of hydrophilic substitutes through the nonpolar domain of the membrane (Cantor and Schimmel, 1980
). Our finding that there are subtle conformational changes induced in the NeuE peptide after binding C95 or C95-Pparticularly in the C-terminal segment, which becomes more compactsuggests that these changes may induce neighboring residues to adopt a more
-helix-like structure. This observation is in accord with the finding that the
-helix content in some serum apolipoproteins increases after binding lipid (Cantor and Schimmel, 1980
; Israelachvili and Pashley, 1982
; Kahn et al., 1992
).
Conformational features of the PIs: a revised C95 model
The 3D structure of C95 determined in this study (Figure 5) has some features in common with an earlier model (Murgolo et al., 1989), yet there are significant differences. The major difference is the length of the PI; our revised structure is considerably shorter, 33 Å in length, compared with 52 Å, as originally proposed. The principal reason for this difference is that our molecular modeling calculations yielded a more energy minimized structure. This resulted from two factors. First, advances in computational speed made since 1989 allowed us to carry out over 1000 iteration steps to calculate our energy minimized structures. Second, the MM2 force field method used by Murgolo et al. (1989)
was an older-generation program that lacked many parameters, specifically force constants for the phosphate and pryrophosphate head groups. In contrast, our 3D structures of C95, C95-P, and C55-P were built by energy minimization with respect to all atoms using the AMBER force field (Weiner et al., 1984
; Chou et al., 1998
; Chou et al., 2000
). In AMBER, the force constants for the phosphate and pyrophosphate groups are well defined.
Given that 33 Å is considerably shorter than the thickness of a biological membrane (4060 Å), this means that if the PIs were oriented perpendicular to the plane of the bilayer, as previously shown for the phosphorylated derivatives (McCloskey and Troy, 1980a,b
; de Ropp and Troy 1984
; Valtersson et al., 1985
; de Ropp et al., 1987
; Murgolo et al., 1989
), they would not span the bilayer. Rather, such a molecule would penetrate to only about the midbilayer region. This important structural feature of the PIs should be considered when proposing models to explain the function of PIs in glyconconjugate synthesis and translocation processes.
Molecular motions of the PIs: why does NeuE bind to the central coil region of the PIs?
The extensive poly-cis double bond geometry in the PIs likely endows them with the flexibility to fold into coils and to adopt the compact, tripartite structures shown in Figure 5. Because the central coil region (28 Å for C95 and C95-P and 19 Å for C55) is longer than both the head (15 Å for C95 and C95-P and 10 Å for C55 ) and tail (13 Å for C95 and C95-P and 8 Å for C55) regions (Table IV), the molecular motions of the coil region for all three PIs was predicted to be slower than the head and tail region. de Ropp et al. (1987) measured the T1 time of the acetyl ester head group and the tail group for 2H-labeled PIs using 2H NMR. These studies showed that the head and tail groups of the nonphos-phorylated PIs had the same T1 and correlation times. In the present study, 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 and found that they were shorter than the protons near the head groups of C95 (Table VI). These results showed that the correlation time of the coil region of C95 was longer than that of the head and tail regions. Thus, the slower motion in the coil region is what likely facilitates the specific binding of NeuE to this domain of the PI molecules.
It has been shown that free C95 can alter membrane fluidity (Valtersson et al., 1985; de Ropp et al., 1987
; Knudsen and Troy 1989a
,b
; Troy, 1991
). Further, the results obtained from differential scanning calorimetry (Valtersson et al., 1985
), fluorescence depolarization (Vigo et al., 1984
), and 31P-NMR (Valtersson et al., 1985
; de Ropp et al., 1987
; Knudsen and Troy, 1989a
; Troy, 1991
) also demonstrated that C95-P can modulate fluidity of a PC bilayer and induce a bilayer to nonbilayer (HexII) structure, even at concentrations as low as 15 mol% (Knudsen and Troy, 1989a
). Our present findings confirm these earlier reports and show further that the PI-induced fluidity in phospholipid membranes is less pronounced after the PIRS peptides bind the PIs. The T1 time for both the coil (poly-CH2 and poly-CH3) and head group region of C95 or C95-P, for example, are significantly lowered after binding NeuE peptide (Tables V and VI). Binding thus decreases the overall motional rates of the PIs. These findings support our hypothesis that the C95-P-induced alteration in bilayer structure may be modulated or at least partially stabilized when PIRS-containing proteins bind to polyisoprenols.
The results in Tables V and VI further show that the T1 values of protons in the head group of C95-P (Figure 1) were significantly longer than those of C95, indicating greater motion within this region of the phosphorylated PI in comparison with the free alcohol. This is in accord with our previous conclusion that C95 and C95-P have different orientations in membranes, possibly because of the strong charge repulsion between the phosphate head group of C95-P and phospholipids (McCloskey and Troy, 1980a,b
). The exclusion interaction caused by these negatively charged groups likely increases the motional rate of the head group and protons in the
-isoprene unit. In contrast, the free hydroxyl head group on C95 is too distantly removed from the phosphate group of the phospholipids to have much effect. As a consequence, the head group and
-isoprene unit in C95 have slower motional rates than that of C95-P.
Conformational differences between the NeuE peptide when bound to C95 and C95-P
Both the 1D 1H NMR and 2D NOESY spectra of NeuE changed significantly after binding C95 or C95-P. No cross-peaks were observed, for example, in the NH-NH, NH-ßH, NH-H, or NH-
regions of the NOESY spectrum for the free NeuE peptide, using 240 ms mixing time, and only 19 cross-peaks appeared in the NH-
region. In contrast, 46 cross-peaks were observed in the NOESY spectrum when NeuE bound C95, and 70 cross-peaks were observed when the peptide bound C95-P. This suggested that the NeuE peptide had different motional properties when bound to C95 or C95-P. This supposition was verified by determining the T1 values for different protons in the peptide in the presence and absence of C95 or C95-P (Tables V and VI). On the basis of these results, we conclude that the different motional rates reflect a different conformation and/or orientation of the NeuE peptide when bound to C95 or C95-P.
Conformational difference between C95 and C95-P after binding NeuE
Our earlier studies showed that free C95 in model membranes did not distribute homogeneously in a PC bilayer but rather underwent pronounced reversible self-association in which little interaction with the phospholipid acyl chains occurred (McCloskey and Troy, 1980a,b
). C95-P, in contrast, remained monomolecularly dispersed. On this basis, we concluded that C95 and C95-P would have different conformations and/or orientations in the membrane, which we now recognize could be modulated by binding a PIRS peptide. Accordingly, differences in orientation and motional rates between C95 and C95-P could be an important factor in affecting the position or extent of NeuE binding. Thus, our energy minimization and simulated annealing calculations, which led to the construction of the structural models for the C55-PNeuE, C95NeuE, and C95-PNeuE complexes (Figures 11 and 12), were significant for two reasons. First, they revealed different conformations for the free and bound PIs; second, they showed that conformational changes occurred in NeuE after binding PI.
Energetics of PIsPIRS binding: van der Waalsinteractions are the major contributions to PI:PIRS docking
Because of the extreme hydrophobicity of both NeuE and the PIs, strong hydrophobic interactions would be predicted to dominate the interactive forces. Such hydrophobic interactions can influence the binding between two molecules over distances extending up to 100 Å (Israelachvili and Pashley, 1982). The docking energy between PIs:NeuE would thus be expected to differ with various binding sites on a PI molecule. Our results show that the minimum docking energy was -10 kcal/mol for a single NeuE peptide when bound to the opposite side of the head group of the central coil of C95 and -7 kcal/mol when bound to the coiled region on the head group side of the PI (Figure 12). Both of these binding interactions involved protons on the L1 and L6 residues of the NeuE peptide (Table VII). In addition, we found that minimum docking energies were obtained when L1 and L6 (for binding to C95) or I3, L6, and I7 (for binding to C95-P) interacted with the coiled region of the PIs (Table VII). Therefore, the specificity of binding between PIs and the PIRS peptide was further supported by measurements of the docking energies. These results indicated that van der Waals interaction were likely the major contribution to the specific binding between PIs and PIRS peptides because the van der Waals distances for these docking pairs is generally <3.0 Å (Tables VII, VIII, IX).
Possible bifunctional role of the PI carrier lipids in glycosylation/translocation reactions
Our molecular modeling showed that one PI molecule could bind more than one NeuE peptide, with differences in the docking energy between the two sides of the PI molecule (Figure 12). This suggests that it may be possible for a single PI molecule to bind several PIRS-containing glycosyltransferase/translocator proteins within a multienzyme complex. For example, a single C55-P molecule within the polysialyltransferase complex of E. coli K1 could bind to the PIRS motif in several NeuE and/or KpsM molecules to tether the transmembrane domain of these proteins in a complex that may link biosynthetic and translocation events (Troy, 1992).
We found that one C95-P or (C55-P) molecule could bind at least three NeuE peptides. The preferred binding site for the first peptide (N1) on C55-P (-9 kcal/mol) was also in the coiled region on the opposite side of the PI head group. When the second NeuE peptide (N2) was docked to the head group side of the PI, and the third peptide (N3) to the middle of the PI between N1 and N2, the docking energies were -6 and -9 kcal/mol, respectively. However, the position of docking of the three peptides made a significant difference in the docking energies. Although the energy minimized docking energy for binding of the first NeuE to the PIs was always 910 kcal/mol, if N2 was docked in close apposition to N1, and N3 close to N2, then the calculated docking energies for the N2 and N3 peptides were -12 and -17 kcal/mol, respectively. This suggested a positive cooperation in binding between the different NeuE peptides. Such cooperation may result from the fact that when the first NeuE is bound to the PI, the docking energy contains contributions from only van der Waals interactions between the peptide and PI. However, when the second and third peptides were moved around the initial NeuEPI binding complex to determine the minimum docking energy, the binding energy for these peptides also contained contributions from electrostatic interactions between the two peptides, in addition to the van der Waals interactions. The significantly more negative docking energies resulting from each sequential NeuE binding further supported a positive cooperativity between NeuE peptides, a conclusion in accord with our finding that binding induced a conformational change in both NeuE (Figures 7, 8) and the PI (Figure 12).
The tethering of several NeuE and/or KpsM molecules could serve to form a channel or to alter bilayer structure such that nascent or newly synthesized polysialic acid chains could transit from the inner to the outer leaflet of the plasma membrane. The different orientations of PIs in the membrane (McCloskey and Troy, 1980b) could also affect the number of PIRS-containing polypeptides that bind to the PI, thus mediating or stabilizing cooperative interactions to influence biosynthetic and translocation processes. Although it is recognized that the transmembrane movement of lipid-linked oligosaccharides is of fundamental importance in N-linked glycosylation, identification of the putative "flippases" has proven elusive (Helenius et al., 2002
). A potential candidate for this reaction in yeast was recently identified as the Rft 1 protein. Rft 1 is an evolutionarily conserved protein that is postulated to be required for the translocation of Man5- GlcNAc2-P-P-Dol across the endoplasmic reticulum membrane (Helenius and Aebi, 2001
). Because no information is available regarding how this protein may be involved in this process, it seems premature to describe it as being involved in the flipping reaction. It would also seem important to determine if Rft 1 interacts with C95/C95-P before concluding that no additional factors are required for Rft 1 function (Helenius and Aebi, 2001
). The idea that "flippases" and the "flipping reaction" is how PI-linked sugar chains actually traverse membranes appears to be an assumption, so good an assumption that some regard it as fact, even though it is an idea that excludes other possibilities.
It is conceivable that a C95-Pprotein complex could also play a regulatory role in the N-linked glycosylation pathway in eukaryotic cells. For example, the OST complex involves nine nonidentical protein subunits, several of which contain potential PI binding sequences. OST catalyzes transfer of C95-P-linked Glc3Man9GlcNAc2-oligosaccharides to Asn-X-Ser/Thr sequons in nascent polypeptide chains as they are translocated across the endoplasmic membrane (Kelleher et al., 1992; Knauer and Lehle, 1999
; Kim et al., 2000
). The transmembrane domain of some OST proteins have been postulated to stabilize their interactions with other OST proteins, presumably via proteinprotein interactions (Kim et al., 2000
). Our finding that a PIRS peptide forms a binding complex with C95-P suggests that this binding may be another way to stabilize these multiprotein subcomplexes to facilitate synthesis and/or transport of lipid-linked glycans across membranes. The detailed molecular mechanism(s) of how these synthetic and translocation processes occur, however, is not understood in any biological system and thus awaits further study.
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Materials and methods |
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Synthesis of the PIRS peptides
Peptides containing the conserved 13 amino acids of the NeuE and Dpm1 PIRS were synthesized by California Peptide Research (Napa, CA) using standard t-BOC chemistry. The resin containing the peptide was dried under high vacuum, and the peptide cleaved from the resin by 90% HF at 0°C. The released peptides were purified by reverse-phase high-performance liquid chromatography and were estimated to be at least 80% pure. The extremely hydrophobic peptides were not soluble in aqueous solutions but were soluble in deuterated DMSO (DMSO-d6) where they were stored as a stock solution at -20°C. The sequence of the NeuE and Dpm1 peptides used in these studies was as follows:
Preparation of PIRS peptides, PIs, and mixtures of PIRS and PIs in DMSO-d6
To study the interaction between PIRS peptides and PIs, aliquots of the stock solutions of NeuE and C95 and NeuE and C95-P in DMSO-d6 and CDCl3 were mixed in a molar ratio of 2:1. The solvent was removed under a stream of N2. Trace amounts of DMSO-d6 and CDCl3 were removed by overnight lyophilization under high vacuum. Lyophilized samples were dissolved in 0.5 ml of DMSO-d6 by vortexing for 10 min. The final concentration of peptide was 2.3 mM and that of the PIs 0.5 mM. Similar methods were used to prepare the free NeuE and Dpm 1 peptide and PIs alone in DMSO. The viscosity of DMSO (10 M) at 18°C is 8 cP, which approximates that of biological membranes at 37°C (Eto and Rubinsky, 1992). The tendency of hydrophobic peptides to aggregate is also diminished in this membrane mimetic solvent (Wakamatsu et al., 1990
). Several studies have shown that the 2D 1H-NMR derived solution structure of hydrophobic transmembrane peptide helicies, for example, those of bacteriorhodopsin, are similar in DMSO as the corresponding peptide region in the crystal structure of the protein (Albert and Yeagle, 2000
; Katragadda et al., 2000
; Yeagle et al., 2000
). Thus, hydrophobic peptides can adopt the same structure in DMSO as in the native protein.
Preparation of deuterated DMPC vesicles
The solvent from an aliquot of the stock solution of deuterated DMPC was removed under a stream of N2. Traces of solvent were removed under high vacuum overnight. One half milliliter of D2O was added to the dried sample, which was vortexed for 10 min to achieve complete suspension of the multilamellar vesicles (MLVs). Small unilamellar vesicles (SUVs) were prepared by sonication of the MLVs on ice for 5 min under an inert atmosphere of argon, as previously described (McCloskey and Troy, 1980a,b
). Sonication was carried out using an XL2020 Sonicator (Heat Systems-Ultrasonics Incorporated, Farmingdale, NY) fitted with a microprobe. After sonication, the resulting clear dispersion was centrifuged at 35,000xg for 30 min, and the supernatant containing the SUVs was used for the NMR experiment. The resulting SUVs were relatively homogeneous in size, with an average diameter of approximately 280 Å, as observed in the electron microscope (Zeiss EM 109) after negative staining with 1% uranyl acetate, as previously described (McCloskey and Troy, 1980a
).
Incorporation of the PIRS peptide (Dpm1; NeuE) and PIs in DMPC vesicles
The following deuterated DMPC membrane vesicles containing either Dpm1 or NeuE peptide were prepared in the presence and absence of the PIs: (1) Dpm1:DMPC and NeuE:DMPC; (2) DMPC and the PIs, C95:DMPC and C55-P:DMPC; and (3) DMPC and the mixture of either PIRS peptides and PIs, C55-P:Dpm1:DMPC and C55-P:NeuE:DMPC. Vesicles were prepared by adding each component to a small flask to give the desired molar ratios, as described in the text. The bulk of the solvent was removed under a stream of N2 gas. The samples were lyophilized overnight, after which 0.5 ml D2O were added. After incubation at room temperature for 2 h, each sample was vortexed for 10 min to achieve complete suspension of the MLVs. Similarly, SUVs were prepared by sonication of the MLVs, as already described. After sonication, the samples were centrifuged at 13,000xg for 30 min, and the SUVs in the supernatant fraction were transferred to a 5-mm NMR tube.
Determination of the 3D structure of C95, C95-P, and C55-P and the Neu-E peptide: 1H NMR masurements
1H NMR spectroscopy was carried out at 18°C and 25°C on a 500 MHz Nicolet (General Electric) spectrometer. 1H shifts were measured relative to the methyl proton resonance of internal sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSS). 1D proton spectra were recorded with a sweep width of 8000 Hz and 16 k 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 PI in both DMSO-d6 and the DMPC membranes. A presaturated pulse sequence was used to inhibit the water resonance peak.
Correlated spectroscopy (COSY), NOESY (Macura and Ernest, 1980; Wuthrich, 1986
), total correlation spectroscopy (TOCSY) (Bax and Davis, 1985
), and double-quantum filtered COSY (DQF-COSY) (Rance et al., 1983
) experiments were carried out on the 500 MHz Nicolet (General Electric) spectrometer. For NOESY experiments, mixing times of 120, 240, 400, and 480 ms were used. For TOCSY experiments, spin-locking fields of 8 kHz and 65 and 80 ms mixing time were used. 3J
N coupling constants were measured using DQF-COSY and incorporating 1D NMR spectra. All 2D spectral widths were 8000 Hz. The data size in the time domain was 512 points in t1 and 2048 points in t2. For each t1 value, 32 transients were accumulated. Data were processed with a combination of exponential and shifted sine-bell window functions for each dimension. The observed 512x2k complex data matrices were zero-filled to 2kx2k (COSY, NOESY, and TOCSY) or 1Kx4K (DQF-COSY). For the DQF-COSY data, the final digital resolution after zero-filling was 1.8 Hz/point. Because of the relatively low solubility of the NeuE peptide in DMPC, it was difficult to obtain a complete 1D and 2D NMR spectra of the peptide in these vesicles alone. Because the peptide was soluble in DMSO, the complete 1D and 2D NMR spectra were determined in this membrane-mimetic solvent at 18°C.
NMR studies of the interaction of PIRS peptides and polyisoprenols
All 1H NMR spectroscopic studies of the binding of PIRS peptides to the PIs were carried out on the 500 MHz Nicolet spectrometer.
Structure refinement and calculations
Experimentally generated distance constraints derived from NOE intensities and torsion angle constraints derived from coupling constant information were used for restrained molecular dynamics and energy minimization calculations. The NOE distance constraints were classified as strong (1.82.5 Å), medium (1.83.3 Å), and weak (1.85.0 Å). The lower bounds for interproton distance were set to the sum of the van der Waals radii of the two protons. Molecular modeling calculations for the NeuE peptide were carried out on a Silicon Graphic Krebs computer using DGII (NMRchitect, Biosym/MSI, San Diego, CA). The 3D structures of C95, C95-P, and C55-P were built by energy minimization using the AMBER force field, where the force constants for the phosphate and pyrophosphate groups are well defined (Weiner et al., 1984; Chou et al., 1998
; Chou et al., 1991
; Chou et al., 2000
). Due to the limited cross-peaks in the NOESY spectra of the PIs, it would have been difficult to obtain a more accurate molecular model using the NOE data and DGII calculations alone. However, Murgolo et al. (1989)
had approximated the structure of C95 based on molecular mechanics and SAXS. Their energy minimization was carried out using the MM2 force field program. Therefore, we were able to use their SAXS results and energy minimization using AMBER with our NMR results to determine an energy minimized structure for C95, C95-P, and C55-P.
Calculations for the docking energies between PIRS peptides and PIs were carried out on the Silicon Graphic Krebs Computer using the Insight II program (Biosym). The objective of the binding calculation (nonbond energy) was to evaluate the interaction energy of many orientations of a PI molecule relative to the PIRS peptide, while searching for orientations that resulted in low interaction energies. Insight II provided information for calculating the interaction energy between two molecules using explicit electrostatic or van der Waals energies, or a combination of the two energies. In our calculations, 8 Å was used as the specified intermolecular cutoff parameter.
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Acknowledgements |
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Footnotes |
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2 To whom correspondence should be addressed; e-mail: fatroy{at}ucdavis.edu
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Abbreviations |
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References |
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