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Vol. 18, Issue 5, 1928-1942, May 2007

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Conformational Dynamics of the Major Yeast Phosphatidylinositol Transfer Protein Sec14p: Insight into the Mechanisms of Phospholipid Exchange and Diseases of Sec14p-Like Protein Deficiencies

Margaret M. Ryan^*, Brenda R.S. Temple, Scott E. Phillips^*, and Vytas A. Bankaitis^*

*Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Research Center; and R. L. Juliano Structural Bioinformatics Core Facility, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090

Submitted November 20, 2006; Revised January 30, 2007; Accepted February 27, 2007
Monitoring Editor: Reid Gilmore

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Molecular dynamics simulations coupled with functional analyses of the major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p identify structural elements involved in regulating the ability of Sec14p to execute phospholipid exchange. The molecular dynamics simulations suggest large rigid body motions within the Sec14p molecule accompany closing and opening of an A₁₀/T₄/A₁₁ helical gate, and that "state-of-closure" of this helical gate determines access to the Sec14p phospholipid binding cavity. The data also project that conformational dynamics of the helical gate are controlled by a hinge unit (residues F₂₁₂, Y₂₁₃, K₂₃₉, I₂₄₀, and I₂₄₂) that links to the N- and C-terminal ends of the helical gate, and by a novel gating module (composed of the B₁LB₂ and A₁₂LT₅ substructures) through which conformational information is transduced to the hinge. The ₁₁₄TDKDGR₁₁₉ motif of B₁LB₂ plays an important role in that transduction process. These simulations offer new mechanistic possibilities for an important half-reaction of the Sec14p phospholipid exchange cycle that occurs on membrane surfaces after Sec14p has ejected bound ligand, and is reloading with another phospholipid molecule. These conformational transitions further suggest structural rationales for known disease missense mutations that functionally compromise mammalian members of the Sec14-protein superfamily.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Phosphatidylinositol transfer proteins (PITPs) play important biological roles in defining the identities of individual lipid signaling pools that regulate specific biological processes. It is in this capacity that PITPs are suggested to represent components of lipid metabolic nanoreactors that promote specific signaling reactions (Ile et al., 2006). How PITPs couple exchange of phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) monomers to physiological function remains unresolved (Phillips et al., 2006). PITPs fall into two families (i.e., the Sec14 family and the metazoan PITP family) on the basis of primary sequence homology and structural fold. Members of one family share no primary homology or structural similarity with members of the other family (Bankaitis et al., 1989; Dickeson et al., 1989; Sha et al., 1998; Yoder et al., 2001).

The SEC14 gene product (Sec14p) is the major PITP of budding yeast, and it is required for efficient transport of secretory cargo from the yeast trans-Golgi network (TGN; Bankaitis et al., 1989, 1990; Cleves et al., 1991a). Genetic and biochemical evidence demonstrates Sec14p coordinates lipid metabolism with activities of proteins required for biogenesis of transport vesicles on yeast TGN membranes (Cleves et al., 1991b; McGee et al., 1994; Xie et al., 1998; Li et al., 2002; Yanagisawa et al., 2002; Phillips et al., 2006). Sec14p is of additional interest in that it is the prototype for a eukaryotic protein superfamily of Sec14-like proteins consisting of >500 known members distributed across the Eukaryota (Phillips et al., 2006). Sec14-like proteins regulate membrane trafficking, membrane biogenetic pathways, or both, that include polarized membrane trafficking in yeast (Carmen-Lopez et al., 1994; Nakase et al., 2001; Rudge et al., 2004) and in higher plants (Vincent et al., 2005). Other Sec14-like proteins are implicated in control of specific stress responses (Kearns et al., 1998; Monks et al., 2001). Finally, loss-of-function mutations in members of the Sec14 superfamily are associated with inherited human disorders (Aravind and Koonin, 1999; Bomar et al., 2003; Meier et al., 2003; Panagabko et al., 2003; Stocker and Baumann, 2003).

Although not formally proven, phospholipid binding and exchange are presumed to be of relevance to Sec14p function in vivo (Ile et al., 2006; Phillips et al., 2006). This reaction is of biochemical interest in that Sec14p ejects bound phospholipid upon encountering a membrane surface and reloads with another phospholipid molecule before disengaging from that surface. Studies in purified systems using defined liposomes and recombinant Sec14p demonstrate the exchange reaction involves efficient abstraction of phospholipid from a stable membrane bilayer and that Sec14p executes this interfacial reaction in the absence of cofactors or ATP (Phillips et al., 1999). Thus, Sec14p couples conformational change to a cycle of phospholipid exchange. The crystal structure of detergent-bound Sec14p suggests how this may occur. The Sec14p hydrophobic cavity is bounded by a hybrid -/3₁₀-helix proposed to function as a gate (Figure 1). The conformational status of this gate is proposed to determine whether Sec14p is in an "open" or a "closed" conformation (Sha et al., 1998; Phillips et al., 1999).

View larger version (63K): [in this window] [in a new window]   Figure 1. Schematic representation of the Sec14p crystal structure. (A) Ribbon and -carbon backbone renditions of the Sec14p monomer. Structural elements include -helices (A1–A12), 310-helices (T1–T8), and -strands (B1–B6). Relevant substructures include helix tripod motif (copper), the A7T3 hybrid helix (gray), -helix A9 (purple), the A10T4 A11 hybrid helix, A12, T5–T8 (green), the 1–6 -strands (gold), and loops (black). The opening of the Sec14p hydrophobic pocket is oriented toward the reader. (B) Sec14p rendition as described in A except viewed from the posterior. Residue G266 is represented by a blue sphere.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Preparation of Starting Structures
The 2.5-Å resolution coordinates of the open Sec14p conformation (PDB 1AUA [PDB] ; Sha et al., 1998) was obtained from the Protein Data Bank (Berman et al., 2000). The two bound -octylglucoside molecules were removed, and crystallographic waters were retained. Hydrogen atoms to both amino acid residues and water molecules were added using the LEaP module of AMBER 8.0 (Case et al., 2004). LEaP was also used to place eight sodium ions at positions of minimum electrostatic potential to neutralize the protein system. The system was solvated in 10,414 TIP3P water molecules (Jorgensen et al., 1983) with a buffer distance of 12.5 Å around the solute. In this manner, an octahedral box with initial dimensions of 81.5 x 81.5 x 81.5 Å, and containing a total of 36,156 atoms, was generated as input to the MD simulations. For purpose of simplicity, we loosely refer to this starting Sec14p structure as apo-Sec14p to reflect it is not a phospholipid-bound conformer.

Glycine 266 was replaced with an aspartate while selecting the lowest energy rotamer by using the Biopolymer module of Insight II (www.accelrys.com) to generate a starting structure for simulations of Sec14p^G266D. This protein system was neutralized with nine sodium ions and solvated in 10,420 TIP3P molecules in a manner similar to the wild-type structure. The final Sec14p^G266D system contained a total of 36,180 atoms in an octahedral box with initial dimensions similar to the Sec14p system.

Molecular Dynamics Simulation
MD simulations were carried out using the SANDER module of AMBER version 8.0. (Case et al., 2004) with the ff03 forcefield of Duan et al. (2003) as defined in parm99.dat and frcmod.ff03 parameter files (Cornell et al., 1995). Long-range electrostatic interactions were estimated using the particle mesh Ewald method (Toukmaji et al., 2000), whereas bonds involving protons were constrained using the SHAKE algorithm (tolerance = 00001 Å). A 2-fs time step was used throughout the simulation. A 10-Å cutoff was applied to Lennard–Jones interactions, and the nonbonded list was updated every 25 steps. Periodic boundary conditions were applied. All MD production runs were carried out at constant temperature and pressure.

Protein systems were minimized for 5000 steps. The first 2000 steps were of steepest descent followed by 3000 steps of conjugate gradient minimization. The minimization was followed by 20-ps constant volume dynamics with heating from 50 K to 300 K. Pressure and temperature were then held constant for 100 ps for the system to reach a density of 1 g/cm³. This was followed by a 40-ps regime at constant volume with reheating from 200 to 300 K, yielding a total equilibration time of 160 ps. The production run was continued for more than 32 ns, and snapshots of the coordinates were written out every 1 ps.

Molecular Dynamics Data Analyses
The PTRAJ module of AMBER 8.0 was used for trajectory processing and analyses of root mean square deviation (rmsd) values, interatomic distances, hydrogen bonding, and atomic positional fluctuations. The and dihedral angles measuring residue backbone conformations were also reported using PTRAJ. Unless otherwise stated, data analyses are reported for the 15- to 32-ns time frames of production simulations so that analyses could be focused on a time window where large conformational changes were evident in the Sec14p molecular dynamics simulation.

Correlation coefficients (Cohen, 1988; Blaikie, 2003) were calculated between local backbone angular conformations and Sec14p "state-of-closure" according to the following equation:

where x_i represents the local backbone angular conformation (_i or _i) of a given residue at time i. The variable y_i represents state-of-closure at time i as measured by distance between the C atoms of residues K₁₉₅ and F₂₃₁. The summations are over all snapshots included in the analysis. Snapshots between 15 and 32 ns were used for the Sec14p analysis, because conformational changes of a gate-like movement (including both opening and closing) occurred during this entire period. For Sec14p^G266D, only snapshots between 2.5 and 4 ns were included in the analysis, because no "gating-like" motion occurred after this period. In addition, the 2.5- to 4.0-ns period included a closing motion without a subsequent opening motion. Correlation coefficients were calculated, and they are reported for every backbone dihedral angle for all residues.

Yeast Strains and Methods
Standard media, genetic techniques, and plasmid shuffle assays were described previously (Ito et al., 1983; Rothstein, 1983; Sherman et al., 1983; Cleves et al., 1991b; Phillips et al., 1999; Li et al., 2002). Strains included CTY182 [MATa ura3-52, his3-200, lys2-801, SEC14], CTY1079 [MATa ura3-52, his3-200, lys2-801, sec14-1^ts spo14::HIS3], CTY558 [MAT ade2 ade3 leu2 his3 ura3-52 sec141::HIS3 YCp(SEC14, URA3)], and CTY303 [MATa ura3-52, his3-200, cki1, sec14P::hisG]. Experiments were performed at 30 and 37°C. All transformants were selected based on acquisition of uracil prototrophy.

Protein Stability
Appropriate derivatives of yeast strain CTY303 carrying SEC14 expression plasmids of interest were grown to mid-logarithmic phase at 25°C and shifted to 37°C for 1, 3, and 5 h. Cell densities were normalized to an OD₆₀₀ = 1.5, protein extracts were prepared by cell disruption with glass beads, and extracts were subsequently analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting.

Site-directed Mutagenesis
The various mutagenic primers and their corresponding reverse complementary sequences are available from us by request. Mutagenesis of SEC14 sequences was performed on a 2.6-kb EcoR1-Sph1 SEC14 fragment resident in either plasmid pRE246 (Alb et al., 1995) or the pQE31-based _HIS6SEC14 plasmid pRE526 (Skinner et al., 1995). Mutagenized SEC14 sequences were confirmed by DNA sequence analysis and correctly mutagenized gene cassettes were individually subcloned into the centromeric and episomal yeast–Escherichia coli shuttle vectors YCplac33 and YEplac195, respectively (Geitz and Sugino, 1988).

Sec14p Purification
Recombinant Sec14p and selected mutant derivatives were purified from E. coli as described previously (Phillips et al., 1999) with modification. Briefly, cultures of E. coli strain KK2186 were grown aerobically at room temperature to an OD₆₀₀ of 0.5 in superbroth medium supplemented with 60 µg/ml ampicillin. Sec14p expression was induced by addition of isopropyl -thiogalactoside to 1 mM final concentration, and cultures were incubated for an additional 5 h with aeration. Cells were harvested by centrifugation at 7000 x g (10 min), and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.1, 300 mM sodium chloride, 1 mM NaN₃, and 0.2 mM phenylmethanesulfonyl fluoride). Lysozyme was added, and the suspension was incubated at room temperature for 10 min with occasional agitation. Cells were disrupted using 0.1- to 0.3-mm glass beads and a bead beater (Biospec Products, Bartlesville, OK), and cell-free lysate was clarified by serial centrifugation at 7000 and 130,000 x g, respectively. The supernatant was applied to Talon Sepharose resin, and bound protein was eluted with a linear imidazole gradient (0–200 mM in lysis buffer). Peak fractions were identified by SDS-PAGE, pooled, and buffer was exchanged with lysis buffer by using Centripore 10 spin columns (Millipore, Billerica, MA).

PtdCho Transfer Assays
[¹⁴C]PtdCho transfer from liposomes to bovine heart mitochondria by using either recombinant protein or yeast cytosol was assayed as described previously (Phillips et al., 1999; Li et al., 2000). When yeast cytosol was used, Sec14p species were expressed in strain CTY303 from the episomal pDR195 vector where Sec14p expression was driven by the PMA1 promoter (Rentsch et al., 1995). This system drives significant overexpression of Sec14p species and increases signal to noise in transfer assays and compensates for trivial effects associated with mutant protein instability.

Limited Proteolysis
Lyophilized trypsin (650275; Calbiochem, San Diego, CA) was reconstituted in 50 mM Tris, pH 7.5, and 10 mM CaCl₂ and stored as 0.1 µg/µl aliquots. Reactions were run in 50 mM Tris, pH 7.5, and 10 mM CaCl₂ with a 100:1 M ratio of recombinant Sec14p (15 µg):trypsin in 50 µl and incubated for 5 min at 25°C. The products were evaluated by SDS-PAGE. Individual proteolytic products were identified by staining with Coomassie blue, excised from destained SDS-polyacrylamide gels, and analyzed by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry at the University of North Carolina Proteomics Core Facility (Chapel Hill, NC).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Helix A₁₀/T₄ Fluctuates between Open and Closed States in Sec14p
The rmsd values comparing a series of structural snapshots, taken at 1-ps intervals of Sec14p during the course of a 32-ns production run, to the first apo-Sec14p snapshot are reported in Figure 2A. A structural transition is apparent for Sec14p at 9 ns, and additional rmsd fluctuations in excess of 1 Å continued throughout the simulation. The fluctuating rmsd values report an ensemble of Sec14p conformers where flexible domains are undergoing large motions. To identify such mobile Sec14p structural elements, the atomic positional fluctuation (apf) for all Sec14p C atoms were monitored during the 15- to 32-ns simulation time window. In all references to amino acid residues herein, we define the initiator Met as residue 1. Plots of the root mean square fluctuation (rmsf) data indicated the largest atomic positional motions of Sec14p occurred within helix A₁₀/T₄ around F₂₃₁ (Figure 2B). Residue F₂₃₁ is located at the C terminus of helix A₁₀/T₄ and exhibited the greatest rmsf value (5.4 Å). The rmsf values that averaged in excess of 2.0 Å included residues 223–238 that span helices A₁₀/T₄ and A₁₁ (referred to as the A₁₀/T₄/A₁₁ helical gate; see below). Similarly, the ₂₇₁DESK₂₇₄ cluster positioned immediately C terminal to helix T₅ in the string motif also showed average rmsf values in excess of 2.0 Å. The significance of these string motif dynamics is discussed below.

View larger version (27K): [in this window] [in a new window]   Figure 2. Mobile regions of the Sec14p molecule. (A) All atom RMSD plot for Sec14p (black) and Sec14pG266D (gray) simulations as a function of simulation time (nanoseconds). (B) Average C atomic position fluctuation plot for Sec14p (black) and Sec14pG266D (gray) simulations as a function of residue (plotted from N to C terminus). Two major areas of distinction between the Sec14p and Sec14pG266D plots are centered around residues K116 and F231. The position of G266 is identified with the arrow for reference.

View larger version (24K): [in this window] [in a new window]   Figure 3. Conformational transitions in the helical gate and Sec14p status-of-closure. (A) Distance monitoring plot between the C atoms for residues R195 and F231 for Sec14p (black) and Sec14pG266D (gray) simulations as a function of time (nanoseconds). (B) C backbone rendition of apo-Sec14p highlighting the relative starting positions of residues R195 (gray sphere) and F231 (black sphere). (C) PtdCho transfer activity of Sec14p cysteine pair mutants. Recombinant His6-Sec14p, His6- Sec14pR195C,F231C, and His6-Sec14pY188C,F231C were purified from E. coli and assayed for PtdCho transfer activity under nonreducing (open bars) and reducing (solid bars) conditions. E. coli cytosol served as background. Activity is expressed as the percentage of total radiolabeled PtdCho transferred in 30 min at 37°C after subtraction of background. Data represent the averages of at least three independent experiments and a working range of 0.25–2.0 µg of recombinant protein. Total input of [14C]PtdCho averaged 37,320 cpm per assay. The average background was 1325 cpm per reaction. (D) Purified recombinant Sec14p was challenged with trypsin and 0.1% SDS as indicated below. Lane 1 represents mass standards and the various forms of Sec14p; the N-terminal Sec14p tryptic fragment (Sec14p'), and the C-terminal Sec14p tryptic fragment (‘Sec14p) are identified at right.

Together, unrestrained MD simulations of apo-Sec14p in explicit solvent reveal an oscillatory pattern for Sec14p between open and closed conformations. The data further suggest that conformational oscillations are associated with "gate-like" motions of the A₁₀/T₄/A₁₁ helices. For purposes of simplicity, we refer to the Sec14p conformation characterized by R₁₉₅-F₂₃₁ interatomic C distance of 10.9 Å as closed, although it is likely this represents a "partially closed" conformation (see Discussion).

Biochemical Evidence for Gate Closure
We sought direct validation that 1) the trajectory of gate closure for Sec14p described by the MD simulation models a relevant conformational pathway and 2) that gate-mediated closure of the Sec14p hydrophobic pocket occurs by constriction of distances between the A₁₀/T₄/A₁₁ helical gate and helix A₉. To that end, opposing pairs of Cys residues were positioned on helices A₉ and A₁₀/T₄, and each pair was evaluated for competence to form a disulfide bond. The double Cys mutant proteins were subsequently purified and assayed in parallel for PtdCho transfer activity under nonreducing and reducing conditions.

Sec14p contains four endogenous Cys residues, all of which are buried within the interior of the protein and are not solvent accessible (Sha et al., 1998). This is reflected by the robust PtdCho transfer activity of Sec14p in both nonreducing and reducing conditions (Figure 3C) and by our inability to modify Sec14p with N-ethylmaleimide without first denaturing the protein (our unpublished data). In the open apo-Sec14p conformation, residues R₁₉₅ and F₂₃₁ are separated by 23.82 Å (Sha et al., 1998). When recombinant Sec14p^R195C,F231C was assayed under nonreducing conditions, a 72% decrease in specific PtdCho transfer activity was recorded for the mutant protein relative to Sec14p (Figure 3C). This diminution of activity was not the result of some trivial and undefined protein denaturation. Sec14p^RF195,231CC PtdCho transfer activity was substantially resuscitated upon introduction of -mercaptoethanol into the transfer assay. These results demonstrate Sec14p tolerates the R₁₉₅C,F₂₃₁C substitutions so long as Sec14p^R195C,F231C is in a reducing environment.

We interpret these data to indicate that the R₁₉₅C and F₂₃₁C side chains efficiently form disulfide bonds in nonreducing environments and that the resultant "forced closure" of the Sec14p hydrophobic pocket is inconsistent with phospholipid transfer activity. That inhibition of Sec14p^R195C,F231C activity under nonreducing conditions was quantitatively constant over a 40-fold range of protein concentration in the assay, strongly argues against formation of intermolecular disulfide bonds (our unpublished data). Such intermolecular reactions are expected to be exquisitely sensitive to protein concentration, whereas formation of intramolecular disulfide bonds should not be so sensitive. As significant distance constraints govern disulfide bond formation (i.e., participating sulfhydryl groups must be within 3 Å of each other; Careaga and Falke, 1992), these data indicate the side chains of helix A₉ residue R₁₉₅ and A₁₀/T₄ residue F₂₃₁ must lie in proximity to each other in the closed Sec14p conformation. In support, a mutant Sec14p^Y188C,F231C (where the interatomic distance for the C atoms of these helix A₉-helix A₁₀/T₄ Cys pairs is 16 Å in the closed Sec14p conformation defined by MD simulation; compared with 10.9 Å in the case of Sec14p^R195C,F231C) exhibited wild-type PtdCho transfer activities in both oxidative and reducing environments (Figure 3C).

Limited proteolysis was used as an independent probe for changes in the proximity of helices A₉ and A₁₀/T₄/A₁₁ as Sec14p transitions from closed to open conformations. Opening of the A₁₀/T₄/A₁₁ helical gate evokes large conformational changes, and we sought to detect proteolytic events unique to open Sec14p conformations. These experiments were aided by the fact that nondenatured Sec14p adopts a compact fold and is highly resistant to proteolytic challenge with trypsin (Figure 3D) and a variety of other proteases (our unpublished data). In the presence of SDS micelles, however, trypsin rapidly and quantitatively cleaves Sec14p to generate two significant degradation products (Sec14p' and 'Sec14p; Figure 3D). In-gel trypsinolysis of each species coupled with MALDI-TOF mass spectrometry unambiguously identified the more rapidly migrating 'Sec14p species as being derived from the C-terminal 109 residues of Sec14p. The N terminus of 'Sec14; is defined by the ₁₉₆EASYISQNYYP ER₂₀₈ tryptic peptide, and all subsequent major tryptic fragments C-terminal to this peptide were identified. Only the short ₂₀₉MGK₂₁₁, ₂₅₀ELLK₂₅₃ and ₂₆₄FGGK₂₆₇ peptides were not detected in these analyses (our unpublished data).

The more slowly migrating Sec14p' represents the N-terminal Sec14p fragment and the most C-terminal tryptic peptide identified from this fragment was ₁₄₆NLVWEYESVVQYR LPACSR₁₆₄. In the intervening 31 residues between Sec14p' and 'Sec14p, the only possible site(s) of trypsinolysis are limited to the amide linkages between helix A₉ residues K₁₈₀/G₁₈₁ and/or R₁₉₅/E₁₉₆, and A₈ residues R₁₆₄/A₁₆₅. Although we have not yet been able to distinguish several viable possibilities for pattern(s) of cleavage (i.e., whether only one or several of these positions define the precise site(s) of cleavage; we know similar trypsin cleavage patterns are obtained for Sec14p and Sec14p^R195C), we note all three candidate positions surround the opening of the hydrophobic pocket in the open Sec14p conformer. We interpret these data to suggest SDS micelles induce transition of Sec14p from the closed to the open conformation with the result that the K₁₈₀, R₁₉₅, and/or R₁₆₄ amide bonds are rendered exquisitely susceptible to trypsin cleavage. Together, the Cys-scanning and trypsinolysis data independently indicate that transition from the open to the closed Sec14p conformation involves closure of the A₁₀/T₄/A₁₁ helical gate with respect to helix A₉.

A Hinge for Rigid Body Motions of the A₁₀/T₄/A₁₁ Helical Gate
Distance monitoring between the C atoms of the highly conserved A₁₀/T₄ residue F₂₂₁ and the B₄ residue I₂₁₄ suggest restricted motion of the N-terminal region of the helix A₁₀/T₄ with respect to the -strands. Similarly, interatomic C distance monitoring between F₂₂₁ and helix A₉ residue A₁₈₇ suggest that relatively constant distances averaging ca. 14.9 Å are maintained between these A₉ and A₁₀/T₄ residues in Sec14p throughout the simulation (Supplemental Figure S2, A and B). A superimposition of average structures from different time frames of the MD simulation is depicted in Supplemental Figure S3. This superimposition illustrates the displacement of helix A₁₀/T₄ in the transition of Sec14p from an open conformation to a closed conformation during the simulation and that the N-terminal aspect of A₁₀/T₄ remains relatively fixed during this transition.

To identify candidate structural elements that might be involved in supporting the transitions between open and closed Sec14p conformations, a set of correlation coefficients between Sec14p state-of-closure parameters and backbone (, ) angle variations were calculated for each residue along the polypeptide chain (Supplemental Figure S4). Distances between the R₁₉₅ and F₂₃₁ C atoms in each snapshot reported the state-of-closure parameter, whereas (, ) angles in each snapshot reported the backbone conformation for each residue. Changes in the (, ) angles of F₂₁₂ and Y₂₁₃ (both reside within the B₄ strand) as well as K₂₃₉ (immediately precedes B₅), I₂₄₀ (N-terminal residue of B₅), and I₂₄₂ (immediately follows B₅) correlated most strongly with the state-of-closure for the A₁₀/T₄/A₁₁ helical gate (less than –0.34 or >0.34). Interestingly, the B₄ and B₅ structural elements flank the A₁₀/T₄ and A₁₁ helices, i.e., these residues occupy both the appropriate positions and exhibit the appropriate conformational motions to hinge the A₁₀/T₄/A₁₁ gate (Figure 4A). For purposes of definition, we identify the overall gating region as residues G₂₁₀-I₂₄₂, and we refer to residues F₂₁₂, Y₂₁₃ as the FY component of the hinge unit and residues K₂₃₉, I₂₄₀, I₂₄₂ as the KII component, respectively.

View larger version (26K): [in this window] [in a new window]   Figure 4. Functional analysis of Sec14p hinge residue mutants. (A) Hinge residues (gray spheres) are identified in the context of a Sec14p -carbon backbone trace at left. The hinge is depicted in greater detail at right where the specific residues of the hinge are identified and rendered as black spheres. The gating helix is the rightmost structural element in these depictions. (B) PtdCho transfer activity of purified recombinant Sec14p (open bars) and Sec14pF221A,S222A (solid bars) as a function of protein concentration (see Materials and Methods). Activity is expressed as the percentage of total [14C]PtdCho transferred after subtraction of background (determined by a mock assay using an equivalent concentration of E. coli cytosol). Data represent the averages of at least three independent experiments. (C) PtdCho transfer assays for yeast cytosol derived from YEp(SEC14), YEp(sec14Y213A) and YEp(sec14Y213A,F221A,S222A) derivatives of the sec14 strain CTY303. The first condition serves as positive control. Background values measured for the negative control [YEp(URA3) cytosol] were subtracted from values measured for YEp(SEC14), YEp(sec14 Y213A) and YEp(sec14Y213A,F221A,S222A) cytosols. With regard to the PtdCho transfer assays in B and C, total input of [14C]PtdCho averaged 15,755 cpm per assay. The background averaged 642 cpm per reaction. In these assays, under the conditions of protein expression used (see Materials and Methods), saturation is achieved at 0.5 µg of purified wild-type Sec14p and 50 µg/ml Sec14p-containing yeast cytosol. The data are presented to emphasize the strong decrement in transfer activity of the mutant proteins.

Together, the MD simulations suggest the structural elements that undergo large-scale motions during gating of the Sec14p hydrophobic pocket move as a rigid body. The data further suggest that such rigid-body conformational motion is facilitated by "hinge-like" local backbone angular changes in the KII component of the hinge that interdigitates with the N terminus of the A₁₀/T₄/A₁₁ helical gate and the FY component of the hinge that resides within the phospholipid binding pocket itself.

Hinge Function and Efficient Sec14p Activity
To assess whether the hinge unit is a functionally important element, the ability of Sec14p derivatives compromised for this element to catalyze PtdCho transfer was assessed in vitro. With regard to the KII hinge component, Sec14p^F221A,S222A is defective in interdigitation of hinge residue I₂₄₂ with the N terminus of the A₁₀/T₄/A₁₁ helical gate and exhibits an 80% reduction in PtdCho transfer activity relative to Sec14p when the corresponding purified recombinant proteins are assayed (Figure 4B). With regard to the FY component of the hinge, the single mutant Sec14p^Y213A protein similarly exhibits a 40% reduction in PtdCho activity in vitro (Figure 4C), but combination of individual defects in the KII and FY hinge components levies more dramatic defects in general hinge function than those observed for either individual defect alone. Sec14p^{Y213A,F221A,S222A} exhibits no measurable PtdCho transfer activity in vitro (Figure 4C) and is nonfunctional as a Sec14p in vivo (Table 1), even though it is a stable protein both in vivo and in cytosol fractions used in transfer assays.

In contrast to the oscillations between open and closed conformations evident in the Sec14p simulation, however, rmsf calculations indicated the A₁₀/T₄/A₁₁ helical gate failed to undergo the large-scale conformational motions throughout the 15- to 32-ns simulation time window that were observed for Sec14p. This is reflected in the reduced rmsf value (1.5 Å) for residue F₂₃₁ in Sec14p^G266D relative to the value of 5.4 Å calculated for F₂₃₁ in Sec14p MD simulations (Figure 2B). Distance-monitoring analyses indicate that, after 3.4 ns, the interatomic distances between C atoms of R₁₉₅ and F₂₃₁ converged to an average value of 10.5 Å. This interatomic distance was then maintained throughout the remainder of the 32-ns simulation (Figure 3A). These measurements suggest apo-Sec14p^G266D collapses to a closed structural conformation and cannot efficiently reopen. Closure of the helical gate in the Sec14p^G266D simulation recapitulated the trajectory observed in the unrestrained Sec14p MD simulation. That is, helix A₁₀/T₄ residue K₂₂₉ receded from residue E₂₅₀ on helix A₁₂ at the same time that helix A₁₀/T₄ residue F₂₃₁ approached residue R₁₉₅ on helix A₉ (Supplemental Figure S1C).

The rmsf values in the immediate vicinity of the G₂₆₆D missense substitution were altered in the Sec14p^G266D simulation relative to those observed for that region in Sec14p. An additional region of C fluctuation also became apparent in the Sec14p^G266D simulation. This new region of conformational mobility involved residues D₁₁₅-R₁₁₉ that reside within the loop element between -strands B₁ and B₂. This unique conformational mobility was maximal for residue K₁₁₆ which was displaced 1.7 Å in Sec14p^G266D compared with 0.8 Å for Sec14p. The significance of these observations is discussed below.

Hinge Function and Gate Closure in Sec14p^G266D
The unrestrained MD simulations of apo-Sec14p^G266D suggested this mutant protein is defective in gate opening. We therefore inspected the conformational dynamics of the FY and KII hinge components to assess their functionality in the context of Sec14p^G266D. As for Sec14p, distance monitoring plots of the F₂₂₁ and I₂₁₄ C atoms suggest that rigidity of the N-terminal region of helix A₁₀/T₄ with respect to the -strands is maintained (Supplemental Figure S2). However, in contrast to Sec14p, distance monitoring of F₂₂₁ and A₁₈₇ C atoms reveal the N-terminal region of helix A₁₀/T₄ approaches helix A₉ during the first 7.0 ns of simulation (Supplemental Figure S2). An average interatomic distance of 8.5 Å is then maintained throughout the remainder of the Sec14p^G266D MD production run. This is in contrast to the average interatomic distance of 15 Å for the F₂₂₁ and A₁₈₇ C atoms maintained in the Sec14p simulation. The contrast between the conformational dynamics of Sec14p and Sec14p^G266D is highlighted by superimposition of the two average structures (Supplemental Figure S3).

Insight into the mechanism of the gate opening defect was gleaned from correlations of backbone (, ) angle variations with R₁₉₅-F₂₃₁ distance monitoring plots that report state-of-closure of the A₁₀/T₄/A₁₁ helical gate. These analyses identified candidate bonds, rotations around which permit conformational changes of the helical gate. The strong correlations between (, ) angle changes and gate closure we observed in the Sec14p MD simulations were preserved in the Sec14p^G266D context for hinge residues K₂₃₉ and I₂₄₂ (–0.48 and –0.59, respectively; Supplemental Figure S4). Unlike the case of Sec14p, however, changes in (, ) angles for hinge residues F₂₁₂ and Y₂₁₃ were no longer correlated with closure of the A₁₀/T₄/A₁₁ helical gate. These collective data suggest the FY component of the hinge functions primarily to open the Sec14p A₁₀/T₄/A₁₁ helical gate, whereas the KII hinge component functions in gate closure.

A Novel Gating Module That Regulates Opening and Closing of the A₁₀/T₄/A₁₁ Helical Gate
How does the G₂₆₆D missense substitution, which resides in the string motif, compromise C-terminal hinge function given these two structural elements are not in proximity to each other? As described below, the 3₁₀-helix T₅ (i.e., the structural element directly impacted by G₂₆₆D) is one component of a novel gating module that we posit regulates opening and closing of the A₁₀/T₄/A₁₁ helical gate (overall distance monitoring schematic is illustrated in Figure 5). This gating module, designated the Sec14G-module, is made up of two independent substructures that are themselves interdigitated via a hydrogen bond (H-bond) network. The first substructure (A₁₂LT₅) includes helix A₁₂, extends slightly beyond 3₁₀-helix T₅ and encompasses residues Q₂₄₈–S₂₆₈. The second substructure is made up of the B₁ and B₂ -strands along with the intervening loop region. It includes residues P₁₀₈-E₁₂₅ and is designated B₁LB₂. Sec14p residues ₁₁₄TDKDGR₁₁₉ of the B₁LB₂ motif cluster with Q₂₅₄ of the A₁₂LT₅ substructure to form a polar, solvent-exposed patch on the Sec14p surface. The function of the Sec14G-module in the context of Sec14p and Sec14p^G266D simulations is compared below. For purposes of comparison, we begin with a dynamics analysis of the hydrogen bond network that involves helix T₅. We then consider the dynamics of the B₁LB₂ component and its interaction with the loop separating helix A₁₂ from 3₁₀-helix T₅.

View larger version (36K): [in this window] [in a new window]   Figure 5. General distance-monitoring schematic of the Sec14p G-module. The Sec14G-module consists of two independent substructures. The first substructure, A12LT5, includes helix A12 and extends slightly beyond helix T5. The second substructure, B1LB2, consists of the 1- and 2 -strands and the intervening loop region. The Sec14G-module conformation depicted here is that which exists in the open Sec14p conformer. Sec14p residue G266 is highlighted as a blue sphere, and relevant interatomic distances to be monitored are identified by the dashed lines. Reference residues that report status-of-closure include helix A10/T4 residue F231 (magenta sphere) and A9 residue R195 (green sphere).

View larger version (29K): [in this window] [in a new window]   Figure 6. Backbone hydrogen bond network of the helix T5 component of the Sec14G-module. All backbone interactions are read from the amide nitrogen to the backbone carbonyl oxygen. (A) Schematic diagram of the hydrogen bond network helix T5 and vicinal residues of Sec14p (top). The two consecutive 4 1 hydrogen bonds are characteristic of 310-helices. Helix capping residues are designated as Nc and Cc. Backbone hydrogen bond interactions are indicated by the dashed lines. An atomic stick rendition of that network within Sec14p is presented at bottom. (B) Schematic diagram of the hydrogen bond network helix T5 and vicinal residues of Sec14pG266D is presented at top. Backbone hydrogen bond interactions are indicated by the dashed lines. An atomic stick model of that network within Sec14pG266D is presented at bottom.

Interface between A₁₂LT₅ and B₁LB₂ in the Sec14G-Module
Several categories of H-bond interactions are evident between the A₁₂LT₅ and B₁LB₂ substructures in Sec14p MD simulations (Figure 5). B₁LB₂ residues T₁₁₄, D₁₁₅, and K₁₁₆ interact with A₁₂LT₅ residues Q₂₅₄ and N₂₅₉ to stabilize the Sec14G-module. R₁₁₉ is a candidate for several intra- and interstructural side chain solvent interactions as well. The panels displayed in Supplemental Figure S7 overlay distance-monitoring plots for the indicated residue pairs within the Sec14G-module. Those results suggest helix A₁₂ residue Q₂₅₄ engages in two H-bond interactions; the backbone interaction with K₁₁₆, and a side chain interaction with D₁₁₅. A side chain to main chain H-bond interaction between residues N₂₅₉ and T₁₁₄ further stabilizes this configuration. As detailed below, rearrangements in this H-bond network correlate with dynamics of the A₁₀/T₄/A₁₁ helical gate.

Sec14G-Module Conductance Correlates with Motions of the A₁₀/T₄/A₁₁ Helical Gate
Gate motions (as reported by distance monitoring between the C atoms of R₁₉₅ and F₂₃₁) correlate with the atom-to-atom H-bond switch involving the Q₂₅₄-D₁₁₅ side chains. Although the side chain interactions are maintained throughout the Sec14p MD simulation, the interactions alternate between the two H-bond acceptor atoms of the D₁₁₅ side chain (OD1 and OD2) (Figure 7A). In the open conformation, H-bonds from Q₂₅₄ NE2 form with either D₁₁₅ OD1 or OD2. When Sec14p transitions to closed conformations, only the Q₂₅₄ NE2::D₁₁₅ OD1 H-bond is evident. A Q₂₅₄ NE2 H-bond switch from D₁₁₅ OD1 to D₁₁₅ OD2 is also registered at the onset of every gate-opening event that occurs once the R₁₉₅ and F₂₃₁ C atoms approach to within 14 Å of each other.

View larger version (29K): [in this window] [in a new window]   Figure 7. Sec14G-module transitions and gate dynamics. (A) Interatomic distances between the C atom of residues R195 and F231 report status of A10/T4/A11 gating helix closure (gray). Distances between the Q254 side chain amine and D115 OD1 (red) and D115OD2 (black) are also plotted. A hydrogen bond interaction between the side chains of Q254 and D115 persists throughout the simulation, but this interaction switches between OD1 and OD2 of residue D115 when Sec14p is in the open conformation, and it is restricted to OD1 when Sec14p is in the closed conformation. (B) Correlation of Sec14G-module to the fluctuating interaction between residues V262 and S268. Segments of time exist (3.5–8.9, 8.9–17.2, and 17.2–22.9 ns) when V262 and S268 (green) are bound or unbound, defining clusters that show transitions occurring at times when the side chains of Q254 and D115 (black) switch between the OD1 and OD2 atoms of D115 as well as transitions in the opening and closing of the A10/T4/A11 helical gate (R195-F231; gray). (C) First 5 ns of unrestrained Sec14pG266D MD simulation of the status of three hydrogen bond interactions between the B1LB2 and A12LT5 substructures during the transition from the open-to-closed Sec14p conformers at 3 ns. The T114–N258 interaction breaks at 0.882 ns but reforms again. Main chain hydrogen bonding interaction (blue) between the K116 amide and the Q254 carbonyl is lost during the transition and fails to reestablish during the simulation.

Rearrangements in the T₅ Helical Component of Sec14p^G266D
The closed Sec14p^G266D conformation is suggested to exhibit significant rearrangements within the helix T₅ backbone conformation. These rearrangements minimize the steric problems otherwise imposed by the G₂₂₆D missense substitution, while maximizing favorable interactions of the G₂₆₆D side chain with solvent. The projected consequence of the G₂₆₆D substitution for the string motif is loss of four H-bonds normally found within the Sec14p T₅ helical component, and formation of a new H-bond not present in Sec14p (Figure 6B). The G₂₆₅–V₂₆₂ interaction is weakened, and the V₂₆₂–S₂₆₈ and S₂₆₈–L₂₆₀ H-bonding interactions are subsequently lost in Sec14p^G266D (Supplemental Figure S6, B–D). In Sec14p, the K₂₆₇ carbonyl engages in an H-bond with the A₂₅₇ amide. By contrast, that specific H-bond interaction breaks early in the Sec14p^G266D simulation and is replaced by an alternate interaction of the A₂₅₇ amide with the backbone carbonyl of the mutant D₂₆₆ residue (Supplemental Figure S6G). This alternate A₂₅₇–D₂₆₆ interaction is quite long-lived; it persists for almost 13 ns. The F₂₆₄–P₂₆₁ and N₂₅₉–P₂₅₆ interactions observed for Sec14p are preserved in the Sec14p^G266D MD simulations (Supplemental Figure S6, A and F).

Disengagement of Sec14G-Module Structural Elements in Sec14p^G266D and Closure of the A₁₀/T₄ Helical Gate
The loops of the two Sec14G-module substructures move apart during the simulated transition of Sec14p^G266D to the closed conformation, and all H-bonds that link A₁₂LT₅ and B₁LB₂ of the Sec14G-module are broken (Supplemental Figure S7, A–D). The transition from open to closed conformations occurs early (between 3 and 4 ns) in the Sec14p^G266D simulations. During this transition period, side chain distances between Q₂₅₄ and D₁₁₅ increase in a manner inconsistent with maintenance of H-bond interactions. The distance monitoring plot between these two specific amino acids then remains erratic throughout the remaining 28 ns of the MD simulation (Supplemental Figure S7, B and C). The N₂₅₉ side chain to T₁₁₄ main chain interaction breaks first (at 0.882 ns of the simulation; snapshot 882), but the interaction forms again (Figure 7C). The next dissociation is the backbone H-bond interaction between K₁₁₆ and Q₂₅₄, and this bond is not recovered again in the simulation. Loss of this H-bond interaction suggests a rationale for the unique site of conformational flexibility that involves residues of the ₁₁₄TDKDGR₁₁₉ motif (particularly K₁₁₆) that is apparent in the Sec14p^G266D apf plot (Figure 2B). The K₁₁₆–Q₂₅₄ dissociation is accompanied by strong correlation between changes in (, ) angles of B₁ residues Y₁₁₁ and H₁₁₂ and closure of the A₁₀/T₄/A₁₁ helical gate (Supplemental Figure S4). These specific correlations uniquely occur in Sec14p^G266D simulations and not in Sec14p MD production runs.

H-Bond Interactions between Loops of the Sec14G-Module Are of Functional Consequence
The MD simulation data implicate residue ₁₁₄TDKDGR₁₁₉ of the B₁LB₂ substructure as playing an important role in activity of the Sec14G-module. Specifically, the data suggest ₁₁₄TDKDGR₁₁₉ helps maintain the association of the B₁LB₂ and A₁₂LT₅ substructures and that this component of the B₁LB₂ motif serves as an inflection point, or swivel, that transduces B₁LB₂ conformational motions to helix A₁₀/T₄/A₁₁ gating function (see Figure 8A for relative positions of K₁₁₆ and G₂₆₆). The MD simulations suggest the backbone H-bond between K₁₁₆ amide (B₁LB₂) and the Q₂₅₄ carbonyl (A₁₂LT₅), and that side chain interactions between D₁₁₅ (B₁LB₂) and Q₂₅₄, play important roles in execution of these functions. One prediction of this hypothesis is that removal of the backbone H-bond formed by K₁₁₆ to the Q₂₅₄ carbonyl and side chain H-bond interactions between residues D₁₁₅ and Q₂₅₄ will be of functional consequence. This prediction was tested in silico with a Sec14^K116P mutant. The K₁₁₆P missense substitution obviates formation of the relevant backbone H-bond with the Q₂₅₄ carbonyl. A 32-ns Sec14p^K116P MD simulation reproduced key features of the Sec14p^G266D simulations described above. Distance monitoring plots for interatomic R₁₉₅-F₂₃₁ C distances show Sec14p^K116P begins to transition toward the closed state at 10 ns of simulation and that a closed state is achieved at 14 ns (average interatomic R₁₉₅-F₂₃₁ C distance of 10.5 Å; Figure 8B). Sec14p^K116P remains closed for the remainder of the simulation.

View larger version (25K): [in this window] [in a new window]   Figure 8. B1LB2–A12LT5 interactions and efficient Sec14p function. (A) -Carbon backbone rendition of Sec14p highlighting residues K116 (gray sphere) and G266 (black sphere). (B) Distance monitoring plot between the C atoms for helix A9 residue R195 and helix A10/T4 residue F231 that report helical gate status-of-closure for Sec14pK116P (black trace) as a function of simulation time (nanoseconds). The same plot for Sec14p is presented as the gray trace. (C) PtdCho transfer activity of cytosol from yeast strains expressing Sec14pK116G, Sec14pK116P, Sec14pD115A, Sec14pD115G, Sec14pD115A,K116P, and Sec14pD115A,K116G as sole Sec14p species. Transfer assays were performed as described in the legend to Figure 4. Total input of [14C]PtdCho averaged 18,803 cpm per assay, whereas assay background averaged 950 cpm per reaction. (D) Stabilities of mutant Sec14p proteins at 30 and 37°C. Cytosol was prepared from the sec14 cki1 yeast strain CTY303 expressing individual Sec14p species from the YEplac195-derived overexpression vector as for phospholipid transfer assays (see Materials and Methods). Aliquots of each cytosol were split and incubated in parallel at 30 or 37°C for 30 min. Reactions were terminated by boiling in SDS (1% final concentration), resolved by SDS-PAGE, transferred to nitrocellulose, and decorated with monoclonal anti-Sec14p antibody. Sec14p species were visualized by the enhanced chemiluminescence system developed by GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom). The Sec14p species are identified at top, and 10 µg of total protein was loaded for each sample.

To fully ablate the interaction between the loops of the B₁LB₂ and A₁₂LT₅ substructures, the K₁₁₆P and D₁₁₅A missense mutations were combined. The resultant Sec14p^D115A,K116P exhibits no measurable PtdCho transfer activity in vitro, whereas Sec14p^D115A,K116G retains wild-type levels of PtdCho transfer activity (Figure 8C). Although we present only the PtdCho transfer data, essentially the same results were obtained in PtdIns transfer assays (our unpublished data). These biochemical data are recapitulated by functional complementation analyses in vivo. Sec14p^K116G, Sec14p^D115A, Sec14p^D115G, and Sec14p^D115A,K116G all score as functional in complementation tests, whereas Sec14p^D115A,K116P is nonfunctional in those in vivo assays (Table 1). Finally, we note the ₁₁₅DKDGRPV₁₂₁ motif is conserved in even distant members of the Sec14 family, e.g., the plant SSH PIP binding proteins (Kearns et al., 1998). It is of functional importance in those contexts as well. The relatively conservative D₁₁₅N substitution inactivates the Sec14-like activities of the soybean PITP Ssh2p (our unpublished data).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Herein, are described a series of conformational transitions that we propose accompany phospholipid exchange by the major yeast PITP Sec14p. These findings were obtained from a combination of unrestrained MD simulations of Sec14p and Sec14p^G266D conformational dynamics and functional analyses that test the predictive power of the simulations. Because simulations derive from a starting structure that approximates an open conformer of an apo-Sec14p, the conformational trajectories described most likely simulate those that occur on membrane surfaces once Sec14p has ejected bound phospholipid and is poised to reload with another phospholipid molecule. The major intramolecular motion simulated for Sec14p involves closing and opening of the A₁₀/T₄/A₁₁ helical gate, i.e., the structural element proposed to regulate access to the hydrophobic phospholipid binding cavity of Sec14p. The conformational dynamics of the helical gate are projected to involve large rigid body motions within the Sec14p molecule, and these motions are controlled by two additional structural elements: 1) a hinge unit that interfaces with the N- and C-terminal ends of the helical gate and controls status-of-closure of the A₁₀/T₄/A₁₁ helical gate; and 2) a gating module, or G-module through which conformational information is transduced to the hinge. A normal mode simulation of Sec14p conformational dynamics that coarsely depicts the motions described in these studies is provided in the Supplemental Figure S8.

The possibility that helix A₁₀/T₄ gates the phospholipid binding cavity of Sec14p was first raised when the crystal structure of an open Sec14p conformer bound to two detergent (-octylglucoside; OG) molecules was solved (Sha et al., 1998; Phillips et al., 1999). Subsequent crystallographic studies of closed conformers of distant members of the Sec14 protein superfamily provided additional support for this idea. In those structures, the cognate A₁₀/T₄ and A₉ helices lie adjacent to each other, thereby sealing the opening to the hydrophobic cavity of Sec14-like proteins (Stocker et al., 2002; Min et al., 2003). The Sec14p MD simulations report oscillating motions of the A₁₀/T₄/A₁₁ helical gate that are consistent with this structural element regulating access of phospholipid to the Sec14p interior. Moreover, the transitions between the closed and open states in the MD simulations involve large movements that occur on fast time scales. The rapidity of these substantial conformational transitions suggests Sec14p is designed to undergo an intrinsic high-frequency "breathing" regime when it is in the apo-conformer on membrane surfaces. These breathing motions may prime the apo-Sec14p for efficient reloading with phospholipid. The rapid motion of the A₁₀/T₄/A₁₁ helical gate is consistent with the rapid rate with which Sec14p catalyzes PtdIns and PtdCho exchange reactions in vitro.

That the MD simulation is approximating conformational motions relevant to Sec14p phospholipid exchange activity is strongly supported by three lines of experimental evidence. First, trypsinolysis and Cys pair cross-linking experiments demonstrate that holo-Sec14p exists in a closed conformation in solution and that open and closed Sec14p conformers are distinguished by the distances between helix A₉ and the A₁₀/T₄/A₁₁ helical gate. The Cys pair experiments further demonstrate that covalent forced closure of the A₁₀/T₄/A₁₁ helical gate is incompatible with phospholipid exchange. These forced closure data provide a powerful first demonstration that mobility of the A₁₀/T₄/A₁₁ helical gate is required for phospholipid exchange by Sec14p. Third, both in vitro and in vivo data demonstrate that a functional hinge unit is essential for Sec14p to catalyze phospholipid transfer in vitro, and for Sec14p functionality in cells.

The MD simulations suggest state-of-closure of the A₁₀/T₄/A₁₁ helical gate is controlled by N- and C-terminal hinges that flank the gate itself. The N-terminal aspect of the helical gate is projected to remain reasonably fixed during transitions between open and closed conformations, whereas the C-terminal region is simulated to swing open and shut, much like a windshield wiper blade. We find it most interesting that the hinge residue F₂₁₂ contacts the acyl chain of OG in the detergent-bound Sec14p structure, which we loosely refer to as the apo-Sec14p conformer, whereas a combination of genetic, biochemical, and crystallographic data identify an involvement of the hinge residue K₂₃₉ in binding of the inositol headgroup of PtdIns (Phillips et al., 1999; our unpublished data). These physical interactions raise the attractive possibility that activities of the hinge subcomponents are instructed not only by intramolecular transitions within the Sec14p molecule but also by the phospholipid ligand itself. Given that the MD simulation data suggest a primary role for the KII component of the hinge in gate closure, and residue K₂₃₉ resides within this component, the ligand-dependent regulation of conformational transitions may restrain the helical gate from reopening. Such a restraint could thereby direct apo-Sec14p from a "shallow breathing" conformational dynamics regime down a trajectory to a stable closed conformer. In that regard, the unrestrained MD simulation for Sec14p almost certainly approximates a "partially closed" structure. The closest approach of the helix A₁₀/T₄ residue F₂₃₁ C atom to the residue R₁₉₅ C atom on helix A₉ in the Sec14p MD simulation is 10.21 Å (snapshot 27653). The corresponding interatomic distance in the closed phospholipid-bound conformer of a yeast Sec14-like protein must be smaller given our ability to induce disulfide bond formation in Sec14p^R195C,F231C.

An unanticipated outcome from comparisons of MD simulations of Sec14p and Sec14p^G266D was identification of a novel gating module, the Sec14G-module, that we propose transduces conformational information to the hinges, and, ultimately, to the A₁₀/T₄/A₁₁ helical gate. This Sec14G-module includes the A₁₂LT₅ and B₁LB₂ composites, and these are linked by an intricate H-bond network. The data suggest a primary function of the Sec14G-module is conductance of conformational change to the hinges to open the A₁₀/T₄/A₁₁ helical gate. A movie depicting key elements of this conductance is presented in the Supplemental Figure S9A. This finding begs reconsideration of the defects associated with the sec14-1^ts mutation, and of the role of the Sec14p string motif itself. Originally, it was suggested the string motif functions merely to stabilize the Sec14-fold and that the sec14-1^ts mutation results in structural destabilization of the protein (Sha et al., 1998). Although the stability of the sec14-1^ts gene product (Sec14p^G266D) is clearly affected, the mutant protein is functionally defective even under conditions where stability is not yet compromised. The Sec14p^G266D MD simulations suggest this intrinsic flaw reflects an inability of Sec14p^G266D to efficiently open the A₁₀/T₄/A₁₁ helical gate because of a defect in the B₁LB₂ substructure of the Sec14G-module. In this regard, the MD simulations reported herein make a strong prediction with respect to the types of conformational differences we expect to discern in the G-modules of open and closed conformers of Sec14p. Specifically, the twist of the ₂ strand, and, subsequently, the ₁ strand around the D₁₁₅/K₁₁₆/Q₂₅₄ swivel depicted in the Supplemental Figure S9B, is expected in the closed phospholipid-bound form of Sec14p. This transition from open to closed conformations will also alter the relative orientations of the loops of the B₁LB₂ and A₁₂LT₅ substructures to each other in a specific way. Determination of whether these key predictions are fulfilled is forthcoming given recent progress in the crystallization of phospholipid-bound forms of the yeast Sec14p homologue Sfh1p (Schaaf et al., 2006).

With regard to the B₁LB₂ substructure of the Sec14G-module, we are compelled to comment on the recent work of Griac and coworkers. They reported the D₁₁₅G missense substitution ablates Sec14p PtdCho transfer activity and simultaneously effects a strong reduction in PtdIns transfer activity (Tahotna et al., 2007). Although we concur that Sec14p^D115G reduces Sec14p PtdCho and PtdIns transfer activity, and now provide the first mechanistic explanation for why this missense substitution exerts these effects, we cannot agree with the claim of Griac and coworkers that Sec14p^D115G is devoid of PtdCho transfer activity (Tahotna et al., 2007). In our hands, Sec14p^D115G exhibits readily detectable PtdCho transfer activity, particularly when precautions are taken to ensure that levels of this polypeptide are not limiting in the phospholipid transfer assay. Among other issues, we note Griac and coworkers used crude yeast cytosol clamped at a rather low concentration in the assays upon which they base their claim, and they prepared those cytosols from yeast where they apparently assumed that Sec14p^D115G and Sec14p levels were similar. We find Sec14p^D115G (as well as Sec14p^D115A and Sec14p^K116P) produced under such conditions is subject to significant postlysis lability at 37°C. Because Tahotna et al. (2007) do not report what effect increased Sec14p^D115G input has on measured transfer activities, or actual levels of mutant protein under conditions of assay, we suspect these issues form the basis of the dissonance between our conclusions and theirs. Obviously, if Sec14p^D115G is not absolutely devoid of PtdCho transfer activity (and in our hands, it retains measurable activity), the claim of Tahotna et al. (2007) that PtdCho transfer activity is dispensible for Sec14p function in vivo is premature. This issue of threshold activity assumes additional import when one considers that physiological levels of Sec14p are approximately an order of magnitude in excess of those required for in vivo function (Salama et al., 1990). It is for this reason that mutant Sec14p derivatives with strong reductions in in vitro PtdIns and PtdCho transfer activity score as functional in genetic complementation assays (case in point, the sec14-1^ts gene product at permissive temperatures; Bankaitis et al., 1990).

The importance of the structural elements identified in this study, and particularly of the B₁LB₂ substructure, in conformational dynamics of Sec14-like proteins is amply emphasized by inspection of distant members of the Sec14 protein superfamily. Human Sec14-like proteins include the vitamin E binding proteins -tocopherol transfer protein (TTP) and SNPF, the neurofibromin 1 (NF1) ras GTPase activating protein, and cellular retinaldehyde binding protein (CRALBP). Either crystal structures, or structural models, are available for each (Stocker et al., 2002; Min et al., 2003; Liu et al., 2005; D'Angelo et al., 2006). We find the structural elements geraine to this study (i.e., the helical gate, the hinge, and the A₁₂LT₅ and B₁LB₂ substructures of the Sec14G-module) are represented in each of these proteins. In Sec14p, TTP, NF1, and SNPF, the loop between cognate 1 and 2 elements (B₁LB₂) orients itself such that the respective -strands bend away from the interior of the hydrophobic pocket and toward the posterior of the protein. A backbone H-bond (K₁₁₆-Q₂₅₄ in Sec14p) is observed between the cognate B₁LB₂ and A₁₂LT₅ substructures in three of the four structures. With regard to that H-bond interaction, TTP is the single exception where the cognate K₁₁₆ is substituted with a proline. Thus, TTP represents a natural version of the K₁₁₆P context that partially compromises Sec14p. Why is TTP tolerant of this K P substitution, but Sec14p is less tolerant? Inspection of the TTP crystal structure suggests the C-terminal helix of this protein provides structural support to the atypical TTP G-module, thereby obviating the necessity of a backbone hydrogen bond between the cognate TTP B₁LB₂ and A₁₂LT₅ motifs (data not shown).

Are the cognate helical gate, hinge and A₁₂LT₅/B₁LB₂ substructures of the G-module of functional consequence for these distant members of the Sec14 superfamily? It seems so. TTP, NF1 and SNPF, and CRALBP insufficiencies cause various human diseases, and the responsible disease mutations have been identified at the primary sequence level. Strikingly, a number of these mutations lie either within the hinge regions that flank the helical gate or within the B₁LB₂ substructure of the cognate G-module itself (Figure 9). Given the small size of the B₁LB₂ element (17 residues), it is impressive how well represented this motif is with regard to incidence of mutation. We suggest the disease mutations highlighted in Figure 9 resemble Sec14p^G266D in functional crippling of the G-modules of their respective proteins. These defects in turn hinder opening of the corresponding helical gates that control access of the individual binding substrates to the hydrophobic ligand-binding cavities of each of these Sec14-like proteins. With regard to the atypical G-module of TTP, a common truncation mutation found in families affected by severe ataxia with vitamin E deficiency is caused by a single base-pair insertion (Ouachi et al., 1995; Benomar et al., 2002). This insertion results in a frame shift that alters the last 30 residues of TTP (Figure 9), thereby compromising the C-terminal TTP helix we posit stabilizes the atypical G-module of this Sec14-like protein.

View larger version (46K): [in this window] [in a new window]   Figure 9. Relationship between disease mutations in Sec14-like proteins and structural elements involved in conformational transitions. Primary sequences of Sec14p, human TTP, and human NF1 were aligned on the basis of primary sequence homology and structural criteria. CRALBP was aligned according to an TTP-based structural homology search (Liu et al., 2005). Relevant secondary structures for each conformational element are indicated at top. Key Sec14p residues in this study and disease-causing mutations in the indicated Sec14-like proteins are highlighted.

	ACKNOWLEDGMENTS

 
Oligonucleotide primer synthesis and DNA sequence analyses were performed at the University of North Carolina Lineberger Comprehensive Cancer Center Genome Analysis and Nucleic Acids Core Facility. The mass spectrometry was performed at the University of North Carolina–Duke Michael Hooker Proteomics Center, and we thank Carol Parker for assistance and guidance in these analyses. Finally, we are grateful to Jan Hermans (Department of Biochemistry and Biophysics, University of North Carolina) for helpful advice regarding the MD simulations. MD production runs were performed at the R. L. Juliano Structural Bioinformatics Core Facility. This work was supported by National Institutes of Health grant GM-44530 (to V.A.B.).

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-11-1024) on March 7, 2007.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Vytas A. Bankaitis (vytas{at}med.unc.edu)

Abbreviations used: TTP, -tocopherol transfer protein; OG, -octylglucoside; apf, atomic positional fluctuation; CRALBP, cellular retinaldehyde binding protein; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; MD, molecular dynamics; NF1, neurofibromin 1; PAGE, polyacrylamide gel electrophoresis; PtdCho, phosphatidylcholine; PtdIns, phosphatidylinositol; rmsd, root mean square deviation; rmsf, root mean square fluctuation.

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