Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors[S]

Sec14-like phosphatidylinositol transfer proteins (PITPs) play important biological functions in integrating multiple aspects of intracellular lipid metabolism with phosphatidylinositol-4-phosphate signaling. As such, these proteins offer new opportunities for highly selective chemical interference with specific phosphoinositide pathways in cells. The first and best characterized small molecule inhibitors of the yeast PITP, Sec14, are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and a hallmark feature of NPPMs is their exquisite targeting specificities for Sec14 relative to other closely related Sec14-like PITPs. Our present understanding of Sec14::NPPM binding interactions is based on computational docking and rational loss-of-function approaches. While those approaches have been informative, we still lack an adequate understanding of the basis for the high selectivity of NPPMs among closely related Sec14-like PITPs. Herein, we describe a Sec14 motif, which we term the VV signature, that contributes significantly to the NPPM sensitivity/resistance of Sec14-like phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer proteins. The data not only reveal previously unappreciated determinants that govern Sec14-like PITP sensitivities to NPPMs, but enable predictions of which Sec14-like PtdIns/PtdCho transfer proteins are likely to be NPPM resistant or sensitive based on primary sequence considerations. Finally, the data provide independent evidence in support of previous studies highlighting the importance of Sec14 residue Ser173 in the mechanism by which NPPMs engage and inhibit Sec14-like PITPs.


Small molecule inhibitors
Small molecules of interest were purchased from ChemBridge Chemical Store (San Diego, CA.), dissolved in DMSO to a fi nal stock concentration of 20 mM, and stored in the dark at Ϫ 20°C.

Unbiased cell-based screen
Spontaneous NPPM R mutants were generated using yeast strain CTY1079 ( MAT a ura3-52 lys2-801 his3 ⌬ -200 spo14 ⌬ SEC14 ) or the isogenic pdr5 ⌬ strain, YKM03, and seeded onto YPD plates individually supplemented with the appropriate NPPM at concentrations of 10 M and 2 M, respectively. Approximately 1 × 10 7 cells from 45 independent overnight cultures were each seeded onto a YPD agar plate, and the seeded plates were incubated at 30°C for 96 h. One colony was picked from each plate and purifi ed by two rounds of dilution streaking for isolated colonies. The NPPM R phenotype of each mutant colony was verifi ed on YPD agar supplemented with NPPM and the colonies were expanded to generate individual frozen stock cultures.

Amplifi cation and DNA sequencing of SEC14 genes from isolated NPPM R yeast isolates
Genomic DNA from each independently isolated NPPM R mutant was prepared and the SEC14 gene amplifi ed via PCR using the DKO98 and 99 primer pair (supplementary Table 2). The SEC14 nucleotide sequences were determined in both directions (Eton), and aligned using the multiple sequence alignment program, Clustal Omega, with wild-type SEC14 as query sequence ( 27,28 ).

Protein expression construct generation
NPPM R missense mutations and VV bar code mutants were generated by site-directed mutagenesis using pET28b ( His 8 -SEC14 ) as mutagenesis substrate. Site-directed mutations were generated using QuikChange II (Stratagene) and confi rmed by nucleotide sequencing. SEC14 CA was amplifi ed from genomic DNA via two rounds of PCR using the DKO1,2 and DKO14,15 primer sets (supplementary Table 2) and subcloned into pET28b, taking advantage of plasmid Nco I and Sac I restriction sites . SEC14 KL was amplifi ed using oligonucleotides KL100,101 and subcloned into the Sac II and Sph I restriction sites of plasmid pVB16. An internal Nco I restriction site in SEC14 KL was destroyed by incorporating a sense mutation using the KL92,93 mutagenic primer pair followed by a second round of amplifi cation using primers KL90,91 to incorporate an N-terminal His 8 tag. That PCR product was subcloned as an Nco I-Sac I restriction fragment into pET28b. The SEC14 CG gene was similarly amplifi ed using oligonucleotides CG86,87 as primers, subcloned into pVB16, and a natural Nco I site was removed by site-directed mutagenesis. The modifi ed gene was amplifi ed in a second round using CG89,90 as primer pair and the amplifi ed gene was subcloned into pET28b as an Nco I-Sac 1 restriction fragment. Primer sequences are listed in supplementary Table 2. Protein expression plasmids used in this study are listed in supplementary Table 3.
An attractive property of NPPMs is their exquisite selectivity for Sec14 as a target. Even though yeast expresses fi ve other Sec14-like PITPs ( 13 ), none of these proteins are at all sensitive to inhibition by NPPMs. This selectivity is, in most cases, accounted for by the mechanism by which NPPMs are bound by Sec14. Available data project that NPPM invades the hydrophobic lipid-binding pocket of Sec14 during a lipid-exchange cycle where the inhibitor engages both residues within PtdIns-and PtdCho-acyl chain space and residues essential for coordination of the PtdCho headgroup. Computational docking simulations, coupled with rational mutagenesis experiments, indicate the latter binding interactions are crucial. The NPPM-activated aryl halide moiety is projected to engage residue Ser173, a key component of the PtdCho headgroup-coordinating substructure, via a halogen bond ( 14 ). Because four of the other fi ve yeast Sec14-like PITPs (Sfh2-Sfh5) do not conserve this PtdCho-coordinating unit ( 9 ), the NPPM resistance of those proteins is readily explained by their lack of the structural elements essential for NPPM binding.
The Sec14 specifi city of NPPM remains completely obscure in other cases. For example, the Sec14-like PITP, Sfh1, is highly homologous to Sec14. Sfh1 has both PtdInsand PtdCho-binding/transfer activities, as it shares with Sec14 the same PtdIns-binding motif and, more strikingly, the same PtdCho-coordinating substructure that is critical for NPPM binding ( 9,13,22 ). Yet, Sfh1 lipid exchange activities are impervious to NPPM challenge ( 14 ). Moreover, Sec14-like PtdIns/PtdCho-transfer proteins from other fungal pathogens are even more similar to Sec14 than is Sfh1, but are similarly resistant to inhibition by NPPMs (see Results). Those exceptional cases indicate that, despite a detailed and experimentally well-supported description for how NPPMs dock into the Sec14 lipidbinding pocket ( 14 ), there remain substantial gaps in our knowledge regarding the mechanism of Sec14 inhibition by NPPMs. Herein, we exploit unbiased approaches to identify a new Sec14 motif, the VV signature, that contributes to NPPM sensitivity. The data empower prediction of which Sec14-like PtdIns/PtdCho-transfer proteins are likely to be NPPM resistant or sensitive by extending our understanding of the underlying mechanisms that govern the sensitivities of Sec14-like PITPs to NPPMs.

Yeast media and methods
Yeast complex and minimal media and genetic methods followed standard procedures (23)(24)(25). Yeast strains used in this study For Glide docking routines, proteins and ligands were prepared using Protein Preparation Wizard and the LigPrep module of Maestro 10.1 Interface of Schrödinger Suite (Schrödinger Suite 2015, Glide version 6.6). Receptor grids were generated without constraints and with standard settings. Docking was performed using XP scoring function. No similarity torsional and inter-molecular interaction (hydrogen bonding or hydrophobic) constraints were used. Ligand was docked fl exibly with nitrogen inversions and ring sampling turned on with post-docking minimization ( 31 ).

Molecular mechanics with generalized born and surface area
Molecular mechanics with generalized born and surface area (MM-GBSA) solvation is a widely applied method to calculate the free energy of the binding of ligands to proteins. A MM-GBSA approach was applied to calculate ligand binding energies and ligand strain energies in wild-type and mutant protein-ligand complexes. The docked solutions generated from GOLD and Glide ligand docking were used for calculating ligand binding energies of complexes. Residues within a 10 Å radius from the ligand were included in the fl exible region with implicit solvent. MM-GBSA is implemented in the Prime module of the Schrödinger software package ( 32 ). Structural coordinates of Sec14::NPPM complexes overlayed on model Sec14 structures from Candida albicans , Candida glabrata , and Kluyveromyces lactis in PDB format are available upon request.

Comparisons of NPPM 6748-481 docking poses in Sec14 versus Sfh1
We sought to capitalize on high resolution Sfh1 crystal structures and a detailed computational model for how Sec14 binds NPPM 6748-481 to produce a NPPM-sensitive Sfh1 for the purpose of direct structural analysis of an Sfh1::NPPM complex. This approach was recommended by the fact that the modeled NPPM-binding pose in the Sec14 hydrophobic pocket is experimentally supported by rational mutagenesis and chemical structure-activity relationship data ( 14 ), that the Sfh1 and Sec14 binding pockets share 79% primary sequence identity and 89% similarity in the residues that form the boundaries of their respective phospholipid binding cavities, and that those two PITPs share essentially identical phospholipid binding properties ( 9 ). The high degree of structural conservation between these two cavities is further emphasized by the 1.06 Å root mean square deviation (RMSD) calculated after superposition of the two cavity structures . In those comparative analyses, the binding pockets were defi ned by the binocular criteria of Sfh1 versus Sec14 structural alignments and residues of interest falling within 4.5 Å of bound 6748-481 (as represented by the experimentally favored Sec14::NPPM 6748-481 pose previously described) ( 14 ).
To query why Sfh1 is naturally resistant to inhibition by NPPMs, 6748-481 was docked into the phospholipid-binding cavity of Sfh1 (see Materials and Methods), and those results were compared with the model pose for 6748-481 bound in the Sec14 hydrophobic pocket. First, the Sfh1 crystal structure was superposed on Sec14 coordinates and the respective phospholipid-binding cavities were related by expression plasmids were incubated at 37°C with shaking until cultures reached the desired cell densities (A 600 = 0.8). Recombinant protein expression was induced with 60 M isopropyl ␤ -D-1-thiogalactopyranoside and cultures were incubated for an additional 18 h at 16°C. Cells were pelleted, resuspended in 300 mM NaCl, 25 mM Na 2 HPO 4 , and 1 mM phenylmethanesulfonylfl uoride (pH 7.8), and subsequently disrupted by two successive passages through a French press at 10,000 psi. Cell-free lysates were clarifi ed by serial centrifugations at 2,800 g (20 min) and 27,000 g (60 min). Clarifi ed lysates were incubated with Co 2+ TALON metal affi nity beads overnight at 4°C with agitation, and washed exhaustively with 300 mM NaCl, 25 mM Na 2 HPO 4 , 5 mM 2-mercapthoethanol, and 5 mM imidazole (pH 7.8). Bound proteins were eluted with a continuous 10-200 mM imidazole gradient in 300 mM NaCl, 25 mM Na 2 HPO 4 , and 5 mM 2-mercapthoethanol (pH 7.8). Peak fractions were pooled, dialyzed against 300 mM NaCl, 25 mM Na 2 HPO 4 (pH 7.8), and 5 mM 2-mercaptoethanol, and concentrated by using Amicon Ultra fi lter centrifugation (EMD Millipore). Protein concentrations were estimated by SDS-PAGE and visual comparisons to BSA titration series, and by A 280 measurements.

PtdIns transfer assays
Assays were performed by previously established methods ( 9,14 ). Recombinant Sec14 proteins were preincubated with acceptor membranes in 300 mM NaCl and 25 mM Na 2 HPO 4 (pH 7.5) and SMI or DMSO (vehicle control), as appropriate, for 30 min at 37°C. Donor membranes (rat-liver microsomes) were added to initiate the assay, and reactions were incubated for an additional 30 min at 37°C.

Preparation of structural fi les for docking simulations
A homology model for the closed Sec14 conformer was generated based on structural templates for both the open Sec14 conformer [Protein Data Bank (PDB) identifi cation, 1AUA] and the closed conformer of Sfh1 bound to PtdIns (PDB ID, 3B7N ). Proteins were prepared for docking using the Protein Preparation Wizard panel in Schrödinger Suite and complete structure was optimized to relieve all atom and bond constraints after adding all side chains and missing atoms ( 29 ).

Docking simulations
Two independent docking platforms were used. Computational docking used the genetic algorithm-based ligand docking program, GOLD (version 5.2.1), which exhaustively explores ligand conformations and provides limited fl exibility to protein side chains with -OH groups by reorienting the H-bond donor and acceptor moieties. The GOLD scoring function is based on favorable conformations documented in the Cambridge Structural Database, and on empirical results of weak chemical interactions. The active site was defi ned by a single solvent accessible point near the center of the protein active site, a radius of ‫ف‬ 10 Å, and the GOLD cavity detection algorithm. To maintain consistency in docking, Sfh1 crystal structure was superposed on Sec14 coordinates and the binding cavity was defi ned using the same centroid that was used to defi ne the Sec14 binding pocket. This ensured that the binding site interaction grids generated before docking shared relatable spatial coordinates and allowed direct comparisons of Sfh1 and Sec14 6748-481 docking results. GOLD docking was unconstrained to obtain unbiased results and to explore all possible ligand binding modes. Ligand was docked in independent runs, the 10 best solutions were produced for each run, and early termination of ligand docking was switched off. All other parameters used default settings ( 30 ). acquire NPPM sensitivity. To that end, structural and primary sequence alignments of Sfh1 and Sec14 identifi ed six divergent residues in the cohort of amino acids that line the binding pocket and lie within 4.5 Å of the "bound" 6748-481. Except for F 156 (as numbered in Sfh1) most of the nonconserved residues are positioned toward the hydrophobic subregion of the proposed NPPM binding site ( Fig. 2A ). Those six residues were converted en bloc to the cognate residues in Sec14 (F 153 V, L 176 M, I 193 V, V 196 A, A 197 S, and V 227 F) to generate what we term the Sfh1 6X mutant. Computational docking experiments were then run using the same parameters and settings as those employed in the Sfh1/Sec14 comparative docking experiments. Gratifyingly, the highest ranked docking solution was much more similar to the favored Sec14::6748-481 pose (RMSD = 1.0 Å), and the distal fl uoro-benzyl group now intercalated into the hydrophobic cleft where it engaged in stacking interaction with the newly converted F 230 ( Fig. 2A ). While, the chloro-nitrophenyl headgroup of 6748-481 still adopted the 180° rotamer conformation relative to the Sec14::6748-481 pose, we considered this a conformer that could rotate the activated aryl-halide functional group to a pose nearly identical to that which we predict describes bound 6748-481 in the Sec14 lipid-binding cavity ( 14 ).
To test whether the reengineered protein was sensitive to inhibition by NPPM, a Q 204 A missense substitution was incorporated into the Sfh1 6X protein. This was done because Q 204 A endows Sfh1 the desirable properties of stimulated lipid exchange activity in vitro and enhanced Sec14-like properties in vivo ( 14,22 ). Those features made it an ideal experimental scaffold for manipulating, and subsequently monitoring, the NPPM-resistance/sensitivity properties of Sfh1 and its variants. Surprisingly, while purifi ed recombinant Sfh1 Q204A,6X exhibited robust [ 3 H]PtdIns transfer activity in vitro, this activity remained indifferent to a high concentration of 6748-481 (20 M, ‫ف‬ 100 times Sec14 IC 50 ; Fig. 2B ).

Genetic screen for Sec14 variants resistant to NPPM
The inadequacy of the working Sec14::NPPM 6748-481 docking solution in guiding successful engineering of an NPPM-sensitive version of Sfh1 indicated that our defi ning a reference centroid within the superposed cavity environments. As had previously been done with Sec14 ( 14 ), the Sfh1 pocket environment was defi ned as the sampling space for 6748-481 docking simulations. The reference centroid ensured that the Sec14 and Sfh1 interaction grids shared relatable spatial coordinates, thereby allowing direct comparisons of Sfh1 and Sec14 6748-481 docking results. Comparison of the highest-scoring 6748-481 dock pose indicated the Sfh1 solution shared the same coordinate space within the hydrophobic pocket, and a similar conformation as that calculated for Sec14. That is, the chloro-nitrophenyl group of 6748-481 was oriented toward the polar subregion of the pocket ( Fig. 1A ). However, unlike the case of the Sec14::6748-481 complex, where the chloro-nitrophenyl headgroup coordinates with the S 173 -OH group, the Sfh1::6748-481 complex has the NPPM headgroup rotated 180° around the single phenyl-acyl rotatable bond that links the chloro-nitrophenyl headgroup moiety with the ketone functional group of the NPPM. This same rotamer was also represented in potential docking solutions for NPPM 6748-481 with Sec14, and it was initially considered a plausible binding mode pending experimental test ( 14 ). Short MDS runs (10 ns) showed the rotamer readily fl ips its headgroup orientation to adopt the 6748-481 pose in the Sec14 cavity supported by experiment (supplementary Movie 1). Nonetheless, the Sec14 and Sfh1 6748-481 docking poses remain signifi cantly different (RMSD > 3 Å, Fig. 1B ). Of particular note, the distal fl uorobenzyl moiety of 6748-481 is wedged in a deep hydrophobic cleft of the Sec14 lipid-binding cavity where it engages in stacking interactions with residues F 228 and F 212 . In the Sfh1 case, those hydrophobic interactions are not evident ( Fig.  1B ), and this disparity refl ects the fact that the Sec14 and Sfh1 pocket residues diverge most signifi cantly along this subregion of their respective lipid-binding cavities.

Rational structure-based engineering of the Sfh1 pocket is insuffi cient to produce an NPPM-sensitive Sfh1
We sought to use the structural and 6748-481 docking information to design an Sfh1 with a Sec14-like pocket with the expectation that such an Sfh1 variant would now yeast mutant as parental strain to preclude any background of known bypass Sec14 mutants.
Parental cells were seeded onto YPD agar plates individually supplemented with NPPMs, 6748-481, 4130-1276, or 67170-49, to fi nal concentrations of 10 M (supplementary Fig. 1A). While these NPPMs vary in their potencies as Sec14 inhibitors (6748-481 > 67170-49 > 4130-1276), all three NPPMs strongly inhibited yeast cell proliferation when incorporated in growth medium at this concentration. After incubation at 30°C for 96 h, the frequencies of emerging NPPM R colonies were scored. As expected, those frequencies were inversely proportional to NPPM potency as Sec14 inhibitor. Colonies resistant to 4130-1276 or 67179-49 intoxication arose at frequencies of ‫ف‬ 3 × 10 Ϫ 5 and 10 Ϫ 6 per cell per generation, respectively, and those resistant clones started appearing within 48 h of incubation. By contrast, colonies resistant to the most potent inhibitor, 6748-481, appeared only after approximately 96 h of incubation, and those colonies emerged at much lower frequencies ( ‫ف‬ 10 Ϫ 7 per cell per generation) (supplementary Fig. 1B). A parallel 6748-481 resistance selection was also performed in an isogenic pdr5 ⌬ genetic background where the structural gene for the major yeast drug pump was deleted ( 39 ). In that version of the screen, the 6748-481 concentration for selection was lowered to 2 M, as the pdr5 ⌬ parental strain is approximately fi ve times more sensitive to the inhibitor (unpublished observations). The frequencies of the 6748-481 R colonies obtained in this sensitized selection regime were reduced even further ( ‫ف‬ 3 × 10 Ϫ 8 per cell per understanding of the inhibitor binding mechanism was, at best, incomplete or, at worst, incorrect. We therefore sought to gain additional insight regarding mechanisms of NPPM-binding by Sec14 (and NPPM-resistance for Sfh1) via an approach that did not rely on the necessarily targeted logic inherent to structure-based regimes. To that end, an unbiased genetic screen was performed that selected for mutant Sec14s ablated for NPPM sensitivity, while at the same time demanding maintenance of biologically suffi cient levels of protein activity. One important consideration in designing the screen was limiting re-isolation of previously characterized "bypass Sec14" mutations that represented loss-of-function mutations in structural genes of the CDP-choline pathway for PtdCho biosynthesis ( 17 ), the SAC1 gene which encodes the major yeast PtdIns-4-P phosphatase (33)(34)(35) or the sterol-and PtdIns-4-P-binding protein, Kes1/Osh4 ( 19,(36)(37)(38). These bypass Sec14 mutations occur spontaneously at high frequencies [ ‫ف‬ 5 × 10 Ϫ 5 per cell per generation ( 17,33 )], would pass selection by conferring viability to yeast cells defi cient in normally essential Sec14 activities ( 14,21 ), and dominate the results of the screen . We therefore took advantage of the fact that all known bypass Sec14 mechanisms require activity of the normally nonessential phospholipase D enzyme encoded by the SPO14 gene to alleviate the cellular Sec14 requirement, and spo14 ⌬ yeasts fail to yield spontaneously occurring bypass Sec14 mutants at all [<10 Ϫ 10 per cell per generation ( 26 )]. Thus, the NPPM R screen was conducted using an otherwise wild-type spo14 ⌬  Table 4.
single amino acid changes to the Sec14 protein sequence: P 120 Q, V 154 F, V 155 F, S 173 P, R 208 L, G 210 V, and F 212 L. Superposition of the NPPM R missense substitutions onto high resolution Sec14 structural models demonstrated that, consistent with our previous in silico docking solution ( 14 ), all substitutions involved residues positioned in the immediate vicinity of the Sec14 lipid binding pocket ( Fig.  3A ). Indeed, we had previously identifi ed S 173 and F 212 in Sec14::NPPM interaction fi ngerprint analyses where those two residues engaged the activated aryl halide and fl uoro-benzyl moieties of NPPM 6748-481 via polar and hydrophobic/ -stacking interactions, respectively ( 14 ). Recovery of Sec14 S173P was particularly satisfying, as this variant was altered for the very residue identifi ed by our previous docking simulations and rational mutagenesis studies as essential for the halogen-bonding mechanism that we propose governs Sec14::NPPM binding interactions ( 14 ). By contrast, the remaining fi ve residues exhibited generation). As existing Sec14::NPPM docking poses were built using 6748-481 as ligand ( 14 ), subsequent analyses were restricted to yeast isolates resistant to NPPM 6748-481. A total of 45 independently isolated 6748-481-resistant clones were recovered from the two parallel screens, these clones were purifi ed by at least two rounds of single colony isolation, and were analyzed in further detail.

Sec14 missense substitutions that confer NPPM resistance
The SEC14 gene was amplifi ed from each of the isolates by PCR using genomic DNA as template, and the nucleotide sequences of the recovered SEC14 genes were determined in their entirety. Of the genes so analyzed, 35 exhibited wild-type SEC14 sequence, while the remaining 10 carried single missense mutations in SEC14 . Those 10 mutant SEC14 genes represented a total of seven unique SEC14 missense mutations that resulted in the following The mock condition documents the phenotype of an isogenic strain where a SEC14 -less integration cassette was transplaced into the LEU2 locus. That expression of each Sec14 protein was suffi cient to rescue sec14-1 ts growth defects at the restrictive temperature of 37°C is demonstrated by comparison of the growth profi les in the left (30°C) and center (37°C) panels of the integrants relative to mock controls. The NPPM R phenotypes are displayed in the right panel. The plates were incubated for 48 h at the indicated temperatures before imaging. The NPPM481-resistance phenotypes were scored at 30°C. Note that R 208 L and F 212 L were weak mutants. Scale bar, 1 cm. C: Indicates that spontaneous mutants generated in response to 6748-481 confer pan-NPPM resistance to Sec14. The experiment is the same as above in (B) except that the plates were supplemented with  Table 5. transfer assays spiked with 20 M NPPM, but all those proteins nevertheless retained measurable sensitivities to the NPPM at this concentration [ ‫ف‬ 100 times IC 50 Sec14 ( 14 )].
In line with the weakness of the associated NPPM R phenotype, Sec14 F212L was the least resistant to inhibition by NPPM481 at this concentration of inhibitor. The PtdIns transfer activity of this mutant exhibited an IC 50 (642.7 ± 1.2 nM) that was only modestly increased relative to that of wild-type Sec14 protein (287.8 ± 1.1 nM).

A VV signature correlated with NPPM sensitivity
Of the seven Sec14 residues identifi ed in the unbiased NPPM-resistance screen, we noted that P 120 , S 173 , R 208 , G 210 , and F 212 are precisely conserved between Sec14 and Sfh1. However, V 154 and V 155 are not conserved at all ( Fig. 4A ). The Sec14 V 154 V 155 motif is diverged to F 156 A 157 in Sfh1 and, strikingly, this very V 154 F substitution conferred upon Sec14 an essentially complete resistance to inhibition by NPPMs (see above). We therefore investigated the possibility that the Sec14 V 154 V 155 motif is a signature of NPPMsensitive Sec14-like PtdIns/PtdCho-transfer proteins. To that end, primary sequence alignments highlighting residues that line the Sec14 and Sfh1 phospholipid-binding pockets were generated. Those alignments were subsequently compared with the corresponding residues projected to line the phospholipid-binding cavities of Sec14 orthologs from C. albicans (Sec14 CA ), C. glabrata (Sec14 CG ), and K. lactis (Sec14 KL ). The essentially complete conservation, across this set of proteins, of the distinct substructure that coordinates PtdCho-binding emphasizes, yet again, the close similarities of the various pocket architectures ( Fig. 4A ). Even in this extended analysis, fi ve out of seven residues identifi ed in the NPPM R screen (P 120 , S 173 , R 208 , G 210 , and F 212 ) are absolutely preserved across this cohort of Sec14-like PITPs. By contrast, while the Sec14 V 154 V 155 motif is conserved between Saccharomyces cerevisiae and C. glabrata (V 152 V 153 in the latter), K. lactis exhibits an F 152 V 153 version, whereas the motif is diverged to M 154 C 155 in C. albicans . ( Fig. 4A ). Although the Sec14 V 154 V 155 motif is not predicted to interact directly with bound NPPM 6748-481 ( Fig. 4B ), we noted that the cognate V 154 polymorphisms in K. lactis and C. albicans Sec14 proteins (F and M, respectively) had the effect of extending hydrophobic side chains deeper into the hydrophilic microenvironment that defi nes the PtdCho-headgroup coordination substructure of Sec14 ( Fig. 4C ).
To test the predictive power of the VV signature for NPPM sensitivity of a Sec14 protein, the corresponding SEC14 genes were amplifi ed from genomic DNAs of the corresponding fungal species, the amplifi ed products were subcloned into the appropriate transplacement cassettes, and the genes of interest were individually integrated into the S. cerevisiae genome. All of those Sec14 orthologs scored as functional proteins when expressed in yeast, as judged by their abilities to rescue growth of sec14-1 ts mutants on YPD agar at a restrictive temperature of 37°C ( Fig. 5A ). When these yeast strains were challenged with 20 M NPPM 6748-481, the Sec14-and Sec14 CG -expressing strains failed to proliferate, whereas yeast expressing either weak scores in those analyses (V 154 and R 208 ) or were excluded from the interaction fi ngerprint list entirely (P 120 , V 155 , and G 210 ).
To verify that these seven missense substitutions were suffi cient to confer NPPM 6748-481 resistance to Sec14, each corresponding mutation was individually incorporated into the genome of the naïve parental strain ( SPO14 + in this case) and resistance to NPPM 6748-481 was subsequently queried as an unselected phenotype. Survey of NPPM R as an unselected phenotype circumvented the possibility of mistaking that phenotype for a bypass Sec14 phenotype. In every case, the mutation of interest was suffi cient to render the recipient yeast strain resistant to the NPPM 6748-481 concentration used as selection fi lter in the NPPM R screen, irrespective of whether the naïve recipient was of a SPO14 + ( Fig. 3B ) or a spo14 ⌬ (supplementary Fig. 2) genetic background. However, in both genetic backgrounds, we noted that the NPPM R phenotype of the SEC14 F212L allele was very weak. Each SEC14 R allele held the added feature of conferring an unselected NPPM 4130-1276 and 67170-49 resistance to naïve yeast cells and, when interrogated in the context of these NPPMs, the resistance phenotype associated with the SEC14 F212L allele was clear ( Fig. 3C ). We inferred from the collective data that the mutant Sec14 R s were rendered insensitive to challenge with any of these three NPPMs.

SEC14 R gene products exhibit NPPM R lipid exchange activities
As Sec14 is required for yeast cell viability ( 17,27,33 ), the NPPM R screen demanded that any Sec14 R proteins passing through the screen must retain suffi cient PtdInsand PtdCho-exchange activities for biological function, but that those activities would be intrinsically resistant (at least to a substantial degree) to inhibition by NPPM. To examine those biochemical properties directly, representative Sec14 R s were produced as recombinant protein in bacteria, purifi ed, and the NPPM-resistance properties tested in vitro. We were unable to analyze Sec14 S173P or Sec14 P120Q in this manner because those two mutant proteins exhibited insuffi cient activity for confi dent analysis when purifi ed from the bacterial host (unpublished observations). As expected, the Sec14 R proteins exhibited a range of basal PtdIns transfer activities (supplementary Table 5).
Given previous analyses of Sec14 S173C and Sec14 S173A substitutions whose design was guided by the Sec14::NPPM 6748-481 docking pose ( 14 ), the Sec14 V154F was of particular interest as it was impervious to challenge with 20 M NPPM 6748-481 in vitro ( Fig. 3D ). Indeed, it exhibited an essentially complete resistance to NPPM 6748-481 challenge at near saturating aqueous concentrations of inhibitor (100 M; data not shown) even though V 154 was not identifi ed by our working Sec14::NPPM 6748-481 pose as a residue critical to the mechanism by which the NPPM is coordinated within the Sec14 lipid-binding cavity ( 14 ). As shown in Fig. 3D  , respectively, were each converted to VV signatures. The NPPM-resistance properties of the mutant proteins were subsequently examined in vitro and in vivo. [ 3 H]PtdIns transfer assays demonstrated that purifi ed Sec14 CA M154V,C155V had now acquired a signifi cant sensitivity to NPPM 6748-481. While this sensitivity was not as marked as that of Sec14, it was dramatic, nonetheless, when gauged against the complete resistance of the parental Sec14 CA to inhibitor. Similarly, whereas partial reconstitution of a VV motif in Sfh1 E126A (by incorporation of the single F 156 V substitution) endowed modest NPPM sensitivity, full reconstitution of a VV signature further enhanced the sensitivity of Sfh1 E126A,F156V,A157V to inhibition by NPPM 6748-481 ( Fig. 5C ). Consistent results were also obtained when the F 152 V substitution was transplaced into Sec14 KL to reconstitute a VV signature in that protein. Although Sec14 CG V152F,V153F also showed a reduced sensitivity to NPPM inhibition, as expected, those data came with the caveat that this mutant protein exhibited poor PtdIns-and PtdCho-transfer activity in vitro (unpublished observations). Thus, these substitutions come with a structural cost and, as described above, the mutant Sec14 variants exhibited a range of basal PtdIns-transfer activities in vitro (supplementary Table 6).
The biochemical results were supported by in vivo experiments where S. cerevisiae strains individually expressing Sec14 KL or Sec14 CA were indifferent to the chemical challenge. We inferred from those results that Sec14 CG was sensitive to NPPM 6748-481, whereas Sec14 KL and Sec14 CA were not ( Fig. 5A ). As control, the Sfh1 E126A protein was similarly analyzed, and proliferation of yeast expressing this protein was insensitive to inhibition by NPPM 6748-481 ( Fig. 5A ). The inferences derived from in vivo data were confi rmed by in vitro [ 3 H]PtdIns transfer assays that directly assessed the intrinsic sensitivities of these Sec14 proteins to NPPM 6748-481. Whereas Sec14 CG activity was strongly inhibited by NPPM 6748-481 to an extent similar to that observed for S. cerevisiae Sec14, activities of the Sec14 CA and Sfh1 E126A proteins were completely impervious to challenge with this compound. Sec14 KL , which has an incomplete VV signature (FV), was also resistant to inhibition by NPPM 6748-481 in vitro ( Fig. 5B ). These collective data report a strong correlation between the VV signature and NPPM sensitivity in closely related Sec14 proteins.

Reconstitution of a VV signature sensitizes Sec14-like PITPs to NPPM
The second approach for examining the relationship between the VV signature and NPPM sensitivity of Sec14like PtdIns/PtdCho-transfer proteins interrogated whether transplacement of a VV signature into a naturally NPPM R Sec14-like PITP sensitizes the protein to this class of inhibitor. Fig. 4. The Sec14 VV motif is not conserved among closely related Sec14 PtdIns/PtdCho-transfer proteins. A: Shown is a partial primary sequence alignment of S. cerevisiae Sec14 with Sfh1 and Sec14 PITPs from C. albicans , C. glabrata , and K. lactis . The residues corresponding to the lipid-binding cavity are highlighted in color. Residues conserved between S. cerevisiae Sec14 and other Sec14-like proteins are colored in orange with nonconserved residues in cyan. At top, the Sec14 VV motif is identifi ed. For reference, PtdCho-binding barcode residues (black circles) and residues altered to produce the Sfh1 6X mutant (green diamonds) are also highlighted at top. Labeled at bottom are residues where substitutions confer NPPM resistance to Sec14 (magenta diamonds). B: The S. cerevisiae Sec14::NPPM 6748-481 dock model is shown. Protein is rendered in gray transparent loop with residues V 154 , V 155 , and S 173 shown in black ball and stick render. NPPM 6748-481 is highlighted in green ball and stick mode. C: A magnifi ed view shows the spatial orientation of the Sec14 V 154 V 155 side chains (in stick render) relative to the model NPPM 6748-481 pose (in green ball and stick mode), residue S 173 is indicated, as is the Sec14 hydrophobic pocket surface (rendered as a gray wire-mesh surface). The cognate VV motif residues from S. cerevisiae (yellow), C. albicans (magenta), C. glabrata (cyan), and K. lactis (orange) are overlayed.
to inhibition by NPPM 6748-481. Reciprocally, conversion of the Saccharomyces Sec14 VV motif to that of the Candida PITP (Sec14 V154M and Sec14 V154M,V155C ) yielded mutant Sec14s that were resistant to NPPM 6748-481 ( Fig. 5D ). Taken together, the data indicate that the VV signature is not only a strong predictor of NPPM sensitivity in Sec14-like PITPs, but that transplacement of a VV signature into a naturally NPPM-resistant Sec14-like PtdIns/PtdCho-transfer protein is suffi cient to confer NPPM sensitivity to that PITP. Sec14 CA M154V,C155V or Sec14 KL F152V were tested for sensitivity to intoxication with NPPM 6748-481. Whereas proliferation of yeast cells expressing the wild-type versions of these Sec14 proteins was resistant to inclusion of this NPPM (20 M) in the culture medium, the growth of cells expressing these same proteins with reconstituted VV signatures was arrested in the presence of inhibitor ( Fig. 5D ). The same result was observed in the case of Sfh1 E126A , where full reconstitution of a VV signature rendered the Sfh1 E126A,F156V,A157V sensitive Fig. 5. The VV motif is a signature of NPPM-sensitive yeast Sec14-like PtdIns/PtdCho-transfer proteins. A (top panels): SEC14 genes from the indicated fungal species were integrated into the LEU2 locus of a sec14-1 ts strain and expressed under the control of the S. cerevisiae SEC14 promoter to generate strains exhibiting physiological levels of Sec14 CA , Sec14 CG , and Sec14 KL . The integrants were subsequently dilution spotted onto YPD plates supplemented with vehicle control DMSO or NPPM, as indicated at top, and incubated for 48 h at the indicated temperatures. The mock condition documents the phenotype of an isogenic strain where a SEC14 -less integration cassette was transplaced into the LEU2 locus. That expression of each heterologous Sec14 protein was suffi cient to rescue sec14-1 ts growth defects at the restrictive temperature of 37°C is demonstrated by comparison of the growth profi les in the left (30°C) and center (37°C) panels of the integrants relative to mock controls. A (right panel): NPPM 6748-481 resistance or sensitivity of the corresponding integrants was scored by spotting cells on YPD plates supplemented with 20 M 6748-481 and incubating at 30°C for 48 h, at which time images were taken. The mock condition documents the phenotype of an isogenic strain where a SEC14 -less integration cassette was transplaced into the LEU2 locus. The SFH1 control (little Sec14-like function in vivo) and the test SFH1 E126A (enhanced Sec14-like function in vivo) were expressed from low-copy centromeric plasmids (YCp) as ectopic expression of this sort was required for visualization of the enhanced Sec14-like properties of Sfh1 E126A . B: Characterization of the in vitro sensitivities of purifi ed recombinant Sec14/Sec14-like proteins and the indicated mutant variants is shown. Protein concentrations were clamped at 287 nM throughout and NPPM 6748-481 concentrations were fi xed at 20 M. Values represent the mean ± SEM of triplicate measurements from three independent experiments. The intrinsic PtdIns-transfer activities measured for each Sec14 PITP and other assay statistics are provided in supplementary Table 6. C: Characterization of the in vitro sensitivities of purifi ed recombinant Sec14/Sec14-like proteins with transplaced VV motifs is shown. Protein concentrations were clamped at 287 nM and NPPM 6748-481concentrations were fi xed at 20 M. Values represent the mean ± SEM of triplicate assay determinations from three independent experiments. The intrinsic PtdIns-transfer activities measured for each Sec14 PITP, the indicated variants, and other assay statistics are provided in supplementary Table 6. D: Figure organization and experimental conditions are as described for (A). The abilities of the indicated SEC14 and SFH1 variants to complement sec14-1 ts growth defects at 37°C (middle panels) and to endow naïve yeast with resistance to NPPM challenge (right panels) were tested. Left panels represent vehicle growth controls under nonchallenge conditions (30°C), whereas the NPPM challenge plates were also incubated at 30°C. Images were taken after 48 h. The mock condition documents the phenotype of an isogenic strain where a SEC14 -less integration cassette was transplaced into the LEU2 locus. mutants failed to confer an NPPM R phenotype to yeast cells ( Fig. 6B ), and this failure was not the trivial consequence of those proteins having been inactivated for PITP activity, as both proteins scored as active by the sec14-1 ts complementation assay ( Fig. 6B ). These collective results indicate that Sec14 tolerates nonconservative changes in its VV motif from both a protein structure and protein function point of view, and with regard to its NPPM sensitivity properties. However, Sec14 does not tolerate incorporation of residues with side chains bulkier than Val, or of charged residues, at those positions from the standpoint of maintaining sensitivity to inhibition by NPPM.

DISCUSSION
A sophisticated appreciation of how NPPMs bind and inhibit Sec14 activity is essential for further progress in several important areas, including rational design of SMIs with desirable properties, e.g., increased potency and a suitably broadened range of activity among closely related Sec14-like PITPs, etc. Our previous efforts to understand Sec14::NPPM binding interactions relied on rational loss-offunction approaches. That is, functional properties of Sec14 and of the NPPM were altered, using computational docking model-guided logic, with a view toward compromising protein::inhibitor interactions ( 14 ). Herein, we exploit an unbiased NPPM-resistance genetic screen to identify a new Sec14 motif, termed the VV signature, that contributes to NPPM sensitivity. We further demonstrate that suitable transplacement of a VV signature into naturally NPPM-resistant Sec14-like PtdIns/PtdCho-transfer proteins confers an NPPM-sensitivity onto those PITPs. The collective data both extend our appreciation of the determinants that govern Sec14-like PITP sensitivities to NPPMs and facilitate primary sequence predictions of which Sec14-like PtdIns/PtdChotransfer proteins are likely to be NPPM resistant or sensitive. Moreover, these data further emphasize the importance of S 173 in the mechanism by which NPPMs engage and inhibit Sec14-like PITPs.
A hallmark feature of NPPMs as PITP inhibitors is their exquisite specifi city. Even very closely related Sec14-like PITPs exhibit differential sensitivities to these inhibitors, and the detailed structural pose for NPPM-binding to Sec14 does not offer a satisfactory structural basis for explaining those variable sensitivities. The Sec14-like Sfh1 makes a case in point. Unlike Sec14, this protein is completely resistant to NPPM challenge even though it preserves all obvious structural features required for NPPM-binding, as defi ned by the model Sec14::NPPM pose ( 14 ). As described herein, attempts to capitalize on the proposed Sec14::NPPM 6748-481 pose to engineer an NPPM-sensitive Sfh1 failed. In those analyses, we presumed that the NPPM pose closely approximated the stably bound confi guration of the inhibitor within the Sec14 lipid-binding cavity.
There are several potential mechanisms by which a Sec14-like PITP can be intrinsically resistant to NPPM inhibition, however. First, the PITP may simply fail to bind the

Degrees of freedom in the Sec14 VV signature
Given that divergence from the VV signature exerted signifi cant effects of the sensitivity of Sec14-like PtdIns/ PtdCho-transfer proteins to NPPM 6748-481, we wished to better assess the degrees of freedom permitted in the VV motif associated with NPPM sensitivity of the PITP. To that end, a series of single missense substitutions was incorporated into the Sec14 V 154 V 155 motif, and the NPPM sensitivities of the individual mutants were tested by integrating the mutant SEC14 alleles into the genome of a naïve yeast strain and subjecting the transplacement mutants to an NPPM challenge. Yeasts expressing the Sec14 V154F , Sec14 V154E , Sec14 V154Y , Sec14 V155F , Sec14 V155A , or Sec14 V155Y proteins were completely resistant to NPPM 6748-481, indicating that those polypeptides were functional PITPs with the property of being NPPM R ( Fig. 6A ). The former property was demonstrated in sec14-1 ts growth rescue experiments at 37°C, and in plasmid shuffl e experiments where rescue of the lethal sec14 ⌬ allele was scored ( Fig. 6B ). By contrast, expression of the Sec14 V154A and Sec14 V155E Fig. 6. Functional analysis of the VV motif in Sec14. SEC14 genes carrying the indicated for V 154 and V 155 missense substitutions (Phe, Ala, Glu, Tyr) were integrated into the LEU2 locus of a sec14-1 ts strain. The mock condition documents the phenotype of an isogenic strain where a SEC14 -less integration cassette was transplaced into the LEU2 locus. A: Integrants were interrogated for in vivo function by scoring rescue of sec14-1 ts growth defects at the restrictive temperature of 37°C (left panel) or complementation of the normally lethal sec14 ⌬ allele (right panel). Left panel: The YPD plates upon which the cells were spotted were incubated at 37°C for 48 h and images taken. Right panel: A colony color-based plasmid shuffl e assay was used for determining whether the mutant Sec14 proteins could fulfi ll all biological functions of Sec14. Each mutant SEC14 gene was integrated into the MET17 locus of an ade2 ade3 sec14 ⌬ strain carrying a Yep ( SEC14 , LEU2 , ADE3 ) plasmid [strain CTY558 ( 9 )]. The mock condition documents the phenotype of an isogenic strain where a SEC14 -less integration cassette was transplaced into the MET17 locus. Plates were incubated at 30°C for 48 h and images were taken. The ability of mutant Sec14 expression to rescue lethality of the sec14 ⌬ allele is recognized by loss of the Yep ( SEC14 , LEU2 ) plasmid upon relief of nutritional selection for LEU2 selection by spotting cells onto YPD agar. Plasmid loss is recognized by appearance of white colony segregants from the background of red plasmid-bearing colonies. All mutant proteins retained biological activity in this assay. B: Integrants expressing the indicated mutant Sec14 proteins in sec14-1 ts genetic background were examined for NPPM 6748-481 sensitivity by dilution spotting on DMSO and drugsupplemented YPD agar. Plates were incubated at 30°C for 48 h and images taken. For both panels, the mock condition documents the phenotype of an isogenic strain where a SEC14 -less integration cassette was transplaced into the LEU2 locus. exchangeable ligand and, therefore, a poor inhibitor of Sec14 F212L .
Of the remaining six missense substitutions, fi ve can be interpreted in the context of the previously proposed Sec14::NPPM docking pose that is anchored by a strong halogen bond interaction between the headgroup halide and residue S 173 ( 14 ). Obviously, recovery of S 173 P in the unbiased genetic screen is particularly satisfying in that regard, and this mutant is essentially indifferent to NPPM challenge. However, the strong NPPM-resistance phenotypes associated with the R 208 L, G 210 V, and P 120 Q missense substitutions can be interpreted in terms of a highly conserved "S 173 coordination envelope" that governs the essential nature of the S 173 interaction with NPPM ( Fig. 7 ). The R 208 backbone engages in an H-bond with the backbone amide group of S 173 , whereas the G 210 backbone amide coordinates with the S 173 backbone carbonyl oxygen. Similarly, the S 173 backbone amide is coordinated with the carbonyl oxygen of P 120 ( Fig. 7 ). Thus, these three residues not only reside within 4.5 Å of the NPPM binding site, but make direct contact with residue S 173 and contribute to the spatial positioning of its side chain. Even subtle conformational perturbations in this S 173 coordination envelope are expected to be of suffi cient magnitude to upset the strict geometric requirements for formation of the critical S 173 -NPPM halogen bond and thereby compromise NPPM-binding, even though PtdCho-binding is not catastrophically affected given that these remain functional proteins in cells.
The remaining two Sec14 residues, V 154 and V 155 , are of particular interest on several counts. First, these represent divergent residues among the Sec14-like PtdIns/PtdCho-transfer proteins, in contrast to the other fi ve residues identifi ed in the screen, which are highly conserved. Second, Sec14 V154F resembles Sec14 S173P in its complete indifference to NPPM-481, and Sec14 V155F is also signifi cantly resistant to NPPM challenge. Third, and most strikingly, NPPM sensitivity among Sec14-like PITPs tracks closely with this VV signature SMI. Such defects could be manifested anywhere along the NPPM binding trajectory from initial association of SMI with the protein's surface to assumption of its resting pose within the PITP lipid-binding cavity. In such cases, the computed Sec14::NPPM pose would be of limited utility in deciphering mechanisms of resistance. Alternatively, NPPM binding might occur, but could be of suffi ciently reduced affi nity that the inhibitor becomes an exchangeable ligand (like PtdIns and PtdCho) so that SMI binding is readily reversible. In such circumstances, subtle differences in Sec14like PITP pocket geometries could well determine NPPM sensitivity/resistance. Subtle differences of this nature might be diffi cult to recognize when examination of the problem relies strictly on a dock model in the absence of a high resolution Sec14::NPPM crystal structure.
Unbiased genetic screens cast a wider net in terms of what types of NPPM-resistant Sec14 variants one might recover, and these strategies powerfully complement structural approaches. Using such screens, we uncovered seven unique missense substitutions (P 120 Q, V 154 F, V 155 F, S 173 P, R 208 L, G 210 V, and F 212 L) that confer varying degrees of NPPM resistance to Sec14. This set is noteworthy on several counts. First, the S 173 P substitution involves the very Ser residue that is essential for the proposed halogenbonding interaction required for stable binding of NPPM-481 ( 14 ). Second, fi ve of the seven substitutions do not involve residues previously highlighted for rational mutagenesis on the basis of the proposed NPPM-481 binding pose (S 173 and F 212 are the exception). Third, while S 173 is the only one of these seven residues projected to contact bound NPPM directly, six of the seven substitutions lie within 4.5 Å of NPPM-481, as it is confi gured in the proposed Sec14::NPPM binding pose (V 155 is the exception). Interpretation of the NPPM resistance phenotypes associated with these missense substitutions provides detailed new insights into what properties determine NPPM resistance/sensitivity in Sec14-like PITPs.
Taken together, the results from the genetic screen, while highlighting new residues for consideration, nonetheless strongly support the proposed Sec14::NPPM docking pose ( 14 ). What is striking is that, contrary to the structure-based logic that drove engineering of the Sfh1 6X variant, all but one of the residues identifi ed in the screen lie in the subregion of the Sec14 lipid-binding cavity projected to coordinate binding of the NPPM-481 activated aryl-halide headgroup. Only one (F 212 ) lies in the hydrophobic region of the cavity proposed to coordinate the distal fl uorobenzene moiety of bound NPPM-481, and the F 212 L substitution is the weakest of the substitutions recovered in terms of conferring NPPM resistance to Sec14. Residue F 212 resides on the cavity fl oor and is projected to engage in -stacking interactions with the NPPM fl uorobenzene moiety, as the NPPM intercalates into the narrow hydrophobic cleft bounded by residues F 212 , M 177 (also on the cavity fl oor), and F 228 (on the helical gate). We suggest the F 212 L substitution disorders that hydrophobic cleft and thereby weakens NPPM binding by interfering with its normal intercalation into this substructure. It is a plausible notion that this substitution results in NPPM becoming an Fig. 7. The Ser173 coordination envelope. The boundaries of the Sec14 S 173 coordination envelope are defi ned by the dotted circle (red). Residues that coordinate S 173 via H-bond interactions (magenta) are shown in stick render with carbon atoms in gray, oxygen in red and nitrogen in blue. The NPPM::6748-481 pose is shown in green and the binding pocket surface is rendered as a gray wire-mesh. and transplacement of this signature into resistant Sec14like PITPs confers signifi cant NPPM sensitivity to those otherwise naturally NPPM-resistant PITPs. Residues V 154 and V 155 reside at the conserved kink in the long ␣ -helix A 8 that frames one side of the hydrophobic cavity of Sec14like proteins ( 9,40,41 ). Both residues are positioned in proximity to S 173 , and V 154 lies within the S 173 coordination envelope. Although neither V 154 nor V 155 are projected to make any direct contacts with bound NPPM, one reasonable interpretation of NPPM R substitutions at these positions is that these perturb the S 173 coordination envelope. In support of this, structural modeling experiments show the V 154 F, V 154 M, and V 154 E substitutions, associated with NPPM-resistance, intrude into the hydrophilic microenvironment of the lipid binding pocket that coordinates the S 173 ::NPPM interaction ( Fig. 7 ). Consistent with this idea, substitution of V 154 with a small nonpolar residue (Ala) does not perturb the S 173 coordination envelope in this manner and does not compromise NPPM binding by Sec14. Why bulky amino acid substitutions for V 155 can endow NPPM resistance to Sec14 is less obvious, but indirect perturbation of the S 173 coordination envelope is a tenable possibility given the close proximity of this residue to the coordination envelope boundary. Another possibility is that the VV motif directly binds NPPM during the binding trajectory of the SMI into its most stable confi guration within the hydrophobic cavity. Polymorphisms within the VV motif may thereby suffi ciently weaken Sec14 affi nity for the NPPM to render it an easily exchangeable ligand and, therefore, an ineffectual inhibitor.
A detailed understanding of how NPPMs bind and inhibit Sec14 activity requires high resolution crystal structures of those complexes, and such information is essential for progress in the rational design of NPPMs with increased potencies and suitably broadened ranges of activity among closely related Sec14-like PITPs. The latter goal is desirable, as it would provide new avenues for crystallization of PITP::NPPM complexes. That is, the engineering of NPPM binding activity to mutant versions of naturally NPPM-resistant Sec14-like PITPs, which efficiently crystallize with bound ligands [e.g., Sfh1 ( 9 )]. High resolution structures of such complexes are required for understanding how NPPMs inhibit Sec14-like PITPs, and directly determining whether the proposed S 173 -NPPM halogen-bonding interaction precisely describes the mechanism by which NPPMs inhibit Sec14-like PtdIns/PtdCho-transfer proteins.