s2fieldsofstudy: Biology Chemistry

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The ATPase activity of BfpD is greatly enhanced by zinc and allosteric interactions with other Bfp proteins.

Type IV pilus biogenesis, protein secretion, DNA transfer, and filamentous phage morphogenesis systems are thought to possess similar architectures and mechanisms. These multiprotein complexes include members of the PulE superfamily of putative NTPases that have extensive sequence similarity and probably similar functions as the energizers of macromolecular transport. We purified the PulE homologue BfpD of the enteropathogenic Escherichia coli bundle-forming pilus (BFP) biogenesis machine and characterized its ATPase activity, providing new insights into its mode of action. Numerous techniques revealed that BfpD forms hexamers in the presence of nucleotide. Hexameric BfpD displayed weak ATPase activity. We previously demonstrated that the N termini of membrane proteins BfpC and BfpE recruit BfpD to the cytoplasmic membrane. Here, we identified two BfpD-binding sites, BfpE(39-76) and BfpE(77-114), in the N terminus of BfpE using a yeast two-hybrid system. Isothermal titration calorimetry and protease sensitivity assays showed that hexameric BfpD-ATPgammaS binds to BfpE(77-114), whereas hexameric BfpD-ADP binds to BfpE(39-76). Interestingly, the N terminus of BfpC and BfpE(77-114) together increased the ATPase activity of hexameric BfpD over 1200-fold to a V(max) of 75.3 mumol of P(i) min(-1) mg(-1), which exceeds by over 1200-fold the activity of other PulE family members. This augmented activity occurred only in the presence of Zn(2+). We conclude that allosteric interactions between BfpD and BfpC and BfpE dramatically stimulate its ATPase activity. The differential nucleotide-dependent binding of hexameric BfpD to BfpE(39-76) and BfpE(77-114) suggests a model for the mechanism by which BfpD transduces mechanical energy to the biogenesis machine.

Type IV pili (Tfps) 1 are filamentous appendages found on the surface of Gram-negative bacteria. Tfps are major virulence factors for many important human pathogens including Vibrio cholerae (1), Pseudomonas aeruginosa (2), Neisseria gonorrhoeae (3) and enteropathogenic Escherichia coli (EPEC (4)), mediating attachment to host cells and formation of microcolonies. These filaments are also involved in motility (5)(6)(7)(8), biofilm formation (9), and horizontal gene transfer (10 -13). Tfps are predominantly helical polymers of the pilin subunit that are defined by their shared structural and biochemical features and a highly conserved biogenesis machinery (14,15). Formation of Tfps is a complex process requiring many proteins besides the pilin protein, including the prepilin peptidase (which processes prepilin into pilin), prepilin-like proteins, proteins with nucleotide-binding motifs, and cytoplasmic membrane and outer membrane proteins. Evidence suggests that these proteins compose a sophisticated molecular machine that allows pilin polymerization, filament stabilization, and surface translocation. Most of the proteins involved in the assembly of Tfps have extensive sequence similarity with proteins of protein secretion, DNA transfer, and filamentous phage transport systems (16,17), suggesting that these systems possess similar structures and modes of action. Thus, Tfp biogenesis is a useful model for a variety of systems that transport biological macromolecules across membranes.
EPEC is a major cause of severe infantile diarrhea and a leading cause of infant mortality in developing countries (18). The BFP of EPEC is a particularly good system to study Tfp biogenesis because all of the genes necessary for BFP biogenesis are known. The 14-gene bfp operon contains 13 genes required for BFP biogenesis and function (4, 19 -26). The bfpA gene encodes prebundlin (27,28), which is processed into bundlin (the pilin protein) by the protein product of bfpP (the prepilin peptidase (19)). The precise functions of the other components of the biogenesis machinery are unknown. The bfpB gene encodes an outer membrane lipoprotein of the secretin family (20) that is hypothesized to form a pore in the outer membrane through which the BFP is extruded. The bfpD and bfpF genes encode putative cytoplasmic nucleotide-binding proteins BfpD and BfpF (22,23), which are thought to provide energy for BFP biogenesis and retraction, respectively. The bfpE gene encodes a polytopic cytoplasmic membrane protein with four transmembrane domains, a large cytoplasmic N terminus, two periplasmic domains, and a small cytoplasmic domain (26). We recently demonstrated that the N terminus of BfpE interacts with BfpD and the cytoplasmic N terminus of the bitopic cytoplasmic membrane protein BfpC, which also interacts with BfpD (29). Together, BfpC and BfpE recruit BfpD to the membrane (29).
BfpD is a member of the PulE superfamily of putative nucleotide-binding proteins involved in bacterial type II and IV secretion, Tfp assembly, natural competence, and assembly of archeal flagellae (17). These proteins contain several conserved sequence motifs, including Walker A and B boxes involved in ATP binding and hydrolysis (30). However, ATPase activity has been confirmed for only a few of them, mostly from type IV secretion systems (31)(32)(33)(34)(35)(36)(37)(38). Furthermore, this ATPase activity has been relatively weak, between 0.71 and 61 nmol of inorganic phosphate (P i ) released min Ϫ1 mg Ϫ1 of protein. Mutation of key residues in the Walker A box of PulE superfamily members abolishes activity of the cognate systems, for example, PulE (39,40), EpsE (41), OutE (42), PilB (43), and BfpD (4). Therefore, these proteins most likely share the function of supplying energy from ATP hydrolysis to drive organellar assembly or substrate translocation.
The characterization of the ATPase activity of BfpD is crucial for understanding its role in BFP biogenesis and mechanism of action. In this paper we describe a detailed study of the ATPase activity of BfpD in which we address its activity in the context of the BFP biogenesis machine, i.e. in the presence of components of the machinery with which it interacts. The results of our studies suggest a model for conversion of chemical energy into mechanical energy to power extraction of bundlin from the cytoplasmic membrane for assembly into BFP filaments.
The gel filtration column was calibrated with blue dextran (2,000 kDa), thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa; Amersham Biosciences). The Stokes radius (R) of each form of BfpD was determined from a plot of the partition coefficient (K av ) versus log R of standards as previously described (45). K av was calculated using the equation where V e is the elution volume, V o is the void volume, and V t is the bed volume.

Zonal Sedimentation Analysis
Purified BfpD (20 g) was applied to the top of a 2.5-ml 20 -40% sucrose gradient formed in 20 mM Tris-HCl buffer, pH 7.6, 100 mM NaCl, 10 mM MgCl 2 , with 1 mM ATP as appropriate. Gradients were centrifuged at 245,000 ϫ g for 12 h at 4°C. Fractions (100 l) were collected, precipitated with trichloroacetic acid, and subjected to SDS-PAGE and immunoblotting. Molecular mass standards run in parallel gradients were visualized by SDS-PAGE and Coomassie staining. Sedimentation coefficient (s 20,w ) values were determined by sedimentation relative to protein standards.
Molecular mass values were calculated from the equation molecular mass ϭ 6Nas 20,w 20,w /1 Ϫ 20,w in which N is Avogadro's number, a is the Stokes radius, 20,w is the viscosity of water at 20°C, is the partial specific volume of the protein calculated from its amino acid composition, and 20,w is the density of water at 20°C as described (45).

Electron Microscopy
Negative Staining-Carbon-coated copper grids were floated on 5-l drops of 100 g ml Ϫ1 BfpD in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 10 mM MgCl 2 , 1 mM ATP␥S. After 5 min the sample was wicked off, and grids were floated consecutively on 3 drops (60 l) of 3% uranyl acetate, with blotting in between. Grids were floated on the third drop for 1 min, then blotted and air-dried. Images were recorded on a Philips/FEI CM100 electron microscope operating at 100 keV in low dose mode at ϫ52,000 magnification and a defocus of 500 nm.
Cryo-electron microscopy-Five-l drops of BfpD were applied to Quantifoil holey grids (Quantifoil Micro Tools GmbH) that had been glow-discharged in the presence of amyl amine. After 1 min the grids were blotted for 2.5 s and then plunged into an ethane slush cooled with liquid nitrogen, using the Vitrobot (FEI). Grids were transferred to a Gatan 626 cold stage (Gatan), and images were recorded on a Philips/ FEI CM120 electron microscope operating at 120 keV in low dose mode at ϫ50,000 magnification and a defocus of 1 m.

ATPase Assay
The ATPase activity of hexameric BfpD, purified in the presence of ADP and the absence of MgCl 2 , was measured as the P i released by the hydrolysis of ATP using a colorimetric malachite green assay (46). ATPase activity was determined from the initial rate of P i release, which was linear with time over a period of at least 1 h (Fig. 2B). Assays were carried out in 40 mM Tris-HCl, pH 7.0, 150 mM NaCl, 10 mM ZnCl 2 , and 5% glycerol at 37°C in a 50-l reaction volume. The metal dependence of the ATPase activity of BfpD was examined by substituting the ZnCl 2 in the reaction buffer with MnCl 2 , CaCl 2 , CuCl 2 , MgCl 2 , FeCl 2 , CoCl 2 , CdCl 2 , and NiCl 2 . In experiments to determine the influence of BfpC and BfpE on ATPase activity, different combinations of the N terminus of BfpC (6:1 molar ratio of BfpC to BfpD), BfpE 39 -76 (6:1 molar ratio of BfpE to BfpD), and BfpE 77-114 (3:1 molar ratio) were incubated with hexameric BfpD at 37°C for 30 min prior to initiation of the reactions.
Reactions were initiated by the addition of ATP to a 5 mM final concentration and allowed to proceed for 0, 5, 10, 15, 20, 25, and 30 min. For substrate saturation experiments, ATP was varied from 0.1 to 10 mM. Reactions were terminated and released P i quantified by the addition of 800 l of freshly prepared color reagent (0.034% malachite green, 0.1% Triton X-100, and 1.05% ammonium molybdate in 1 M HCl; filtered to remove insoluble material). After 1 min at room temperature, color development was stopped by addition of 100 l of 34% citric acid. Samples were incubated at room temperature for 30 min, and then the absorbance at 660 nm was measured. Values were calibrated against KH 2 PO 4 standards and corrected for P i released in the absence of BfpD and the absence of ATP. Data from substrate saturation experiments, where ATP concentration was varied, were fit by nonlinear regression to the Michaelis-Menten equation with Origin software (Microcal) and the parameters K m and V max were calculated. Similar values were obtained using Lineweaver-Burk and Eadie-Hofstee plots.

Yeast Two-hybrid Experiments
We used the Matchmaker GAL4 two-hybrid system 3 (Clontech) following the protocol suggested by the manufacturer. The cloning of DNA fragments encoding BfpD and the N terminus of BfpE into pG-BKT7 (GAL4 DNA-binding domain vector) and pGADT7 (GAL4 activation domain vector) has been described previously (29). In addition, PCR-amplified fragments encoding amino acids 1-38, 39 -76, 77-114, 1-76, and 39 -114 of the N terminus of BfpE (for primers see Table I) were cloned into these vectors. Saccharomyces cerevisiae strain AH109 was co-transformed with these constructs by the lithium acetate procedure and a minimum of 30 transformants for each vector combination were used to test protein interactions. The transformants were plated on medium containing X-␣-Gal and lacking tryptophan, leucine, histidine, and adenine and assayed for ␣-galactosidase activity to detect and quantify interactions.

Isothermal Titration Calorimetry
Isothermal titration calorimetry (ITC) experiments were carried out using a VP-ITC Microcalorimeter (Microcal). Peptides of BfpE 39 -76 (LYKFTSDEGRKKDNPDAFALQRWLIAVRNGKTLAEAMR) and BfpE 77-114 (GWVPFDELSIISAGEISGNVHQALDDIIYMNDTKKKVK), synthesized and high performance liquid chromatography-purified by Biopeptide, were titrated with hexameric BfpD purified in the presence of ATP␥S or ADP. Peptide corresponding to BfpE 39 -76 (0.018 mM) or BfpE 77-114 (0.009 mM) in 20 mM Tris-HCl buffer, pH 7.6, containing 100 mM NaCl, 10 mM MgCl 2 and either 1 mM ATP␥S or 1 mM ADP was loaded into the sample cell. BfpD (0.12 mM), in the same buffer as the peptide, was loaded into the injection syringe and titrated into the sample cell. A typical titration series involved 35 injections of 3 l of titrant. The baseline was allowed to stabilize for 210 s between injections to permit adequate time for reaction and equilibration. All experiments were performed at 37°C and the cell was stirred continuously at 280 rpm. For control experiments to determine the heats of dilution of BfpD, the peptides were substituted by buffer. The heat produced for each injection of BfpD into peptide or buffer was obtained by integration of the area under each peak of the titration plots with respect to time. The heats of reaction were calculated by subtraction of the integrated heats of dilution of BfpD from the heats corresponding to the injection of BfpD into peptide. Titration data, corrected for heat of dilution, were analyzed with Microcal Origin using a single-site model.

Fluorescence Measurements
The fluorescent nucleotide analogues 2Ј,3Ј-O-trinitrophenyl (TNP)-ATP and TNP-ADP were used to study nucleotide binding to BfpD. Experiments were performed at 37°C using a spectrofluorometer with bandwidths of 4 nm. Monomeric BfpD (0.72 M) was incubated for 30 min with various concentrations of TNP-nucleotide in 20 mM Tris-HCl buffer, pH 7.6, containing 100 mM NaCl and 10 mM MgCl 2 . The fluorescence emission was measured at 535 nm upon excitation at 408 nm (total fluorescence). Enhancement of fluorescence (⌬F) was calculated as the difference between total fluorescence and the fluorescence measured in incubations of TNP-nucleotide in buffer. The enhanced fluorescence in the presence of BfpD, proportional to the quantity of BfpDbound TNP-nucleotide, was plotted as a function of TNP-nucleotide concentration. Data were fitted to the equation where ⌬F is the change in fluorescence intensity of TNPnucleotide at a concentration [S], ⌬F max is the maximum change in fluorescence intensity, and K d is the dissociation constant. Fitting was performed using Origin and values for ⌬F max and K d were obtained.
The stoichiometry of TNP-nucleotide binding to BfpD was derived using the method of Stinson and Holbrook (47). Briefly, 1/(1 Ϫ ␣) was plotted against [S]/␣, where ␣ is the fractional saturation of the TNPnucleotide-binding sites (⌬F/⌬F max ). A straight line with a slope of 1/K d and an x-intercept of the concentration of TNP-nucleotide-binding sites was obtained. The stoichiometry of binding was then calculated by dividing the x-intercept with the BfpD concentration.

Proteinase K Digestion
All protease digestions were carried out at room temperature in 20 mM Tris-HCl buffer, pH 7.6, 100 mM NaCl, 10 mM MgCl 2 , containing 1 mM ATP␥S or 1 mM ADP as appropriate. Hexameric BfpD (5 g), purified in the presence of ATP␥S or ADP, was preincubated with BfpE 39 -76 (5 g), BfpE 77-114 (5 g), or neither of these peptides at room temperature for 10 min. Proteolysis was initiated by addition of proteinase K to give final concentrations of 0, 0.05, 0.1, and 1 mg ml Ϫ1 and the reaction was allowed to proceed for 30 min. Proteinase K digestion was terminated by adding Laemmli buffer (48) and boiling the samples for 10 min. Finally, proteolytic products were subjected to SDS-PAGE and detected by silver staining.

RESULTS
BfpD Is a Hexamer-BfpD synthesized with a N-terminal His tag in E. coli strain BL21pLysS,pRPA405 was purified to homo-geneity using two chromatographic steps (for BfpD purity, see Fig. 6). BL21pLysS,pRPA405 produces functional BfpD as plasmid pRPA405, encoding the His-tagged BfpD, complements the bfpD mutant UMD926 to restore autoaggregation, a phenotype that correlates with biogenesis of functional BFP (results not shown). The elution profile of BfpD during Sephacryl S-300 gel filtration chromatography, the second purification step, was dependent on ATP and MgCl 2 (Fig. 1A). In the absence of both ATP and MgCl 2 and in the absence of ATP but the presence of MgCl 2 , BfpD eluted as a sharp peak with an estimated molecular mass of 60 kDa (see below). Because this is the molecular mass of BfpD obtained when it is calculated from the amino acid sequence or estimated by SDS-PAGE, we concluded that BfpD exists as a monomer in the absence of ATP. In the presence of ATP and the absence of MgCl 2 and in the presence of both ATP and MgCl 2 , BfpD eluted as two sharp peaks with molecular masses of 60 (the monomeric form) and 369 kDa (see below). The homogeneity of the His-tagged BfpD within the faster peak was confirmed by SDS-PAGE and silver staining, indicating that BfpD forms a homohexamer in the presence of ATP. Interestingly, the majority of BfpD was in the hexameric form (88% hexameric, 12% monomeric) in the presence of both ATP and MgCl 2 , whereas approximately half of the BfpD was in this form (48% hexameric, 52% monomeric) in the presence of ATP and absence of MgCl 2 . Similar results were obtained when ATP was replaced with ATP␥S, TNP-ATP, ADP, and TNP-ADP, suggesting that nucleotide binding, but not its hydrolysis, is required for BfpD hexamerization.
To accurately determine the molecular masses of the two BfpD forms, parameters obtained from gel filtration (Stokes radii) and zonal sedimentation (sedimentation coefficients) were analyzed (Table II). These indicate that the BfpD in the slower peaks was monomeric BfpD (molecular mass ϭ 60 kDa), whereas that in the faster peaks was hexameric (molecular mass ϭ 369 kDa). In addition, DLS analysis of the high molecular mass form of BfpD in the presence of ATP, ATP␥S, or ADP showed that BfpD is a hexamer (Table III).
Electron microscopy studies revealed that hexameric BfpD had a ring-like structure ( Fig. 1, B-E). The electron micrograph of negatively stained BfpD in Fig. 1B shows ring-like structures in which subunits are evident. The diameters of these rings measure ϳ11.5 nm. Furthermore, the rings in the electron micrographs of frozen-hydrated BfpD (Fig. 1, C-E) display 6-fold symmetry, suggesting a hexamer structure consistent with the gel filtration, zonal sedimentation, and DLS results. Taken together, these findings demonstrate that BfpD assembles to form hexamers in the presence of ATP, ATP␥S, TNP-ATP, ADP, and TNP-ADP.
Hexameric BfpD Is an ATPase-We detected the ATPase activity of BfpD using a colorimetric malachite green assay to measure the amount of P i released upon incubation with ATP. BfpD purified in 40 mM Tris-HCl buffer, pH 7.0, containing 150 mM NaCl and 1 mM ADP was used for these ATPase assays. Use of ADP instead of ATP in the BfpD purification produces hexameric BfpD and has the advantage of eliminating high background P i levels. ADP does not inhibit BfpD ATPase ac- The italicized portion of the primer sequence indicates the restriction site present in a PCR product prepared with that primer. b The underlined portion of the sequence anneals to the gene indicated.
tivity as in assays with increasing concentrations of ADP (up to 1000-fold molar excess over the final ADP concentration in the reactions) and BfpD purified in 40 mM Tris-HCl buffer, pH 7.0, containing 150 mM NaCl and 1 mM ATP there was no effect on BfpD activity (data not shown).
In preliminary experiments to optimize the ATPase assay for BfpD, we examined the effects of different reaction conditions on the ATPase activity of hexameric BfpD. In experiments measuring the ATPase activity at different pH values, using 40 mM sodium acetate buffer for pH 5.0 to 6.0, 40 mM Mops for pH 6.5 to 7.0, and 40 mM Tris for pH 7.0 to 9.0, maximal activity occurred at pH 7.0 (data not shown). In additional experiments, the optimal concentration of NaCl was found to be 150 mM (data not shown). Furthermore, ATPase activity required divalent cations; no activity was detected in the absence of divalent cations ( Fig. 2A) and activity was strongly inhibited in the presence of the divalent cation chelator EDTA (data not shown). Interestingly, hexameric BfpD exhibited the highest activity in the presence of Zn 2ϩ (Fig. 2A). Substitution of Zn 2ϩ by Mg 2ϩ and Mn 2ϩ decreased the ATP hydrolyzing activity to 90 and 84%, respectively, of that obtained with Zn 2ϩ , whereas substitution by Ca 2ϩ , Cu 2ϩ , Fe 2ϩ , Co 2ϩ , Cd 2ϩ , and Ni 2ϩ reduced it to less than 10%. Therefore, ATPase assays were performed in 40 mM Tris-HCl, pH 7.0, 150 mM NaCl, 10 mM ZnCl 2 , and 5% glycerol.
When hexameric BfpD protein was incubated with ATP, release of P i occurred in a time-and BfpD concentration-dependent manner (Fig. 2, B and C), indicating that hexameric BfpD has ATPase activity. That this activity was because of BfpD rather than a contaminating ATPase is demonstrated by the fact that preparations obtained by an identical purification scheme from cells containing plasmid lacking bfpD did not display ATPase activity. Interestingly, monomeric BfpD only began to display ATPase activity after 30 min (data not shown). Using gel filtration chromatography, we found that monomeric BfpD hexamerizes in the presence of nucleotide, such as the ATP added to initiate ATP hydrolysis (data not shown). Therefore, we believe that the ATPase activity exhibited by monomeric BfpD was actually that of nascent hexamers. Finally, BfpD did not hydrolyze any other nucleotides (ADP, AMP, GTP, CTP, and UTP) tested in substrate specificity experiments (data not shown).
To determine the Michaelis-Menten constants V max and K m of the ATP hydrolyzing activity of hexameric BfpD, ATPase activity was measured over a range of ATP concentrations, under initial rate conditions. The dependence of the rate of ATP hydrolysis on ATP concentration exhibited typical Michaelis-Menten behavior (Fig. 2D) and showed a linear relationship in Eadie-Hofstee and Lineweaver-Burk plots (data not shown). The V max values obtained from these experiments (Table IV) are consistent with the results shown in Fig. 2, A-C. In conclusion, under optimal conditions, the ATPase activity was calculated as 62.2 Ϯ 2.2 nmol released P i min Ϫ1 mg Ϫ1 of protein (mean Ϯ S.E.). Similar results were obtained using an assay coupling regeneration of ATP to oxidation of NADH (data not shown). Because hexameric, but not monomeric, BfpD displays ATPase activity, it appears that BfpD ATPase activity is activated upon hexamerization.
Either Amino Acids 39 -76 or 77-114 of the N Terminus of BfpE Are Sufficient for Its Interaction with BfpD-We previously demonstrated that BfpD interacts with the N terminus of the polytopic membrane protein BfpE (29). Here we used a yeast two-hybrid system to more precisely define the region in the N terminus of BfpE involved in interacting with BfpD. Full-length BfpD and full-length and fragments of the N terminus of BfpE were expressed as fusions to both the GAL4 DNA-binding (pGBKT7) and activation (pGADT7) domains. S. cerevisiae AH109 was co-transformed with these constructs and subsequently analyzed for transcriptional activation of the three reporter genes, HIS3, ADE2, and MEL1. All of the transformants grew on medium lacking tryptophan and leucine (pG-BKT7 and pGADT7 carry TRP1 and LEU2 selectable marker genes respectively; results not shown), indicating no toxicity as a result of expression of the fusion proteins. Activation of the reporter genes, which is indicative of an interaction between the fusion proteins, was detected as blue colonies on medium containing X-␣-Gal and lacking tryptophan, leucine, histidine, and adenine and quantified by measuring ␣-galactosidase activity. We found reporter gene activation when fusion protein pairs murine p53 and simian virus 40 (Fig. 3). These interactions occurred irrespective of which protein was fused to the activation and which to the DNA-binding domains of GAL4. The remaining transformants did not grow on medium containing X-␣-Gal and lacking tryptophan, leucine, histidine, and adenine and exhibited low levels of ␣-galactosidase activity, similar to the negative control expressing human lamin C and SV40 large T-antigen fusion proteins (Fig. 3). Western analysis showed that the transformants expressed the Bfp fusion proteins at comparable levels (results not shown). Furthermore, S. cerevisiae AH109 cells co-transformed with each construct individually and either "empty" pGBKT7 or pGADT7 grew on the appropriate selective medium, i.e. they did not grow on medium containing X-␣-Gal and lacking tryptophan, leucine, histidine, and adenine and grew as white colonies on medium containing X-␣-Gal and lacking tryptophan and leucine (results not shown). This confirms that the individual fusion proteins cannot activate the reporter genes alone and that the interactions we observed were because of direct interactions between the Bfp domains of the fusion proteins. Thus, it appears that the N terminus of BfpE contains two sites to which BfpD binds, one in BfpE 39 -76 and one in BfpE 77-114 . However, these data do not distinguish whether these sites represent two independent BfpD-binding sites or whether these sites cooperate to form one binding site. Differential Nucleotide-dependent BfpD Affinity for BfpE 39 -76 and BfpE 77-114 -We speculated that BfpD preferentially binds to each of the BfpD-binding sites of the N terminus of BfpE at different stages of catalysis of ATP hydrolysis. To test this hypothesis and to confirm interactions between BfpD and BfpE 39 -76 and BfpE 77-114 , the thermodynamics of the interactions were measured by ITC. For these purposes we synthesized peptides corresponding to BfpE 39 -76 and BfpE 77-114 . Fig. 4 shows calorimetry data for titrations of these peptides with hexameric BfpD in the presence of ATP␥S (a non-hydrolysable ATP analogue) and ADP. Large exothermic enthalpies were observed in titrations of BfpD-ATP␥S into BfpE 77-114 and BfpD-ADP into BfpE 39 -76 . The titration heat was converted to heat mol Ϫ1 as a function of molar ratio and the data were fitted with one set of identical binding site models to give the association constant (K), stoichiometry (molar ratio of BfpD/peptide, n), and enthalpy change (⌬H) upon peptide-BfpD complex   (Fig. 4), even when peptide concentrations were up to 100 times higher (data not shown). Furthermore, we were unable to detect interactions between monomeric (nucleotide-free) BfpD and BfpE 39 -76 or BfpE 77-114 (data not shown). Similarly, no interaction could be detected between monomeric BfpD and the cytoplasmic domain of BfpC (data not shown). In contrast, hexameric BfpD has previously been demonstrated to bind this region of BfpC by ITC (29). To summarize, these results show differential binding of hexameric BfpD to BfpE 39 -76 and BfpE 77-114 depending on the phosphorylation of the nucleotide, i.e. hexameric BfpD binds to BfpE 39 -76 in the presence of ADP and to BfpE 77-114 in the presence of ATP␥S. Interestingly, these data suggest that up to six molecules of BfpE can bind to BfpD that contains ADP, whereas a maximum of three molecules of BfpE can bind to BfpD containing ATP. In addition, the low K d for these interactions suggest that BfpD-BfpE 39 -76 and BfpD-BfpE 77-114 complexes are likely to be biologically relevant.
Hexameric BfpD Binds Six Molecules of the Fluorescent Nucleotide Derivatives TNP-ATP and TNP-ADP-To determine whether the stoichiometry of binding of BfpD-ATP␥S to BfpE 77-114 and BfpD-ADP to BfpE 39 -76 reflects the stoichiometry of binding of BfpD to ATP␥S and ADP, respectively, we used the non-hydrolysable nucleotide analogues TNP-ATP and TNP-ADP to ascertain the parameters of BfpD binding to nucleotides. TNP-nucleotides are weakly fluorescent in aqueous solution, however, their fluorescence is greatly enhanced in a hydrophobic environment such as the nucleotide-binding site of a protein. Upon excitation at 408 nm, each TNP-nucleotide in solution exhibited a characteristic fluorescence emission with a maximum at 558 nm; addition of monomeric BfpD markedly increased the fluorescence and blue shifted the maximum to 535 nm (data not shown). When monomeric BfpD was incubated with the TNP-nucleotides, both TNP-ATP and TNP-ADP bound to BfpD with saturable enhancement in the fluorescence emission (Fig. 5). The data were analyzed as described by Stinson and Holbrook (47) and the K d and the concentration of TNP-nucleotide-binding sites were obtained from the reciprocal of the slope and the x-intercept, respectively, of the resulting plots (data not shown). BfpD bound TNP-ATP (K d 340 nM) with higher affinity compared with TNP-ADP (K d 760 nM). The stoichiometry of binding (molar ratio of TNP-nucleotide/BfpD), calculated by dividing the concentration of TNP-nucleotidebinding sites with the BfpD concentration, was 1.07 for TNP-ATP and 0.98 for TNP-ADP. These results indicate that one molecule of monomeric BfpD binds one molecule of either of these TNP-nucleotides. Because monomeric BfpD hexamerizes in the presence of either TNP-ATP or TNP-ADP, we can assume that one molecule of hexameric BfpD binds six molecules of the TNP-nucleotides, each BfpD monomer of the hexamer binding one TNP-nucleotide molecule. Thus, the inability of hexameric BfpD to bind more than three BfpE 77-114 molecules is not because of an inability of each subunit to bind ATP.

FIG. 3. Yeast two-hybrid analysis of binary interactions between fragments of the N terminus of BfpE and BfpD.
Yeast strain AH109 was co-transformed with expression vectors encoding GAL4 DNA-binding (pGBKT7) and activation (pGADT7) domains fused in-frame to fragments of the N terminus of BfpE and full-length BfpD. For purposes of clarity, only the Bfp protein encoded on vectors pG-BKT7 and pGADT7, respectively, is indicated. Numbers correspond to amino acid residue numbering of BfpE. ϩ and Ϫ are the positive and negative controls, respectively, described in the text. Transformants were plated on medium containing X-␣-Gal and lacking histidine and adenine and assayed for ␣-galactosidase activity to detect transcriptional activation of reporter genes MEL1, HIS3, and ADE2. Transformants that produced blue colonies on selective medium are shown in white, whereas those that did not grow are shown in black. Columns denote the mean Ϯ S.E. ␣-galactosidase activity from three experiments, each performed in triplicate.

FIG. 4. Titrations of hexameric BfpD into BfpE 39 -76 (A) and BfpE 77-114 (B) in the presence of ATP␥S (black) and ADP (red).
Top panels show the raw data for the calorimetric titrations. The area under each peak represents the heat produced at each injection. The lower panels show integrated areas that were corrected for heat of dilution and plotted against the molar ratio (BfpD/peptide), with the best-fit curve for a one set of sites model.

BfpE Binding Increases BfpD Susceptibility to Proteinase K Digestion-
We have previously shown that BfpD and the N terminus of BfpC induce reciprocal conformational changes upon binding to one another (29). We hypothesized that each BfpD subunit of hexameric BfpD-ADP binds one molecule of BfpE 39 -76 , whereas alternate BfpD subunits of hexameric BfpD-ATP␥S bind one molecule of BfpE 77-114 . If this is the case, it suggests that binding of one monomer of BfpD-ATP␥S to BfpE 77-114 induces conformational changes that preclude binding of adjacent monomers. To examine the effect of BfpE binding on the conformation of BfpD occupied by either ATP␥S or ADP, BfpD in the presence of peptides of BfpE 39 -76 or BfpE 77-114 was compared with BfpD in the absence of these peptides with respect to susceptibility to proteinase K proteolysis. Fig. 6 shows silver-stained gels of two representative proteinase K digestion experiments in which hexameric BfpD, purified in the presence of ATP␥S (top panel) or ADP (bottom panel), was incubated alone and together with BfpE 39 -76 or BfpE 77-114 and subjected to limited proteinase K proteolysis. Note that BfpE 39 -76 and BfpE 77-114 migrate too rapidly to be seen on the gels. BfpD-ATP␥S and BfpD-ADP alone were relatively resistant to proteinase K. The presence of BfpE 39 -76 and BfpE 77-114 did not appear to alter the resistance of BfpD-ATP␥S and BfpD-ADP, respectively, to proteinase K. In contrast, BfpE 77-114 significantly increased the sensitivity of BfpD-ATP␥S to digestion by proteinase K. In addition, BfpE 39 -76 modestly but reproducibly increased the sensitivity of BfpD-ADP to proteolysis. These results show that BfpE 39 -76 and BfpE 77-114 only had a detectable effect on the proteinase K sensitivity of the form of BfpD with which our ITC data demonstrates they interact. Furthermore, they suggest that BfpE 39 -76 and BfpE 77-114 induce changes in the conformation of BfpD-ADP and BfpD-ATP␥S, respectively, upon binding, which account for the observed increases in sensitivity to proteinase K digestion. However, consistent with our hypothesis, BfpD-ADP and BfpD-ATP␥S undergo different conformational changes as they display differing proteinase K sensitivities.
BfpD Binding to the N Terminus of BfpC and BfpE 77-114 Dramatically Increases Its ATPase Activity-Because hexameric BfpD interacts with the N terminus of BfpC, BfpE 39 -76 , and BfpE 77-114 (Ref. 29, and this study), we investigated the influence of these proteins on ATPase activity. Hexameric BfpD was preincubated with various combinations of the N terminus of BfpC, BfpE 39 -76 , and BfpE 77-114 at a molar ratio of BfpC or BfpE to BfpD of 6:1, 6:1, and 3:1, respectively, based on reaction stoichiometry determined by ITC, and the ATPase activity was assayed. The results in Fig. 7A demonstrate that the proteins with which hexameric BfpD-ATP␥S interacts, i.e. the N terminus of BfpC and BfpE 77-114 , had a dramatic effect on the ATPase activity of hexameric BfpD. The N terminus of BfpC and BfpE 77-114 together stimulated the ATPase activity more than 1200-fold. The N terminus of BfpC alone increased the ATPase activity to 58.0 mol of released P i min Ϫ1 mg Ϫ1 of protein and BfpE 77-114 further increased it to 75.6 mol of released P i min Ϫ1 mg Ϫ1 of protein. Consistent with these results, the N terminus of BfpC and BfpE 77-114 together increased the V max of BfpD over 1200-fold, with the N terminus of BfpC alone increasing it almost 1000-fold (Table IV). In addition, the N terminus of BfpC reduced the K m 6-fold (Table IV), indicating an increase in the affinity of BfpD for ATP in the presence of BfpC. The stimulation of ATPase activity only occurred in the presence of Zn 2ϩ (Fig. 7B), highlighting the importance of utilizing optimal reaction conditions. Although the total Zn 2ϩ content of E. coli is in the millimolar range, evidence suggests the free Zn 2ϩ concentration is femtomolar (50). Therefore, we examined the ATPase activity of BfpD with and without BfpC and BfpE across a broad range of Zn 2ϩ concentrations. Remarkably, under both conditions there was only a 2% change in activity over Zn 2ϩ concentrations that spanned many orders of magnitude. Thus, the BfpC-and BfpEmediated enhancement of BfpD activity still occurred in the presence of trace levels of Zn 2ϩ (data not shown). In the presence of other divalent cations, ATPase activity was enhanced only 2-fold by the N terminus of BfpC, BfpE 39 -76 , and BfpE 77-114 . Interestingly, BfpE 77-114 alone and BfpE 39 -76 did not stimulate ATPase activity (Fig. 7A). Furthermore, the N terminus of BfpC and BfpE 77-114 had similar allosteric effects on the delayed ATPase activity of monomeric BfpD (data not shown). In conclusion, the N terminus of BfpC and BfpE 77-114 induce conformational changes that greatly enhance the ATPase activity of hexameric BfpD. DISCUSSION Tfp biogenesis, protein secretion, DNA transfer, archeal flagellar assembly, and filamentous phage morphogenesis systems are multiprotein assemblies constructed in part from proteins conserved between these systems. Of the proteins composing these macromolecular transport systems, putative nucleotide-binding proteins of the PulE superfamily are widely distributed and highly conserved (17). The PulE superfamily belongs to the P-loop class of NTPases and is characterized by Walker A (or P-loop) and B boxes, an Asp box, and a His box (32,40,51). Although the general biochemical characteristics of PulE superfamily proteins are well documented, important questions regarding their specific role and mode of action remain unanswered. To decipher the mechanism by which proteins of the PulE superfamily harness energy from ATP hydrolysis for the function of their respective systems, we considered it extremely important to characterize the biochemical activities of proteins of the PulE superfamily in the context of the systems to which they belong. The BFP biogenesis machine of EPEC is an attractive model system because all of the proteins composing this machine and the interactions of the PulE homologue BfpD with the other components have been identified (22,23,29). Therefore in this study, we have characterized the ATPase activity of the EPEC BfpD protein and the effects of conditions related to its biological function on this activity.
We purified BfpD using an approach including Sephacryl S-300 gel filtration chromatography and observed that BfpD eluted as a hexamer as well as a monomer in the presence of nucleotide (ATP, ATP␥S, TNP-ATP, ADP, and TNP-ADP). A combination of gel filtration, zonal sedimentation, DLS, and electron microscopy confirmed that BfpD forms ring-shaped hexamers in the presence of nucleotide (ATP, ATP␥S, and ADP). The ability to form hexameric rings has been reported for other PulE superfamily members (33,34,37,(52)(53)(54) and seems to be a common characteristic of members of this superfamily. These hexameric ring assemblies are reminiscent of those formed by nucleotide-dependent molecular motors such as helicases and F1-ATPases (52,54,55), and membrane fusion ATPases such as N-ethylmaleimide-sensitive fusion protein and p97 (56,57).
The ATPase activity measured for purified hexameric BfpD (V max of 62.2 nmol of P i released min Ϫ1 mg Ϫ1 of protein) is similar in magnitude to the highest values previously measured for other members of the PulE superfamily (ranging from 0.71 to 61 nmol of P i released min Ϫ1 mg Ϫ1 of protein). We considered the weak ATPase activities of these PulE superfamily members to be because of the absence of stimulatory proteins of the cognate systems and/or the reaction conditions used. After optimizing our reaction conditions, we tested whether components of the BFP biogenesis apparatus that interact with BfpD stimulate its ATPase activity. We have previously shown that BfpD interacts with the cytoplasmic N termini of BfpC and BfpE (BfpE 1-114 ) and is recruited to the cytoplasmic membrane by both BfpC and BfpE (29). Here we further define the BfpD-binding sites of BfpE and show that BfpD binds to one of two sites in BfpE depending on the stage of the catalytic cycle. The ATP-bound form of BfpD binds with high affinity (K d ϭ 72 nM) to BfpE 77-114 and the ADP-bound form of BfpD binds with high affinity (K d ϭ 75 nM) to BfpE 39 -76 . We found that the N terminus of BfpC and BfpE 77-114 together stimulate the ATPase activity of BfpD more than 1200-fold to a V max of 75.3 mol of P i released min Ϫ1 mg Ϫ1 of protein. This activity exceeds that of any member of the family studied thus far by over 1200-fold. Moreover, to our knowledge BfpD is the first component of a Tfp biogenesis machinery or similar system whose ATPase activity has been shown to be modulated by other components of the system. The N terminus of BfpC alone increased the ATPase activity almost 1000-fold to a V max of 58.7 mol of P i released min Ϫ1 mg Ϫ1 of protein. However, BfpE 77-114 only increased ATPase activity in the presence of the N terminus of BfpC. Furthermore, BfpE 39 -76 had no detectable effect on BfpD ATPase activity, a result that is not surprising, as BfpD-ATP does not interact with BfpE 39 -76 . The binding of BfpC and BfpE to BfpD induce conformational changes in each of the proteins as indicated by protease sensitivity experiments conducted in this and prior studies (29). The allosteric effects of BfpC and BfpE on BfpD ATPase activity indicate that BfpC and BfpE indirectly play a major role in supplying the BFP biogenesis machine with the energy it requires for its assembly, bundlin polymerization. and/or transmembrane transport. Not only do they recruit BfpD to the biogenesis machinery, they also dramatically increase its ATPase activity. In this way, the ATPase activity of BfpD is regulated to be maximal at the site where energy is needed for BFP formation, making the process of producing BFP energy efficient and minimizing wasted energy expenditure by the free cytoplasmic enzyme. Interestingly, monomeric BfpD preparations exhibited delayed ATPase activity that was greatly increased by the N terminus of BfpC and BfpE 77-114 but not BfpE 39 -76 . This ATPase activity was most likely that of BfpD hexamers formed in the presence of the ATP added in the assay, because monomeric BfpD does not interact with the N terminus of BfpC, BfpE 39 -76 , or BfpE 77-114 . Our data suggest that nucleotide binding induces formation of BfpD hexamers, which are selectively recognized, recruited, and stimulated by membrane-bound BfpC and BfpE, thus maximizing the energy supplied to the BFP biogenesis machine.
We found that hexameric BfpD binds to a maximum of three molecules of BfpE 77-114 in the presence of ATP, but can bind to FIG. 7. BfpC and BfpE increase the ATPase activity of hexameric BfpD by more than 1000-fold. BfpD ATPase assays were carried out in 40 mM Tris-HCl, pH 7.0, 150 mM NaCl, 5% glycerol, 5 mM ATP, and 10 mM ZnCl 2 , in the presence of the N terminus of BfpC, BfpE 39 -76 , and BfpE 77-114 unless otherwise indicated (see "Experimental Procedures"). In A, assays were performed in the presence of the indicated combination of BfpC, BfpE 39 -76 , and BfpE 77-114 . In B, assays were performed in the presence of BfpC and BfpE 77-114 and the Zn 2ϩ in the reaction buffer was replaced with the indicated ion. The results are derived from at least three assays performed in triplicate, each using different BfpD preparations. six molecules of BfpE 39 -76 in the presence of ADP. This difference in stoichiometry is not because of differences in nucleotide saturation, as BfpD is able to bind six molecules of either ATP or ADP. Rather these data suggest that occupancy of the ATPactivated BfpD hexamers by BfpE occurs at every other subunit and induces a conformational change that precludes binding of adjacent subunits.
Hexameric BfpD exhibited the highest ATPase activity in the presence of Zn 2ϩ and the dramatic stimulation of its activity by the N terminus of BfpC and BfpE 77-114 occurred only in the presence of Zn 2ϩ . The crystal structure of a truncated form of the closely related type II secretion ATPase EpsE (missing ninety N-terminal amino acids) of V. cholerae has been solved, revealing a molecule with four domains, N2, C1, C M , and C2, and inferring a missing N1 domain. Electron density most consistent with the presence of a Zn 2ϩ ion was detected in the C M domain, coordinated by four cysteines. This location is remote from the active site. An alignment of BfpD and EpsE reveals that these cysteine residues are conserved. It is not clear therefore, whether Zn 2ϩ is catalytically relevant or merely structurally relevant for either EpsE or BfpD. For example, Zn 2ϩ binding might be necessary for conformational changes that allow binding of BfpE or BfpC. However, this hypothesis is not supported by our ITC data, which were acquired in the presence of Mg 2ϩ , not Zn 2ϩ . Therefore, we propose that the Zn 2ϩ ion might perform a critical catalytic role for BfpD and perhaps for other members of the PulE ATPase family. In our assays, we purified hexameric BfpD in buffer lacking divalent metal ions and measured ATPase activity in the presence of buffer containing various divalent metal ions. We cannot be certain that trace quantities of other metals were not present in any of the buffers used. However, we found using inductively coupled plasma optical emission spectroscopy that the enzyme purified in the absence of Zn 2ϩ already contained an equimolar ratio of Zn 2ϩ (data not shown). Therefore, the fact that the addition of Zn 2ϩ to the enzyme, which already contains Zn 2ϩ , stimulated activity more than 600-fold higher than any other metal, suggests that the Zn 2ϩ plays a catalytic role in addition to a structural role. Furthermore, if the role of Zn 2ϩ were merely conformational, then the structural analogue Cd 2ϩ would be expected to be a reasonable substitute. Instead the activity in the presence of Cd 2ϩ was less than 1/1000 of that in the presence of Zn 2ϩ when BfpC and BfpE 77-114 were also present. Thus, we propose that BfpD and perhaps other PulE ATPases use Zn 2ϩ to perform a catalytic role in ATP hydrolysis, which to our knowledge is unprecedented for ATPases. However, Zn 2ϩ is known to catalyze hydrolysis of phosphate bonds for other enzymes, for example, the hydrolysis of phosphodiester bonds by endonuclease IV (58), and therefore such a role is clearly plausible.
Based on the findings presented here, we envision the role of BfpD in BFP biogenesis is analogous to that of p97 and Nethylmaleimide-sensitive fusion protein in homotypic membrane fusion and organelle biogenesis in that it transduces mechanical force to the BFP biogenesis machine possibly for the extraction of bundlin from the cytoplasmic membrane. This theory is supported by the following findings: (i) nascent bundlin is an integral cytoplasmic membrane protein (59) and therefore must be extracted from the membrane for pilus biogenesis; (ii) the N terminus of BfpE is involved in interactions with other essential Bfp proteins that are necessary for BFP formation (29); (iii) hexameric BfpD exhibits high ATPase activity in the presence of other essential components of the BFP biogenesis machine, consistent with the idea that it is a molecular motor; and (iv) hexameric BfpD displays differential binding to two sites of the N terminus of BfpE depending on the phospho-rylation of its associated nucleotide. This last point indicates that the relative position of BfpD and BfpE change upon ATP hydrolysis. Thus, the resulting chemical energy can be converted to mechanical energy to change the position of BfpE and the proteins to which it binds. Fig. 8 illustrates the proposed mode of action of BfpD, showing the changes it induces in the cytoplasmic membrane subassembly of the BFP biogenesis machine upon hydrolysis of ATP. The figure is simplified to show the action of only a BfpD monomer, but presumably occurs simultaneously at alternating monomers. Hexameric BfpD-ATP interacts with the cytoplasmic N termini of BfpC and BfpE, which also interact with each other. BfpD-ATP binds to BfpE 77-114 , the domain of the N terminus of BfpE closest to the cytoplasmic membrane. Conformational changes in BfpD upon binding to the N terminus of BfpC and BfpE 77-114 greatly increase its ATPase activity. Subsequently, the BfpD-ADP resulting from ATP hydrolysis shifts to BfpE 39 -76 while remaining bound to the N terminus of BfpC. It seems unlikely that BfpD moves to its new binding site in BfpE as its movement is restricted by its interaction with the N terminus of BfpC. Therefore we propose that the N terminus of BfpE is raised allowing BfpD-ADP to interact with BfpE 39 -76 . This movement may force the most distal part of the N terminus of BfpE into the cytoplasmic membrane and perhaps into the periplasm. The interaction between the N terminus of BfpC and BfpE 39 -114 presumably limits the distance the N terminus of BfpE is pushed through the cytoplasmic membrane. According to this model, the BfpD motor energizes BfpE to act as a piston, whereas BfpC serves as a scaffold protein, anchoring BfpD and the N terminus of BfpE in place. Preliminary studies indicate that other essential Bfp proteins bind to BfpE 77-114 , 2 which could be transported across the cytoplasmic membrane in association with the N terminus of BfpE. In this way, BfpD provides the necessary mechanical force for recruitment and assembly of Bfp proteins into the BFP biogenesis FIG. 8. Schematic diagram of the proposed mechanism by which BfpD transduces mechanical energy to the BFP biogenesis machine. Hexameric BfpD-ATP binds to the N terminus of BfpC and to the last third of the N terminus of BfpE. This greatly increases its ATPase activity and the resulting Bfp-ADP releases the distal third of the N terminus of BfpE and instead binds to the middle third while remaining bound to the N terminus of BfpC. This movement drives the distal third of the BfpE N terminus through the membrane. Upon replacement of ADP with ATP, BfpE relaxes, re-establishing its resting state. machine, which results in the extraction of nascent bundlin from the cytoplasmic membrane and incorporation into the growing pilus filament. Finally, BfpD is replenished with ATP and released from BfpE 39 -76 and the cytoplasmic membrane subassembly of the BFP biogenesis machine relaxes, allowing the distal part of the N terminus of BfpE to slip back through the cytoplasmic membrane. Whereas further experiments are necessary to test this model of energy transduction during BFP biogenesis, the insights into the function and mode of action of BfpD we report here increase our understanding of the mechanism of Tfp biogenesis machines and have important implications for similar systems that transport biological macromolecules across membranes. We propose that this model is relevant to the entire PulE superfamily. Orthologues of BfpE (GspF) are highly conserved in systems that include PulE superfamily members (17). Although BfpC orthologues are not common, GspL and GspM proteins, which span the cytoplasmic membrane, interact with each other and recruit PulE family AT-Pases to the cytoplasmic membrane (41,60), are widespread and likely play roles similar to that carried out by BfpC. Furthermore, the unique properties of BfpD suggest that drug therapy targeting the ATPase activity of PulE superfamily proteins and their interactions with proteins of their cognate systems could be designed to disrupt these macromolecular machines and the virulence properties they control.


A Breakthrough in R&D for Neglected Diseases: New Ways to Get the Drugs We Need

New research suggests that long-held beliefs on neglected disease drug development activity are no longer accurate.

R esearch suggests that long-held beliefs on neglecteddisease drug development activity are no longer accurate, and that these inaccurate beliefs have led-and are leading-to poorly designed and targeted government policies and incentives. On a more positive note, this research also highlights opportunities for better-targeted government policies that will more closely match the reality of neglected-disease drug development and the needs of public and industry groups.

Current Perceptions
Current government policies to stimulate development of new drugs for neglected diseases are based on a set of shared understandings.
One of these understandings is that only 13 new drugs have been developed for neglected tropical diseases since 1975, with the main problem being that these diseases are simply non-commercial for companies to invest in [1]. Another is that, although public-private partnerships (PPPs) for drug development have started, they are problematic. (In this article, PPPs are defi ned as public-health-driven not-for-profi t organisations that drive neglected-disease drug development in conjunction with industry groups.) Governments are uncertain which of the plethora of PPPs they should support, particularly as most are thought to be too young to judge their success. At the time this article went to press, the Initiative on Public-Private Partnerships for Health (http:⁄⁄www.ippph.org/) listed 92 PPPs in its database (this number includes all PPP activity, including the small number of organisations that make drugs, vaccines, and microbicides; and one-off partnerings such as donations and cut-price deals).
The general view is that PPPs are inexperienced in drug development, and may eat up public cash without delivering the tools we need, while the real experience and capability in drug development lies with multinational pharmaceutical companies, which must be brought back into the neglected-disease fi eld if we are to achieve success. For example, the Commission for Africa recently stated that we need to increase neglected-disease R&D by "giving large pharmaceutical fi rms incentives to investigate the diseases that affect Africa, instead of focusing on the diseases of rich countries" [2].
The logical outcome of these beliefs is to focus on new policies to commercialise neglected-disease markets for large companies, which seek peak sales of around $500 million per year to justify investment. This commercialisation approach is generally based on supplementing low developing country purchasing power with large-usually billion-dollar-market "pull" incentives, such as transferable intellectual property rights or advanced purchase commitments underwritten by Western governments.
The above all sounds fairly sensible. The only problem is that most of the above statements no longer hold true in the post-2000 world of neglected-disease drug development, and, as a consequence, government policies based on these beliefs are at risk of missing the mark.

The 2005 Reality
The landscape of neglected-disease drug development has changed dramatically during the past fi ve years (I am not

Neglected Diseases
Open access, freely available online The Neglected Diseases section focuses attention either on a specifi c disease or describes a novel strategy for approaching neglected health issues in general. discussing vaccines, only drugs). There were 63 neglecteddisease drug projects under way at the end of 2004, including two new drugs in registration stage and 18 new products in clinical trials, half of which were already at Phase III. Assuming there is suffi cient funding, at standard attrition rates these projects would be expected to deliver eight to nine new neglected-disease drugs within the next fi ve years, even if no further projects were commenced after this time ( Figure  1). This expected yield was calculated using attrition rates from the Tufts Center for the Study of Drug Development [3] combined with PPP-specifi c rates when available. New projects have been commenced since the end of 2004, and activity is expected to further increase as newer PPPs and institutes get into stride.
This increase in activity is not a passing trend, but is a sign of deep-seated structural changes. In particular, it refl ects the formation since 2000 of new pharmaceutical industry neglected-disease institutes, now employing over 200 scientists, and the creation of new drug development PPPs, which now conduct three-quarters of all identifi ed neglecteddisease drug development, sometimes in partnership with these industry institutes.
This renewed activity-at a level unheard of in the past two decades-commenced largely in the absence of signifi cant new government incentives and generally without public intervention. Eighty percent of PPP drug development activity is funded through private philanthropy, while the industry institutes are largely self-funding, although sometimes with PPP project funding input. (One industry institute did benefi t from generous support from the Government of Singapore.) These fi ndings led the Pharmaceutical R&D Policy Project at the London School of Economics, a project funded by the Wellcome Trust, to a closer examination: where is this new activity centred, what is motivating it, and what has suddenly made this "non-commercial" R&D possible? In line with its mandate, the project also sought to assess the performance of the different actors, in terms of both health outcomes and cost-effi ciency. Our fi ndings are set out below.

Neglected-Disease R&D Activity
Multinational drug companies now conduct half of new neglected-disease drug development activity (32 projects), either working with PPPs or working alone (usually with a view to subsequent partnering). In all cases, these companies are working on a non-commercial basis-that is, they are not motivated by commercial returns in neglecteddisease markets-and they have agreed to provide the fi nal products to poor patients in developing countries at notfor-profi t prices. The bulk of this activity is accounted for by the four companies that have formal neglected-disease divisions-GlaxoSmithKline, Novartis, AstraZeneca, and Sanofi -Aventis-while four other companies have less formal neglected-disease activity, conducting perhaps one or two projects each, and generally on a more serendipitous basis.
A further half of the identifi ed 60-plus neglected-disease drug projects are conducted by smaller-scale commercial fi rms working with PPPs, including small pharmaceutical companies, contract research organisations and developing country fi rms, and academic drug developers. The nonacademic part of this activity is on a purely commercial basis, with small fi rms working on a different scale and being motivated by far smaller commercial returns than large multinational pharmaceutical companies. PPPs now spend as much on small company R&D as they do on multinational company projects. The R&D drug landscape for neglected diseases is shown in Figure 2.
Three-quarters of all identifi ed R&D (47 projects) was conducted by drug development PPPs, often working with the large and small pharmaceutical companies mentioned above. Nearly one-third of these projects are at the clinical trial stage, including seven drugs now in Phase III trials, and two further products are in the registration stage-rectal artesunate by the Special Programme for Research and Training in Tropical Diseases (TDR) (http:⁄⁄www.who.int/ tdr), and paromomycin by the Institute for OneWorld Health (http:⁄⁄www.iowh.org) and TDR. Based on standard attrition rates, these PPP portfolios would be expected to yield six to seven new neglected-disease drugs within fi ve years. This is a high proportion of the total eight to nine new drugs expected from all current avenues.
Once these data became clear, we were faced with the question of why pharmaceutical companies were investing in neglected-disease drug development. Current thinking-and our own initial beliefs-told us this was impossible without new commercial incentives, and no such incentives had been introduced.

Motivations and Facilitating Factors
An examination of the 60-plus neglected-disease projects now under way showed that our initial understanding of company motivations and PPPs' role was incorrect. In particular, commercial incentives were largely irrelevant to the decision by multinational companies to re-enter the neglected-disease fi eld, while small companies involved in neglected-disease R&D were indeed responding to existing commercial drivers. In most cases, the involvement or planned involvement of PPPs was crucial to company activity, commercial or otherwise.
Multinational companies. Big companies involved in neglected-disease R&D were not motivated by commercial returns in the neglected-disease market, but rather by longerterm business considerations, including: (1) minimising the risk to their reputation stemming from growing public pressure on companies over their failure to address developing country needs; (2) corporate social responsibility and ethical concerns; and (3) strategic considerations (for example, positioning themselves in emerging developing country markets, or building access to low-cost, high-skilled developing country researchers).
This renewed neglected-disease activity has been made possible by a major structural change in the way multinational  companies approach neglected-disease R&D. Instead of conducting a limited number of more expensive latestage drug development projects (the pre-2000 model), companies have moved upstream to the less expensive and more innovative drug discovery stages-allowing them to control costs and resource inputs to levels more acceptable to shareholders. The resulting drug leads can then be developed in conjunction with public partners. These public partners (usually PPPs) facilitate further development by subsidising clinical trial costs; by providing the necessary public health skills and developing country knowledge for clinical trials, registration, and implementation; and by sharing the risk of trials in important but high-liability patient groups, such as children and pregnant women.
This partnering model, sometimes called the "no profi t-no loss model", allows companies to participate in neglecteddisease research (often providing substantial in-kind inputs) while still protecting shareholder value, and manufacturing and distributing fi nal products to developing country patients at no mark-up. This has three clear advantages. First, it provides a source of high-quality innovative industry drug leads. Second, it uses the public health sector in its area of maximum comparative advantage (developing country clinical trials rather than drug design). Third, it provides fi nal products to poor patients at not-for-profi t prices.
Small companies. Small companies, on the other hand, have largely commercial motivations. Some see neglecteddisease markets, particularly larger markets such as tuberculosis (TB) and malaria, as suffi ciently attractive to warrant investment and will pursue these even without public support. For example, Zentaris (the small company that developed and registered the new anti-leishmaniasis drug, miltefosine), noted that: "While such a market would be negligible for a big pharmaceutical company, it has a good economic scale for us" (personal communication). A second-and potentially much larger-category is that of small fi rms that can use "add-on" neglected-disease R&D to promote their Western commercial goals. Examples of such goals are to expand information on their core commercial compounds, or help to establish proof-ofconcept for a technology that can subsequently be transferred to commercial markets. These fi rms generally seek and need substantial PPP support, including full cost coverage and signifi cant skills input-and are unlikely to continue neglected-disease R&D for developing countries if this support is not forthcoming. Finally, commercial contract research organisations increasingly see neglected-disease R&D as an interesting niche sector, and are now involved on a commercial basis in one-third of PPP projects.
While promising, small company neglected-disease activity remains largely under-exploited. Most small companies continue to be deterred by the substantial barriers to entry that large, disseminated, and unfamiliar developing country markets pose, while fi rms with a primary Western focus can have diffi culty concluding fi nancial agreements with cashstrapped PPPs, particularly if their intellectual property concerns are not adequately addressed.
PPPs. As noted above, the presence of PPPs is probably essential to multinational company participation, and plays a catalytic role in encouraging small Western-focussed companies to expand their remit to neglected-disease indications. However, PPPs also serve other useful functions, particularly from the public perspective, including: (1) integrating and coordinating multiple industry and academic partners and contractors along the drug development pipeline; (2) allocating public and philanthropic funds to the "right" kinds of R&D projects from a public health perspective (for example, since 2000, two-thirds of PPP R&D spending has gone directly to industry, almost equally divided between large and small companies, while a further one-third has been allocated to academics to translate basic research into new drug leads); and (3) managing neglected-disease drug portfolios, including selection and termination of projects based on their relative merits.
By virtue of these functions, PPPs are stimulating alternative approaches in addition to "classical" one-to-one partnerships with multinational pharmaceutical companies (these now represent only about one-third of all PPP projects). Their coordinating and integrating role allows PPPs to develop compounds from many different sources even if there is no interested industry partner. For example, the TB Alliance manages and integrates development of PA-824 (a Chiron compound that the company itself did not want to pursue through the full R&D process) on a purely subcontracted basis and without formal development partners.
Alternatively, by pairing up small Western companies or academics with developing country manufacturers, PPPs can-and do-foster a neglected-disease drug development pipeline that is far cheaper than the traditional commercial approach (see "Cost-effi ciency" section). Nearly onequarter of current PPP projects involve developing country pharmaceutical fi rms as the manufacturing (and sometimes development) partner for a range of compounds from academia, the public domain, or small fi rms. For example, the Medicine for Malaria Venture's synthetic peroxide project brings together academic discovery and early-development partners (Uni Nebraska, Uni Monash, Swiss Tropical Institute) with Ranbaxy (India) as the development and trial manufacturing partner.

Performance of the Different Approaches
Although increased neglected-disease R&D is always welcome, it is also important that this R&D is targeted to optimal health outcomes for developing country patients, and  that it represents the most cost-effi cient use of public and philanthropic funding (that is, that patients see maximum health returns for every dollar spent).
In order to assess performance of the various actors, we devised a range of metrics, including health value of the fi nal products (safety, effi cacy, suitability, and affordability for developing country patients), level of innovation, capacity, drug development times, cost-effi ciency, and cost.
Measurement of the various drug development approaches against these metrics showed that both industry working alone and public groups working alone performed more poorly on most metrics than public-private collaborations. In other words-and perhaps unsurprisingly when we consider the matter more closely-drug development for neglected diseases is optimised by combining industry drug development expertise with public neglected-disease expertise. Below is a summary of outcomes against a sample of metrics.
Health outcomes. The PPP approach delivered the best health outcomes for developing country patients. Eight neglected-disease projects (Artemotil, Paluther, Coartem tablets paediatric label extension, Lapdap, Biltricide, Impavido, Ornidyl, and Mectizan) were conducted in publicindustry collaborations (with TDR). Three of the resulting products had a major impact on developing country health-Mectizan (ivermectin), which halved the global burden of onchocerciasis between 1990 and 2000 [4]; praziquantel, which has helped to control schistosomiasis in Brazil, the Mahgreb, the Middle East, China, and the Philippines [5]; and the TDR-assisted label extension of Coartem tablets for paediatric use, which has delivered Africa its fi rst safe, effective, suitable new anti-malarial for many years. We note that praziquantel's impact was greatly increased by the advent of a cheaper, simpler generic formulation by Shin Poong, which allowed broader rollout than the originator product, Biltricide.
By contrast, virtually all of the 13 drugs developed by industry working alone had a low overall health value for developing country patients (although, as noted above, industry has now largely moved to a partnering approach), with only one drug being widely accessible and useful in the developing world (Zentel/albendazole). These 13 drugs were Zentel, Lariam, Malarone, Mycobutin, Paser, AmBisome, Arsumax, Coartem original registration for adults and children above 10 kg in a four-dose (not sixdose) formulation, Halfan, Priftin, Rifampin, Rochagan, and Vansil. The chief obstacles to wider developing country use of industry-alone drugs were their high price-often due to expensive active pharmaceutical ingredients or high formulation cost-and their poor suitability to the target populations. Examples include the development of intravenous formulations suited to Western patients but diffi cult in poor-country settings, and exclusion of important patient groups, such as children with malaria or HIV-positive patients with TB.
Level of innovation and speed of development. Incremental innovation can offer marked benefi ts to patients. For instance, fi xed-dose combinations of existing drugs can greatly improve ease of use and compliance; follow-on drugs in the same class may improve safety and effi cacy; and paediatric formulations can make childhood treatments simpler and more reliable. However, if we are to effectively manage health outcomes in the long-term then we must also overcome drug resistance, which is a growing problem for many neglected diseases, including malaria, TB, leishmaniasis, and sleeping sickness. To do so, we need to focus R&D on "breakthrough" innovation-that is, novel compounds with a novel mechanism of action against parasites and microbes-as well as on activities that simplify or improve existing therapies from the patient perspective. Measurement of the level of "breakthrough" innovation under each approach shows that PPPs and industry partnering approaches perform best. Nearly half of all PPP projects (49%) and more than half of industry partnering projects (63%) are in the "breakthrough" category, compared to only 8% of drugs developed by industry working alone under the pre-2000 model. The 8% number should not, however, be compared directly with the post-2000 number since the former is based on registered drugs while the latter is for a portfolio of ongoing projects. Given that R&D of "breakthrough" drugs is associated with higher attrition rates, the profi le of fi nished drugs coming out of the post-2000 portfolio is likely to include fewer innovative products. Attrition rates alone, however, cannot account for the much higher share of breakthrough innovation post-2000. The key explanation for this difference is the recent major shift in industry neglecteddisease R&D strategy, as noted above, where the serendipitous approach that characterised the past 25 years has given way to one that is specifi cally focussed toward "breakthrough" innovation. In the long-term, this approach will only deliver high-innovation products, irrespective of attrition rates.
Although the level of innovation is important, it is equally important that innovative R&D projects move quickly to bring new drugs to patients who need them. Time metrics show that PPP drug development trajectories matched or exceeded industry standards (based on data from [3] and [6]). In particular, they were signifi cantly faster than public-alone drug development (see Figures 3 and 4); and they generally exceeded industry trajectories for neglected-disease new chemical entities (although the latter are too few in number to draw signifi cant conclusions).
Cost-effi ciency. The overall cost-effi ciency of PPPs was superior to other approaches-partly, but not only, due to their ability to leverage substantial in-kind inputs from partners and by the exclusion of costs of capital from the  PPP "push" model. The total cost of collective PPP drug development activity from 2000 to 2004 (excluding TDR, for which numbers were not available) was $112 million, including cost of failure for more than 40 projects (ten of these in clinical trials, including four at Phase III). Confi dentiality agreements prevent us from disclosing project costs in many cases, in particular when company partnerships are involved. Full per-project costs were more readily disclosed on projects involving academics, developing country fi rms, and paid sub-contractors, and were remarkably low in most of the cases we examined. For example, Medicines for Malaria Venture's synthetic peroxide project has moved from exploratory, through lead identifi cation, lead optimisation, and pre-clinical, and into Phase I trials at a total cost of $11.5 million. (Costs of completed projects will, of course, be higher.) The industry cost of developing a new chemical entity for Western markets is substantially higher, estimated by the Tufts Institute at $802 million per drug including cost of failure and cost of capital, and at $403 million for outof-pocket R&D costs per drug (including cost of failure) [3]. While indicative, these numbers do not hold fully for neglected-disease drug development, which some companies suggested at interview would be substantially cheaper due to lower developing-country trial costs (for example, around $50 million to take a new compound from discovery through to the start of clinical trials). Even using these lower estimates, however, fi gures to date suggest that PPPs can still be expected to perform signifi cantly better on cost-effi ciency and cost.
Overall performance. It is important to note that these outcomes are not evidence of the capacity of the individual players, but rather of the ability of different R&D approaches to deliver optimal outcomes. A company working in a partnership may be able to deliver a better outcome than the same company working alone, for a number of reasons. Companies working alone (as was generally the case under the pre-2000 model) tend to reduce the cost and risk of neglected-disease drug development by focussing on lessexpensive, less-risky, "adaptive" R&D such as label extensions of veterinary drugs to humans, or new formulations of existing drugs, and/or by working slowly, as staff and funds are prioritised to more commercial programmes. Under the post-2000 partnering model, the same companies can still restrict costs and risks but in a far more productive way, focussing on discovery of breakthrough leads in the knowledge that others are available to help develop these and deliver them to patients.

What Does This Mean for Government Policies?
There is a clear disjunct between the reality of neglecteddisease activity and current government thinking, which is focussed on "commercialising R&D to bring big companies back into the fi eld". This thinking is built fi rmly on the beliefs outlined at the start of this report and is now signifi cantly out of kilter with the industry neglected-disease drug landscape.
Two policy issues stand out. Firstly, there is an urgent need to support the new model of neglected-disease drug development, in particular the PPP approach, which is already generating new drugs, is highly cost-effective, appears to offer the highest health value, and is a crucial factor in continuing cost-effective industry involvement in neglected-disease R&D. On this point, we note-and welcome-the recent G8 commitment to "increasing direct investment … through such mechanisms as Public Private Partnerships … to encourage the development of … drugs for AIDS, malaria, TB and other neglected diseases" [7]. We look forward to seeing the shape of new policies and mechanisms to make this commitment concrete, and encourage policy-makers to ensure that these are designed to incentivise optimal practices within the PPP approach, and to do so in the most costeffective manner. Simply handing over cash may not be the best way.
Secondly, we suggest that policy-makers review their approach to "commercialising" R&D in light of the information above. If big companies tell us that public "commercial" markets are not a catalysing factor in their decision to engage in neglected-disease R&D, then we need to listen carefully to them. Policies to stimulate new multinational company activity are one thing; policies that shift existing industry activity from a not-for-profi t approach to a for-profi t approach are quite another, and may do so at a potential cost of many billions of dollars across all neglecteddisease products. Policy-makers may also want to consider whether commercial incentives should be preferentially targeted toward smaller companies that have a closer fi t with commercial neglected-disease markets, and whether these new incentives should be tailored to encourage industryalone R&D, or to encourage partnered models, which metrics suggest may deliver a better health outcome. The latter is particularly a concern given the relative inexperience of most Western pharmaceutical companies (and in particular small companies) in later-stage clinical development and implementation of tropical disease or TB drugs for use in rural Africa or South Asia, as opposed to their undoubted experience in developing drugs for large-scale US and European disease markets.

Next Steps
The post-2000 renewal of neglected-disease R&D activity is good news for patients with neglected diseases, but it is only a beginning. We hope that this closer analysis will contribute to our store of information, and allow development of policies to encourage and improve these promising new trends in neglected-disease drug development.