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A Tale of the Unexpected
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Gaub, 2006). The information they gained from the pulling could be used to supplement what could be learned from diffraction data. Further, the combination of diffraction data with single-molecule mechanics could be used to learn more about the structural correlates of the measured forces and distances in the molecular mechanics curves of force versus distance. One challenge for the future will be moving to ion channels that cannot be crystallized, but are extremely important for understanding human health, and learning about them. Another direction for the future is moving from single-molecule mechanics to understanding tissue mechanics. As one example of this direction, single-molecule force spectroscopy on bone has recently led to new insights into bone fracture mechanics (Fantner et al., 2005). The hope is that a detailed understanding of tissue mechanics based on single-molecule mechanics can contribute to a fuller understanding of the molecules responsible for macroscopic phenomena such as wound healing and tissue elasticity. In general, atomic or molecular interactions involve forces and energies. Energies can be measured by bulk techniques and are very well-known for almost any combination of atoms and a vast number of molecular interactions. Knowledge of interaction forces, however, is much more difficult to obtain from bulk measurements. This is one of the reasons that pulling with the atomic force microscope to measure molecular mechanics has such a bright future.
Structure 14, March 2006 ª2006 Elsevier Ltd All rights reserved
Paul K. Hansma Department of Physics University of California, Santa Barbara Santa Barbara, California 93106 Selected Reading Binnig, G., Quate, C.F., and Gerber, C. (1986). Phys. Rev. Lett. 56, 930–933. Binnig, G., Rohrer, H., Gerber, C., and Weibel, E. (1982). Phys. Rev. Lett. 49, 57–61. Drake, B., Prater, C.B., Weisenhorn, A.L., Gould, S.A.C., Albrecht, T.R., Quate, C.F., Cannell, D.S., Hansma, H.G., and Hansma, P.K. (1989). Science 243, 1586–1589. Fantner, G.E., Hassenkam, T., Kindt, J.H., Weaver, J.C., Birkedal, H., Pechenik, L., Cutroni, J.A., Cidade, G.A.G., Stucky, G., Morse, D.E., and Hansma, P.K. (2005). Nat. Mater. 4, 612–616. Kessler, M., and Gaub, H.E. (2006). Structure 14, this issue, 521–527. Muller, D.J., Kessler, M., Oesterhelt, F., Moller, C., Oesterhelt, D., and Gaub, H. (2002). Biophys. J. 83, 3578–3588. Quist, A., Doudevski, L., Lin, H., Azimova, R., Ng, D., Frangione, B., Kagan, B., Ghiso, J., and Lal, R. (2005). Proc. Natl. Acad. Sci. USA 102, 10427–10432. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M., and Gaub, H.E. (1997a). Science 276, 1109–1112. Rief, M., Oesterhelt, F., Heymann, B., and Gaub, H.E. (1997b). Science 275, 1295–1297. Schabert, F.A., and Engel, A. (1994). Biophys. J. 67, 2394–2403. Weisenhorn, A.L., Hansma, P.K., Albrecht, T.R., and Quate, C.F. (1989). Appl. Phys. Lett. 54, 2651–2653.
DOI 10.1016/j.str.2006.02.002
A Tale of the Unexpected A human homolog of the prokaryotic phosphate binding protein has been described and its structure determined (Morales et al., 2006 [this issue of Structure]). This protein’s discovery in plasma was unexpected and leads to questions as to what function this type of protein might have in eukaryotes.
Research by its very nature leads us into the unexpected, but once in a while the twist in the tale provides a reminder of this. When one protein is sought but another found, fresh opportunities open up. In a paper in this issue of Structure, Morales et al. (2006) describe such a tale, the discovery and structural characterization of a protein that is the first of its class in eukaryotes. Chabriere and coworkers were investigating paraoxonase (PON1), an enzyme responsible for inactivating various organophosphorus compounds including insecticides and nerve gasses. The fact that a human enzyme is named after an insecticide, paraoxon, just shows that all this is in relatively new territory. The true physiological function of PON1 is apparently unknown, although
it appears to have an important role in prevention of atherosclerosis (Watson et al., 1995; Shih et al., 1998). A hydrolase with such various activities is bound to draw attention. However, the research on this system took an unexpected turn when a novel protein was isolated by copurification with PON1 from human plasma. This discovery led to a number of precise results, including a crystal structure that shows that the new protein is closely related to the periplasmic phosphate binding protein (PBP) of prokaryotes (Luecke and Quiocho, 1990). Thus this human protein was designated HPBP, and it shows a similar fold and binding site to the prokaryotic protein, although there is limited sequence identity. The described HPBP structure has a small oxyanion in the binding site. It presumably binds inorganic phosphate (Pi) in the same way as the prokaryotic protein, via the Venus fly trap model with the two domains engulfing the Pi as they close the binding cleft via a hinge-bend (and twist) (Brune et al., 1998, Mao et al., 1982). In the case of Escherichia coli PBP, a structure has also been obtained of a phosphate-free mutant (Ledvina et al., 1998), which shows a significant opening of the binding cleft. This mechanism produces a high affinity and specificity for Pi, versus, for example,
Structure 392
phosphate esters. The structure is also similar to the lipoprotein version of PBP from mycobacteria (Vyas et al., 2003). In each case the phosphate binds tightly, probably with a submicromolar dissociation constant. In gram-negative bacteria, PBP and other similar ligand binding proteins are periplasmic and interact with membrane bound ABC transporters to enable the import of the cargo ligand. These proteins are generally expressed when the organism is starved of the essential small molecule, and are exported rapidly to the periplasm. In the case of PBP, this process also includes a phosphatase to liberate free Pi from soluble phosphate esters. As such, PBP operates as part of a system to scavenge limited phosphate and enable the ABC transporter to import it, potentially against a concentration gradient. In mycobacteria, the related ligand binding proteins are membrane bound but possibly operate by a similar mechanism. So what is such a protein doing in eukaryotes, specifically in human plasma? Unsurprisingly for a protein that only came to light in this unexpected way, currently the clues are limited. From its sequence, HPBP belongs to a family of proteins called DING that is widely distributed in eukaryotes. The fact that the key amino acids in the phosphate binding site are conserved in this family suggests that this type of binding protein is widespread in eukaryotes; it was previously suggested that members of this family might be related to PBP (Kumar et al., 2004). The HPBP is slightly larger than the archetypical PBP from E. coli. It has several extra loops that may well be involved in its distinct role. It tightly associates with PON1, which in turn associates with high-density lipoprotein. In the plasma itself, inorganic phosphate levels are high enough to saturate HPBP, as its dissociation constant is much less than the likely Pi concentration. In this situation, only a protein with much weaker Pi binding might be expected to be modulated between Pi-free and Pi bound forms. As it is, exposed to the high Pi in plasma, HPBP is always going to have Pi bound. One might not think it necessary to have such a binding protein merely to scavenge for Pi or more distinctly to help transport this small molecule into compartments that otherwise have limited Pi. At least in gram-negative bacteria, the equivalent PBP is apparently not needed and so its expression is repressed when the Pi concentration in the medium is high (Horiuchi et al., 1959, Medveczky and Rosenberg, 1970, Yagil et al., 1976). Intriguingly Morales et al. (2006) raise the possibility of a role in preventing phosphate salt formation, specifically with calcium. Another DING protein has been implicated in prevention of kidney stones by such a mechanism (Kumar et al., 2004).
While PBPs in prokaryotes have a role in Pi import, possibly the direction in plasma may be reversed, helping maintain compartments that are low in Pi. The evidence that HPBP interacts tightly with PON1 gives rise to other possibilities. Does the interaction with PON1 impinge directly on the function of each protein, for example by changing the enzyme specificity? Alternatively, does the interaction form part of a complex control mechanism, perhaps by modulating the affinity of the HPBP or by directing it to particular locations? Do the DING proteins actually interact with ABC transporters? Structural and mechanistic studies on this protein and its interactions may well throw light on potential functions. We can expect some interesting and fresh directions in the future, which may identify a novel role of the small molecule Pi and its binding proteins in eukaryotes. Martin R. Webb MRC National Institute for Medical Research Mill Hill London NW7 1AA United Kingdom
Selected Reading Brune, M., Hunter, J.L., Howell, S.A., Martin, S.R., Hazlett, T.L., Corrie, J.E.T., and Webb, M.R. (1998). Biochemistry 37, 10370– 10380. Horiuchi, T., Horiuchi, S., and Mizuno, D. (1959). Nature 183, 1529– 1531. Kumar, V., Yu, S., Farell, G., Toback, F.G., and Lieske, J.C. (2004). Am. J. Physiol. Renal Physiol. 287, F373–F383. Ledvina, P.S., Tsai, A., Wang, Z., Koehl, E., and Quiocho, F.A. (1998). Protein Sci. 7, 2550–2559. Luecke, H., and Quiocho, F.A. (1990). Nature 347, 402–406. Mao, B., Pear, M.R., McCammon, J.A., and Quiocho, F.A. (1982). J. Biol. Chem. 257, 1131–1133. Medveczky, N., and Rosenberg, H. (1970). Biochim. Biophys. Acta 241, 494–506. Morales, R., Berna, A., Carpentier, P., Contreras-Martel, C., Renault, F., Nicodeme, M., Chesne-Seck, M., Bernier, F., Dupuy, J., Schaeffer, C., et al. (2006). Structure 14, this issue, 601–609. Shih, D.M., Gu, L., Xia, Y.R., Navab, M., Li, W.F., Hama, S., Castellani, L.W., Furlong, C.E., Costa, L.G., Fogelman, A.M., and Lusis, A.J. (1998). Nature 394, 284–287. Vyas, N.K., Vyas, M.N., and Quiocho, F.A. (2003). Structure 11, 765– 774. Watson, A.D., Berliner, J.A., Hama, S.Y., and La Du, B.N. (1995). J. Clin. Invest. 96, 2882–2891. Yagil, E., Silberstein, N., and Gerdes, R.G. (1976). J. Bacteriol. 127, 656–659.
Gaub, 2006). The information they gained from the pulling could be used to supplement what could be learned from diffraction data. Further, the combination of diffraction data with single-molecule mechanics could be used to learn more about the structural correlates of the measured forces and distances in the molecular mechanics curves of force versus distance. One challenge for the future will be moving to ion channels that cannot be crystallized, but are extremely important for understanding human health, and learning about them. Another direction for the future is moving from single-molecule mechanics to understanding tissue mechanics. As one example of this direction, single-molecule force spectroscopy on bone has recently led to new insights into bone fracture mechanics (Fantner et al., 2005). The hope is that a detailed understanding of tissue mechanics based on single-molecule mechanics can contribute to a fuller understanding of the molecules responsible for macroscopic phenomena such as wound healing and tissue elasticity. In general, atomic or molecular interactions involve forces and energies. Energies can be measured by bulk techniques and are very well-known for almost any combination of atoms and a vast number of molecular interactions. Knowledge of interaction forces, however, is much more difficult to obtain from bulk measurements. This is one of the reasons that pulling with the atomic force microscope to measure molecular mechanics has such a bright future.
Structure 14, March 2006 ª2006 Elsevier Ltd All rights reserved
Paul K. Hansma Department of Physics University of California, Santa Barbara Santa Barbara, California 93106 Selected Reading Binnig, G., Quate, C.F., and Gerber, C. (1986). Phys. Rev. Lett. 56, 930–933. Binnig, G., Rohrer, H., Gerber, C., and Weibel, E. (1982). Phys. Rev. Lett. 49, 57–61. Drake, B., Prater, C.B., Weisenhorn, A.L., Gould, S.A.C., Albrecht, T.R., Quate, C.F., Cannell, D.S., Hansma, H.G., and Hansma, P.K. (1989). Science 243, 1586–1589. Fantner, G.E., Hassenkam, T., Kindt, J.H., Weaver, J.C., Birkedal, H., Pechenik, L., Cutroni, J.A., Cidade, G.A.G., Stucky, G., Morse, D.E., and Hansma, P.K. (2005). Nat. Mater. 4, 612–616. Kessler, M., and Gaub, H.E. (2006). Structure 14, this issue, 521–527. Muller, D.J., Kessler, M., Oesterhelt, F., Moller, C., Oesterhelt, D., and Gaub, H. (2002). Biophys. J. 83, 3578–3588. Quist, A., Doudevski, L., Lin, H., Azimova, R., Ng, D., Frangione, B., Kagan, B., Ghiso, J., and Lal, R. (2005). Proc. Natl. Acad. Sci. USA 102, 10427–10432. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M., and Gaub, H.E. (1997a). Science 276, 1109–1112. Rief, M., Oesterhelt, F., Heymann, B., and Gaub, H.E. (1997b). Science 275, 1295–1297. Schabert, F.A., and Engel, A. (1994). Biophys. J. 67, 2394–2403. Weisenhorn, A.L., Hansma, P.K., Albrecht, T.R., and Quate, C.F. (1989). Appl. Phys. Lett. 54, 2651–2653.
DOI 10.1016/j.str.2006.02.002
A Tale of the Unexpected A human homolog of the prokaryotic phosphate binding protein has been described and its structure determined (Morales et al., 2006 [this issue of Structure]). This protein’s discovery in plasma was unexpected and leads to questions as to what function this type of protein might have in eukaryotes.
Research by its very nature leads us into the unexpected, but once in a while the twist in the tale provides a reminder of this. When one protein is sought but another found, fresh opportunities open up. In a paper in this issue of Structure, Morales et al. (2006) describe such a tale, the discovery and structural characterization of a protein that is the first of its class in eukaryotes. Chabriere and coworkers were investigating paraoxonase (PON1), an enzyme responsible for inactivating various organophosphorus compounds including insecticides and nerve gasses. The fact that a human enzyme is named after an insecticide, paraoxon, just shows that all this is in relatively new territory. The true physiological function of PON1 is apparently unknown, although
it appears to have an important role in prevention of atherosclerosis (Watson et al., 1995; Shih et al., 1998). A hydrolase with such various activities is bound to draw attention. However, the research on this system took an unexpected turn when a novel protein was isolated by copurification with PON1 from human plasma. This discovery led to a number of precise results, including a crystal structure that shows that the new protein is closely related to the periplasmic phosphate binding protein (PBP) of prokaryotes (Luecke and Quiocho, 1990). Thus this human protein was designated HPBP, and it shows a similar fold and binding site to the prokaryotic protein, although there is limited sequence identity. The described HPBP structure has a small oxyanion in the binding site. It presumably binds inorganic phosphate (Pi) in the same way as the prokaryotic protein, via the Venus fly trap model with the two domains engulfing the Pi as they close the binding cleft via a hinge-bend (and twist) (Brune et al., 1998, Mao et al., 1982). In the case of Escherichia coli PBP, a structure has also been obtained of a phosphate-free mutant (Ledvina et al., 1998), which shows a significant opening of the binding cleft. This mechanism produces a high affinity and specificity for Pi, versus, for example,
Structure 392
phosphate esters. The structure is also similar to the lipoprotein version of PBP from mycobacteria (Vyas et al., 2003). In each case the phosphate binds tightly, probably with a submicromolar dissociation constant. In gram-negative bacteria, PBP and other similar ligand binding proteins are periplasmic and interact with membrane bound ABC transporters to enable the import of the cargo ligand. These proteins are generally expressed when the organism is starved of the essential small molecule, and are exported rapidly to the periplasm. In the case of PBP, this process also includes a phosphatase to liberate free Pi from soluble phosphate esters. As such, PBP operates as part of a system to scavenge limited phosphate and enable the ABC transporter to import it, potentially against a concentration gradient. In mycobacteria, the related ligand binding proteins are membrane bound but possibly operate by a similar mechanism. So what is such a protein doing in eukaryotes, specifically in human plasma? Unsurprisingly for a protein that only came to light in this unexpected way, currently the clues are limited. From its sequence, HPBP belongs to a family of proteins called DING that is widely distributed in eukaryotes. The fact that the key amino acids in the phosphate binding site are conserved in this family suggests that this type of binding protein is widespread in eukaryotes; it was previously suggested that members of this family might be related to PBP (Kumar et al., 2004). The HPBP is slightly larger than the archetypical PBP from E. coli. It has several extra loops that may well be involved in its distinct role. It tightly associates with PON1, which in turn associates with high-density lipoprotein. In the plasma itself, inorganic phosphate levels are high enough to saturate HPBP, as its dissociation constant is much less than the likely Pi concentration. In this situation, only a protein with much weaker Pi binding might be expected to be modulated between Pi-free and Pi bound forms. As it is, exposed to the high Pi in plasma, HPBP is always going to have Pi bound. One might not think it necessary to have such a binding protein merely to scavenge for Pi or more distinctly to help transport this small molecule into compartments that otherwise have limited Pi. At least in gram-negative bacteria, the equivalent PBP is apparently not needed and so its expression is repressed when the Pi concentration in the medium is high (Horiuchi et al., 1959, Medveczky and Rosenberg, 1970, Yagil et al., 1976). Intriguingly Morales et al. (2006) raise the possibility of a role in preventing phosphate salt formation, specifically with calcium. Another DING protein has been implicated in prevention of kidney stones by such a mechanism (Kumar et al., 2004).
While PBPs in prokaryotes have a role in Pi import, possibly the direction in plasma may be reversed, helping maintain compartments that are low in Pi. The evidence that HPBP interacts tightly with PON1 gives rise to other possibilities. Does the interaction with PON1 impinge directly on the function of each protein, for example by changing the enzyme specificity? Alternatively, does the interaction form part of a complex control mechanism, perhaps by modulating the affinity of the HPBP or by directing it to particular locations? Do the DING proteins actually interact with ABC transporters? Structural and mechanistic studies on this protein and its interactions may well throw light on potential functions. We can expect some interesting and fresh directions in the future, which may identify a novel role of the small molecule Pi and its binding proteins in eukaryotes. Martin R. Webb MRC National Institute for Medical Research Mill Hill London NW7 1AA United Kingdom
Selected Reading Brune, M., Hunter, J.L., Howell, S.A., Martin, S.R., Hazlett, T.L., Corrie, J.E.T., and Webb, M.R. (1998). Biochemistry 37, 10370– 10380. Horiuchi, T., Horiuchi, S., and Mizuno, D. (1959). Nature 183, 1529– 1531. Kumar, V., Yu, S., Farell, G., Toback, F.G., and Lieske, J.C. (2004). Am. J. Physiol. Renal Physiol. 287, F373–F383. Ledvina, P.S., Tsai, A., Wang, Z., Koehl, E., and Quiocho, F.A. (1998). Protein Sci. 7, 2550–2559. Luecke, H., and Quiocho, F.A. (1990). Nature 347, 402–406. Mao, B., Pear, M.R., McCammon, J.A., and Quiocho, F.A. (1982). J. Biol. Chem. 257, 1131–1133. Medveczky, N., and Rosenberg, H. (1970). Biochim. Biophys. Acta 241, 494–506. Morales, R., Berna, A., Carpentier, P., Contreras-Martel, C., Renault, F., Nicodeme, M., Chesne-Seck, M., Bernier, F., Dupuy, J., Schaeffer, C., et al. (2006). Structure 14, this issue, 601–609. Shih, D.M., Gu, L., Xia, Y.R., Navab, M., Li, W.F., Hama, S., Castellani, L.W., Furlong, C.E., Costa, L.G., Fogelman, A.M., and Lusis, A.J. (1998). Nature 394, 284–287. Vyas, N.K., Vyas, M.N., and Quiocho, F.A. (2003). Structure 11, 765– 774. Watson, A.D., Berliner, J.A., Hama, S.Y., and La Du, B.N. (1995). J. Clin. Invest. 96, 2882–2891. Yagil, E., Silberstein, N., and Gerdes, R.G. (1976). J. Bacteriol. 127, 656–659.