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Calculating the Potential of C2 Domains for Membrane Binding
Developmental Cell 132
Calculating the Potential of C2 Domains for Membrane Binding A recent report in Molecular Cell demonstrates the ability of electrostatic calculations to predict the membrane binding properties of structurally characterized C2 domains and assign probable membrane targeting functions to numerous uncharacterized C2 domains. The targeting of cytoplasmic proteins to distinct intracellular membrane compartments is mediated by an expanding list of modular domains frequently found in proteins involved in signal transduction or vesicular trafficking. Whereas FYVE, PX, C1, and a subset of PH domains recognize particular lipid constituents, such as diacyl glycerol and phosphoinositides, most C2 domains function in a less specific manner, interacting with membranes of various phospholipid composition. The C2 domain was first identified as a calcium binding module in protein kinase C. Since that time, over 100 proteins containing these approximately 130 amino acid domains have been identified. Over the last decade, a wealth of structural and functional information has accumulated on C2 domains and the mechanisms by which they associate with intracellular membranes. C2 domains share a common architecture consisting of an eight-stranded antiparallel  sandwich that positions several variable loops on a common membrane interaction surface. The variable loops frequently encode acidic Ca2⫹ binding motifs and account for the functional diversity within the C2 domain family. Many C2 domains, including the C2A domain of Synaptotagmin I (SytI) and the C2 domains of protein kinase C␣ and C (PKC␣ and PKC), associate nonspecifically with anionic phospholipids (Hurley and Misra, 2000). Others, such as the cytoplasmic phospholipase A2 (cPLA2) C2 domain (cPLA2-C2), exhibit a clear preference for neutral, zwitterionic membranes (Davletov et al., 1998; Nalefski and Falke, 1998). Although membrane binding often requires Ca2⫹, some C2 domains, like that of the PTEN tumor suppressor, associate with membranes in a Ca2⫹-independent manner (Lee et al., 1999). Given such diverse functional properties, it is perhaps not surprising that membrane targeting reflects the interplay of multiple energetic contributions. For example, Ca2⫹ binding to SytI-C2A results in an “electrostatic switch” from a negative to a positive potential (see Figure) that is hypothesized to drive nonspecific association with negatively charged phospholipids (Rizo and Sudhof, 1998). The crystal structure of PKC␣-C2 bound to Ca2⫹ and a soluble phosphatidyl serine suggests an additional role for Ca2⫹ as a “protein-lipid bridge” that coordinates anionic groups from both the C2 domain and the phospholipid membrane (Verdaguer et al., 1999). In the case of cPLA2-C2, the partitioning of exposed hydrophobic residues into the lipid bilayer provides the driving force for association with neutral membranes (Perisic et al., 1998). Given the visually dramatic effect of Ca2⫹ on the electrostatic potential of Ca2⫹-dependent C2 domains, the idea that electrostatic interactions could regulate the
affinity and selectivity of membrane binding seems intuitively appealing. But to what extent do nonspecific electrostatic interactions influence the diverse membrane binding properties of C2 domains? Are electrostatic interactions important for just a subset of C2 domains or could they play a general role? In the January issue of Molecular Cell, Murray and Honig employ finite-difference Poisson-Boltzmann (FDPB) calculations to quantitatively and systematically analyze the contribution of electrostatic interactions between C2 domains of known structure and model membranes with defined phospholipid composition (Murray and Honig, 2002). The FDPB method provides a rapid numerical solution to the Poisson-Boltzmann equation (think Coulomb’s Law generalized to any number of charged groups), allowing the electrostatic properties of complex macromolecules to be calculated with high accuracy, provided that the relevant structures are known or can be approximated by homology models (for a concise review, see Honig and Nicholls, 1995). Graphical interfaces, such as the popular program GRASP, developed in the Honig lab, allow the electrostatic properties to be visualized in the context of the corresponding macromolecular structures. FDPB calculations can further be used to assess the electrostatic contribution to the free energy of macromolecular interactions. When these calculations are applied to C2 domains, a remarkably coherent picture emerges of how electrostatic potential and its regulation by Ca2⫹ influences membrane affinity and selectivity. Indeed, the calculations support earlier hypotheses regarding the role of electrostatic interactions and lend new insight into the sometimes-counterintuitive phenotypes of site-specific mutants. As noted above, the well-characterized SytI-C2A associates nonspecifically with negatively charged membranes in the presence of Ca2⫹. FDPB calculations predict that Ca2⫹ binding dramatically decreases the free energy for interaction with negatively charged model membranes. Favorable interactions with the negatively charged model membranes are not observed in the absence of Ca2⫹, while the interaction with neutral PC membranes is unfavorable under all conditions. Thus, the calculations clearly support an electrostatic switch mechanism for SytI-C2A. The C2 domain of PKC provides another interesting case study. Like SytI-C2A, PKC-C2 associates nonspecifically with anionic membranes in a Ca2⫹-dependent manner. Here again, the FDPB calculations are consistent with an electrostatic switch mechanism. Curiously, replacing two negatively charged aspartic acid residues involved in Ca2⫹ coordination with two positively charged arginine residues results in a 30-fold reduction in affinity for anionic membranes, suggesting that PKC-C2 might not employ a simple electrostatic switch mechanism (Edwards and Newton, 1997). The resolution of this apparent paradox lies in the accounting. Although the arginine substitutions result in a gain of ⫹4 units of charge, they also disrupt the binding sites for three Ca2⫹ ions, which contribute a total charge of ⫹6. According to the FDPB calculations, the net loss of ⫹2 charge units should lead to a 50-fold reduction in affinity, in good agreement with the experiments. Several other C2 domains of known structure exhibit
Previews 133
A Ca2⫹-Induced Electrostatic Switch Calculated electrostatic potential of SytI-C2 in the absence (A) and presence (B) of bound Ca2⫹. Positive (blue) and negative (red) potential is contoured at approximately ⫹25 mV and ⫺25 mV, respectively. Adapted from Figure 1 of Murray and Honig (2002).
similar Ca2⫹-dependent electrostatic potentials, including the C2B domain of Rabphilin 3A as well as the C2 domains of phospholipase C␦, PKC␣, and Syt3, indicating that the membrane binding of these C2 domains is also consistent with an electrostatic switch mechanism. Interestingly, the Ca2⫹-independent C2 domains of the PTEN tumor suppressor (known to bind anionic membranes) and phosphatidyl inositol 3-kinase ␥ both exhibit electrostatic potentials similar to the Ca2⫹-bound form of SytI-C2. But what of C2 domains that bind preferentially to neutral, zwitterionic membranes? Here too, it appears that evolution has preserved a role for nonspecific electrostatic interactions. Consider, for example, the C2 domain of cPLA2. In this case, exposed hydrophobic residues in the Ca2⫹ binding loops drive the association with neutral membranes but only in the presence of Ca2⫹. In the absence of Ca2⫹, a substantial negative electrostatic potential opposes penetration into the phospholipid bilayer, due to the energetically unfavorable dehydration of anionic residues. Ca2⫹ binding lowers this barrier to membrane association by neutralizing the electrostatic potential in the vicinity of the nonpolar surface that partitions into the bilayer. Finally, Murray and Honig apply FDPB calculations to homology models of two C2 domains with unsolved structures. Here again, the results are in general agreement with experimental observations. In a structural genomic era, the combination of increasingly better homology models with FDPB calculations will likely prove a
useful bioinformatic tool for C2 domains and, perhaps, other large domain families whose interaction with membranes reflects a nonspecific electrostatic component. Eric Merithew and David G. Lambright Program in Molecular Medicine Department of Biochemistry and Molecular Pharmacology University of Massachusetts Medical School Worcester, Massachusetts 01605 Selected Reading Davletov, B.A., Perisic, O., and Williams, R.L. (1998). J. Biol. Chem. 273, 19093–19096. Edwards, A.S., and Newton, A.C. (1997). Biochemistry 36, 15615– 15623. Hurley, J.H., and Misra, S. (2000). Annu. Rev. Biophys. Biomol. Struct. 29, 49–79. Lee, J.O., Yang, H., Georgescu, M.M., Di Cristofano, A., Maehama, T., Shi, Y., Dixon, J.E., Pandolfi, P., and Pavletich, N.P. (1999). Cell 99, 323–334. Honig, B.H., and Nicholls, A. (1995). Science 268, 1144–1149. Murray, D., and Honig, B. (2002). Mol. Cell 9, 145–154. Nalefski, E.A., and Falke, J.J. (1998). Biochemistry 37, 17642–17650. Perisic, O., Fong, S., Lynch, D.E., Bycroft, M., and Williams, R.L. (1998). J. Biol. Chem. 273, 1596–1604. Rizo, J., and Sudhof, T.C. (1998). J. Biol. Chem. 273, 15879–15882. Verdaguer, N., Corbalan-Garcia, S., Ochoa, W.F., Fita, I., and Gomez-Fernandez, J.C. (1999). EMBO J. 18, 6329–6338.
Calculating the Potential of C2 Domains for Membrane Binding A recent report in Molecular Cell demonstrates the ability of electrostatic calculations to predict the membrane binding properties of structurally characterized C2 domains and assign probable membrane targeting functions to numerous uncharacterized C2 domains. The targeting of cytoplasmic proteins to distinct intracellular membrane compartments is mediated by an expanding list of modular domains frequently found in proteins involved in signal transduction or vesicular trafficking. Whereas FYVE, PX, C1, and a subset of PH domains recognize particular lipid constituents, such as diacyl glycerol and phosphoinositides, most C2 domains function in a less specific manner, interacting with membranes of various phospholipid composition. The C2 domain was first identified as a calcium binding module in protein kinase C. Since that time, over 100 proteins containing these approximately 130 amino acid domains have been identified. Over the last decade, a wealth of structural and functional information has accumulated on C2 domains and the mechanisms by which they associate with intracellular membranes. C2 domains share a common architecture consisting of an eight-stranded antiparallel  sandwich that positions several variable loops on a common membrane interaction surface. The variable loops frequently encode acidic Ca2⫹ binding motifs and account for the functional diversity within the C2 domain family. Many C2 domains, including the C2A domain of Synaptotagmin I (SytI) and the C2 domains of protein kinase C␣ and C (PKC␣ and PKC), associate nonspecifically with anionic phospholipids (Hurley and Misra, 2000). Others, such as the cytoplasmic phospholipase A2 (cPLA2) C2 domain (cPLA2-C2), exhibit a clear preference for neutral, zwitterionic membranes (Davletov et al., 1998; Nalefski and Falke, 1998). Although membrane binding often requires Ca2⫹, some C2 domains, like that of the PTEN tumor suppressor, associate with membranes in a Ca2⫹-independent manner (Lee et al., 1999). Given such diverse functional properties, it is perhaps not surprising that membrane targeting reflects the interplay of multiple energetic contributions. For example, Ca2⫹ binding to SytI-C2A results in an “electrostatic switch” from a negative to a positive potential (see Figure) that is hypothesized to drive nonspecific association with negatively charged phospholipids (Rizo and Sudhof, 1998). The crystal structure of PKC␣-C2 bound to Ca2⫹ and a soluble phosphatidyl serine suggests an additional role for Ca2⫹ as a “protein-lipid bridge” that coordinates anionic groups from both the C2 domain and the phospholipid membrane (Verdaguer et al., 1999). In the case of cPLA2-C2, the partitioning of exposed hydrophobic residues into the lipid bilayer provides the driving force for association with neutral membranes (Perisic et al., 1998). Given the visually dramatic effect of Ca2⫹ on the electrostatic potential of Ca2⫹-dependent C2 domains, the idea that electrostatic interactions could regulate the
affinity and selectivity of membrane binding seems intuitively appealing. But to what extent do nonspecific electrostatic interactions influence the diverse membrane binding properties of C2 domains? Are electrostatic interactions important for just a subset of C2 domains or could they play a general role? In the January issue of Molecular Cell, Murray and Honig employ finite-difference Poisson-Boltzmann (FDPB) calculations to quantitatively and systematically analyze the contribution of electrostatic interactions between C2 domains of known structure and model membranes with defined phospholipid composition (Murray and Honig, 2002). The FDPB method provides a rapid numerical solution to the Poisson-Boltzmann equation (think Coulomb’s Law generalized to any number of charged groups), allowing the electrostatic properties of complex macromolecules to be calculated with high accuracy, provided that the relevant structures are known or can be approximated by homology models (for a concise review, see Honig and Nicholls, 1995). Graphical interfaces, such as the popular program GRASP, developed in the Honig lab, allow the electrostatic properties to be visualized in the context of the corresponding macromolecular structures. FDPB calculations can further be used to assess the electrostatic contribution to the free energy of macromolecular interactions. When these calculations are applied to C2 domains, a remarkably coherent picture emerges of how electrostatic potential and its regulation by Ca2⫹ influences membrane affinity and selectivity. Indeed, the calculations support earlier hypotheses regarding the role of electrostatic interactions and lend new insight into the sometimes-counterintuitive phenotypes of site-specific mutants. As noted above, the well-characterized SytI-C2A associates nonspecifically with negatively charged membranes in the presence of Ca2⫹. FDPB calculations predict that Ca2⫹ binding dramatically decreases the free energy for interaction with negatively charged model membranes. Favorable interactions with the negatively charged model membranes are not observed in the absence of Ca2⫹, while the interaction with neutral PC membranes is unfavorable under all conditions. Thus, the calculations clearly support an electrostatic switch mechanism for SytI-C2A. The C2 domain of PKC provides another interesting case study. Like SytI-C2A, PKC-C2 associates nonspecifically with anionic membranes in a Ca2⫹-dependent manner. Here again, the FDPB calculations are consistent with an electrostatic switch mechanism. Curiously, replacing two negatively charged aspartic acid residues involved in Ca2⫹ coordination with two positively charged arginine residues results in a 30-fold reduction in affinity for anionic membranes, suggesting that PKC-C2 might not employ a simple electrostatic switch mechanism (Edwards and Newton, 1997). The resolution of this apparent paradox lies in the accounting. Although the arginine substitutions result in a gain of ⫹4 units of charge, they also disrupt the binding sites for three Ca2⫹ ions, which contribute a total charge of ⫹6. According to the FDPB calculations, the net loss of ⫹2 charge units should lead to a 50-fold reduction in affinity, in good agreement with the experiments. Several other C2 domains of known structure exhibit
Previews 133
A Ca2⫹-Induced Electrostatic Switch Calculated electrostatic potential of SytI-C2 in the absence (A) and presence (B) of bound Ca2⫹. Positive (blue) and negative (red) potential is contoured at approximately ⫹25 mV and ⫺25 mV, respectively. Adapted from Figure 1 of Murray and Honig (2002).
similar Ca2⫹-dependent electrostatic potentials, including the C2B domain of Rabphilin 3A as well as the C2 domains of phospholipase C␦, PKC␣, and Syt3, indicating that the membrane binding of these C2 domains is also consistent with an electrostatic switch mechanism. Interestingly, the Ca2⫹-independent C2 domains of the PTEN tumor suppressor (known to bind anionic membranes) and phosphatidyl inositol 3-kinase ␥ both exhibit electrostatic potentials similar to the Ca2⫹-bound form of SytI-C2. But what of C2 domains that bind preferentially to neutral, zwitterionic membranes? Here too, it appears that evolution has preserved a role for nonspecific electrostatic interactions. Consider, for example, the C2 domain of cPLA2. In this case, exposed hydrophobic residues in the Ca2⫹ binding loops drive the association with neutral membranes but only in the presence of Ca2⫹. In the absence of Ca2⫹, a substantial negative electrostatic potential opposes penetration into the phospholipid bilayer, due to the energetically unfavorable dehydration of anionic residues. Ca2⫹ binding lowers this barrier to membrane association by neutralizing the electrostatic potential in the vicinity of the nonpolar surface that partitions into the bilayer. Finally, Murray and Honig apply FDPB calculations to homology models of two C2 domains with unsolved structures. Here again, the results are in general agreement with experimental observations. In a structural genomic era, the combination of increasingly better homology models with FDPB calculations will likely prove a
useful bioinformatic tool for C2 domains and, perhaps, other large domain families whose interaction with membranes reflects a nonspecific electrostatic component. Eric Merithew and David G. Lambright Program in Molecular Medicine Department of Biochemistry and Molecular Pharmacology University of Massachusetts Medical School Worcester, Massachusetts 01605 Selected Reading Davletov, B.A., Perisic, O., and Williams, R.L. (1998). J. Biol. Chem. 273, 19093–19096. Edwards, A.S., and Newton, A.C. (1997). Biochemistry 36, 15615– 15623. Hurley, J.H., and Misra, S. (2000). Annu. Rev. Biophys. Biomol. Struct. 29, 49–79. Lee, J.O., Yang, H., Georgescu, M.M., Di Cristofano, A., Maehama, T., Shi, Y., Dixon, J.E., Pandolfi, P., and Pavletich, N.P. (1999). Cell 99, 323–334. Honig, B.H., and Nicholls, A. (1995). Science 268, 1144–1149. Murray, D., and Honig, B. (2002). Mol. Cell 9, 145–154. Nalefski, E.A., and Falke, J.J. (1998). Biochemistry 37, 17642–17650. Perisic, O., Fong, S., Lynch, D.E., Bycroft, M., and Williams, R.L. (1998). J. Biol. Chem. 273, 1596–1604. Rizo, J., and Sudhof, T.C. (1998). J. Biol. Chem. 273, 15879–15882. Verdaguer, N., Corbalan-Garcia, S., Ochoa, W.F., Fita, I., and Gomez-Fernandez, J.C. (1999). EMBO J. 18, 6329–6338.