Electrostatic Control of the Membrane Targeting of C 2 Domains bers

Department of Biochemistry and erally with membranes containing anionic phospholipids Molecular Biophysics (Zhang et al., 1998; Nalefski et al., 2001), while the C2 Columbia University domain from cPLA2 penetrates into the hydrocarbon 630 168 Street core of membranes and prefers electrically neutral, zwitNew York, New York 10032 terionic phospholipid membranes (Davletov et al., 1998; Nalefski and Falke, 1998; Ball et al., 1999; Bittova et al., 1999). In addition, there are C2 domains whose memSummary brane binding does not require calcium (e.g., the C2 domain from the PTEN tumor suppressor [Lee et al., Many proteins involved in signal transduction and ves1999]), while others have been implicated in mediating icle trafficking contain C2 domains whose membrane protein-protein interactions, sometimes in addition to a association is often regulated by calcium. Here, finitemembrane-targeting function (Li et al., 1995). difference Poisson-Boltzmann calculations are used Calcium-induced nonspecific electrostatic interacto describe the electrostatic interactions between C2 tions (“the electrostatic switch” [Shao et al., 1997; Rizo domains of known structure and phospholipid memand Sudhof, 1998]) have been clearly demonstrated to branes. The results explain how calcium binding can provide a major driving force for the membrane associadrive the association of some C2 domains to negation of SytI-C2A (Zhang et al., 1998) and PKC -C2 (Naleftively charged membranes and others to neutral, zwitski et al., 2001). The underlying model is that calcium terionic membranes. Nonspecific electrostatic interbinding increases the electrostatic potential at the bindactions are shown to be a general feature of many C2 ing surface of the C2 domain and increases its attraction domains of known structure, including the calciumto acidic phospholipids. The selectivity for any specific independent C2 domain of the PTEN tumor suppressor. acidic lipid is quite small, suggesting that calcium chelation effects, if present, are of only secondary imporIntroduction tance. Hydrophobic interactions may play an accessory role for SytI-C2A and PKC -C2 (see below), but they C2 domains are independently folded modules of about appear dominant for the case of cPLA2-C2, which binds 130 residues that appear in a diverse set of proteins that to zwitterionic phospholipids through a process involvparticipate in a variety of cellular functions (reviewed in ing penetration of hydrophobic residues in the calcium Ponting and Parker, 1996; Nalefski and Falke, 1996; Rizo binding loops into the membrane interior (Davletov et and Sudhof, 1998; Hurley and Misra, 2000; Nalefski et al., 1998; Nalefski and Falke, 1998; Ball et al., 1999; Bittova al., 2001). The best-studied C2 domains are involved in et al., 1999). However, in this case, the mechanism of signal transduction (e.g., those from protein kinase C calcium regulation is more difficult to understand, since [PKC], phospholipase C [PLC], and cytosolic phospholithere can be, at best, only weak electrostatic attractions pase A2 [cPLA2]) and in membrane trafficking and fusion between the protein and an electrically neutral interface. (e.g., those from synaptotagmin [Syt], double C2 [Doc2], One can then ask whether the electrostatic switch mechand rabphilin), but there are many others whose funcanism is relevant to cPLA2-C2 and, if so, what is the tions have not yet been assigned (e.g., the C2 domains nature of nonspecific electrostatic interactions for this from protein kinase Cand phosphatidylserine decardomain. A related question arises for the membrane boxylase). The C2 domains that have been characterized association of calcium-independent C2 domains, such thus far serve principally as membrane-docking modas that of the PTEN tumor suppressor; that is, do nonules, a role that in many cases is controlled by calcium. specific electrostatic interactions, hydrophobic interacThe structures of over ten C2 domains have been tions, or some other mechanism drive membrane assodetermined to date. All have a quite similar fold, which ciation of these domains? consists of an eight-stranded antiparallel sandwich In this paper, we report calculations of the nonspecific that serves as a scaffold for variable surface loops. Many electrostatic contribution to the binding of various C2 C2 domains bind calcium ions through a cluster of domains to membranes of differing lipid compositions. aspartic acid residues located in the loop region at one The calculations are carried out with the finite-difference end of the domain. Calcium-independent C2 domains Poisson-Boltzmann (FDPB) method, which has proved lack one or more of the calcium-coordinating residues. extremely accurate in its ability to account for many of the electrostatic properties of proteins, nucleic acids, In spite of strong structural similarity, the C2 domain and protein/membrane systems (Davis and McCamfamily shares relatively low sequence identity, which mon, 1990; Honig and Nicholls, 1995). The results of the reflects the functional diversity found among its memcalculations provide strong evidence that nonspecific electrostatic interactions can account for the membrane 1 Correspondence: bh6@columbia.edu association of many C2 domains that have been charac2 Present address: Department of Microbiology and Immunology, terized to date. The calculations explain the membrane Weill Medical College of Cornell University, 1300 York Avenue, New