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J. Biol. Chem., Vol. 278, Issue 48, 48348-48356, November 28, 2003
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From the
Krannert Institute of Cardiology and the Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202 and the Skirball Institute of Biomolecular Medicine and the Department of Cell Biology, New York University School of Medicine, New York, New York 10016
Received for publication, August 27, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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80% efficiency. Optimal cross-linking occurred at spacer chain lengths of 10 and 15 Å for N27C and N30C, respectively. The rapid time course of cross-linking indicated that neither dissociation of PLB pentamers nor binding of PLB monomers to SERCA2a was rate-limiting. Cross-linking occurred only to the E2 (Ca2+-free) conformation of SERCA2a, was strongly favored by nucleotide binding to this state, and was completely inhibited by thapsigargin. Protein sequencing in combination with mutagenesis identified of Lys328 of SERCA2a as the target of cross-linking. A three-dimensional map of interacting residues indicated that the cross-linking distances were entirely compatible with the 10-Å distance recently determined between N30C of PLB and Cys318 of SERCA2a. In contrast, Lys3 of PLB did not cross-link to any Lys (or Cys) of SERCA2a, suggesting that previous three-dimensional models that constrain Lys3 near residues 397–400 of thapsigargin-inhibited SERCA2a should be viewed with caution. Furthermore, although earlier models of PLB·SERCA2a are based on thapsigargin-bound SERCA, our results suggest that the nucleotide-bound, E2 conformation is substantially different and represents the key conformational state for interacting with PLB. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-adrenergic stimulation of the heart reverses PLB inhibition and augments Ca2+ loading of the SR (4), thus producing positive inotropic and lusitropic effects (2, 5). PLB is a single-span membrane protein composed of 52 amino acids, which forms a homopentamer within the SR membrane (1, 4). Recent work suggests that there is a dynamic equilibrium between PLB monomers and pentamers (6), and that the PLB monomer is responsible for binding to SERCA2a (7) and inhibiting it (8, 9). In intact myocardium, -adrenergic receptor stimulation disrupts the inhibitory interaction between PLB and SERCA2a rapidly; PLB phosphorylation, Ca2+ transport, and contractility all increase within seconds after -receptor activation (4, 10). This suggests that PLB monomers must associate and dissociate quickly, over a time course of seconds or faster, to allow for dynamic regulation of SERCA2a and the strength of contractility.
The emerging picture for the mechanism of SERCA2a inhibition by PLB suggests mutually exclusive binding of Ca2+ and PLB to SERCA2a. Chemical cross-linking (7, 11) and immunoprecipitation (12) indicate that PLB binds preferentially to the Ca2+-free conformation of SERCA2a, dubbed E2 (3). The alternative conformation induced by Ca2+, E1, does not appear to be capable of binding PLB (7), but instead is primed for catalyzing the hydrolysis of ATP and subsequent steps leading to Ca2+ transport across the SR membrane (13). By stabilizing SERCA2a in the E2 conformation, PLB slows the transition from E2 to E1 during the catalytic cycle (3), thus impeding overall Ca2+ transport (Fig. 1). Thus, the apparent reduction of Ca2+ affinity is a kinetic effect of PLB on conformational changes, not necessarily a direct effect on Ca2+ binding sites (3). Recently, the crystal structure of SERCA1a, the skeletal muscle isoform of the Ca2+ pump, was determined, both in the E1 state with bound Ca2+ (14), and in the E2 state, bound with the irreversible inhibitor thapsigargin (15). These structures reveal a relatively complex molecule composed of 
1000 amino acids, ten transmembrane helices, and three distinct cytoplasmic domains (16). Although no crystal structure of PLB has yet been reported, numerous biophysical methods have been used to study its structure (17). Because of technical limitations of the various techniques, no consensus has yet been reached for the complete structure of PLB within biological membranes or, more particularly, in association with SERCA2a (1, 17). Nevertheless, there is great interest in understanding the physical basis for Ca2+ pump regulation (1, 2, 5). As a result, several models of the three-dimensional interactions between different residues of PLB and SERCA2a have recently been proposed (18–20), using the E2 conformation of SERCA determined by crystallography (15) or by cryoelectronmicroscopy (21) as a structural template.
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To characterize further the interactions between the PLB monomer and SERCA2a at the molecular level, we initiated a series of studies using cross-linking agents as molecular rulers to measure distances between key amino acid residues of PLB and SERCA2a (7). Using the homobifunctional, thiol cross-linking agent BMH, we demonstrated a highly specific cross-link between Asn30 of PLB changed to cysteine (N30C-PLB) and Cys318 of SERCA2a at the cytoplasmic boundary of M4. The length of the cross-linking spacer chain suggested that these two residues are 10 Å apart. Cross-linking of PLB to SERCA2a occurred only in the absence of Ca2+ and was strikingly potentiated by ATP or ADP, providing strong physical evidence that monomeric PLB binds preferentially to the nucleotide-stabilized, E2 conformation of SERCA2a (7). Significantly, the irreversible SERCA inhibitor thapsigargin completely prevented cross-linking of PLB to SERCA2a (7), supporting previous evidence that the E2-like conformation stabilized by thapsigargin is conformationally distinct from the native E2 conformation (28, 29) (Fig. 1).
Here we report on the use of second generation cross-linking agents, which are heterobifunctional and provide additional constraints on the interaction and dynamics between PLB and SERCA2a. The new cross-linking agents contain a maleimide group at one end for coupling to Asn27 or Asn30 of PLB replaced with cysteine, and an NHS-ester group at the other end, for coupling to nearby lysine residues of WT-SERCA2a (30). We show that these heterobifunctional cross-linking agents work faster and even more efficiently than the homobifunctional agent, BMH, reported on previously (7). Nearly quantitative coupling between PLB and SERCA2a is achieved with heterobifunctional cross-linking agents. At the same time, the heterobifunctional cross-linkers remain highly specific, and react only with Lys328 of SERCA2a when attached to residues 27 and 30 of PLB, with optimal linker lengths of 10 and 15 Å, respectively.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-maleimidoacetoxy)succinimide ester (cross-linking distance 4.4 Å), N-(-maleimidopropyloxy)succinimide ester (6.9 Å), N-(
-maleimidocaproyloxy)succinimide ester (EMCS; 9.4 Å), m-maleimidobenzoyl-N-hydroxysuccinimide ester (9.9 Å), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (11.6 Å), succinimidyl-6-(-maleimidopropionamido)hexanoate (14.3 Å), and N-(
-maleimidoundecanoyloxy)sulfosuccinimide ester (KMUS; 15.7 Å). The homobifunctional cross-linking agent was 1,6-bismaleimidohexane (BMH; 10.2 Å) (7). Thapsigargin was purchased from Sigma. Iodogen was from Pierce. Endo-Asp-N and endo-Lys-C were obtained from Roche Molecular Biochemicals. Mutagenesis and Baculovirus Production—Point mutations in the cDNA encoding canine PLB were made as previously described (8, 22, 24). N27C-PLB and N30C-PLB were expressed on Cys-less PLB background, which is canine PLB with Cys residues 36, 41, and 46 replaced by Ala (7). Wild-type canine SERCA2a cDNA was mutated directly in the transfection vector pVL1393 using the QuikChangeTM XL-Gold system (Stratagene) (7). All cDNA mutations were confirmed by DNA sequencing. Baculoviruses encoding mutated proteins were generated by co-transfecting into Sf21 insect cells mutated cDNAs in pVL1393 with BaculoGoldTM (Pharmingen) linearized baculovirus DNA (7, 8, 24).
Protein Co-expression in Insect Cells and Isolation of Microsomes—Canine SERCA2a and N27C-PLB or N30C-PLB were co-expressed in Sf21 insect cells as described (7, 8, 24). Microsomes were harvested 60 h after initiating baculovirus infections and stored frozen in small aliquots at -40 °C at a protein concentration of 6–10 mg/ml. Protein assay and Ca2+-ATPase activities of microsomes were determined as previously described (8).
Cross-linking—Cross-linking between single Cys residues of N27C-PLB or N30C-PLB and endogenous Lys residue(s) of WT-SERCA2a was conducted using the heterobifunctional cross-linking agents of increasing lengths listed under "Materials." All heterobifunctional agents tested contained a maleimide group at one end for coupling to Cys residues and an NHS-ester group at the other end for coupling to Lys residues (30).
Cross-linking between PLB and SERCA2a was conducted essentially as recently described (7). Reactions were conducted with 11 µg of microsomal protein in 12 µl of buffer A, which contained 40 mM MOPS (pH 7.0), 3.2 mM MgCl2, 75 mM KCl, 3 mM ATP, and 1 mM EGTA. Reactions were started by adding 0.75 µl of cross-linking agent from a 1.6 mM stock solution in dimethyl sulfoxide (0.1 mM final cross-linker concentration) and stopped by adding 7.5 µl of SDS-PAGE sample-loading buffer containing 15% SDS plus 100 mM dithiothreitol. Heterobifunctional cross-linking was conducted for 10 min at room temperature unless otherwise indicated. Homobifunctional cross-linking between Cys residues with 0.1 mM BMH was conducted for 1 h at room temperature (7). After terminating the reactions, samples were subjected to SDS-PAGE and immunoblotting.
To assess Ca2+ effects on cross-linking, ionized Ca2+ was varied by adding CaCl2 to buffer A (7). In some experiments, ATP in buffer A was omitted or was replaced by other nucleotides, as indicated. To determine antibody effects on cross-linking, affinity-purified anti-PLB monoclonal antibody, 2D12, or anti-SERCA2a monoclonal antibody, 2A7-A1, dialyzed in 20 mM MOPS (pH 7) and 150 mM NaCl, were used (7). 5.5 µg of antibody were included with 11 µg of microsomal protein in buffer A.
SDS-PAGE and Immunoblotting—SDS-PAGE was performed in 8% polyacrylamide, and immunoblots were probed with anti-PLB monoclonal antibody, 2D12, to detect free and cross-linked forms of PLB (7). In the experiment of Fig. 9, the immunoblot was also probed with anti-SERCA2a monoclonal antibody, 2A7-A1, to detect Ca2+ pumps not cross-linked to PLB (8). Antibody-binding protein bands were visualized with 125I-protein A, except for the experiment depicted in Fig. 7, in which 125I-2D12 was used directly for PLB visualization (7), to avoid interference from antibodies carried over from the cross-linking reactions. Antibody-binding bands were quantified with a Bio-Rad Personal Fx phosphorimager.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Fig. 2A shows that N27C-PLB and N30C-PLB cross-linked strongly to wild-type SERCA2a in insect cell microsomes with use of heterobifunctional cross-linking agents. When immunoblots were probed with the anti-PLB antibody, 2D12, intense cross-linking signals were obtained at molecular masses of just over 100 kDa (PLB/SER), corresponding to heterodimers formed between PLB and SERCA2a. Some weaker homodimerization of PLB monomers was also detected (PLB2).2 Remarkably, the optimal lengths for heterobifunctional cross-linking were completely different for N27C-PLB and N30C-PLB (Fig. 2A). Specifically, N27C-PLB cross-linked most efficiently to SERCA2a with the cross-linking agents EMCS, m-maleimidobenzoyl-N-hydroxysuccinimide ester, and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, whereas N30C-PLB only cross-linked to SERCA2a with use of the two cross-linkers succinimidyl-6-(-maleimidopropionamido)hexanoate and KMUS (Fig. 2A). The relative intensities of the cross-linking signals from three separate experiments were plotted versus the distances of cross-linker arms and fitted to a Gaussian distribution. Based on the Gaussian fit, N27C of PLB is about 10 Å away from a Lys residue of native SERCA2a (Fig. 2B), whereas N30C cross-links at a distance of 15–16 Å (Fig. 2B).
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In early experiments, we tested the possibility that the natural Lys3 of PLB might cross-link to native Cys or Lys residues of SERCA2a. However, we found no evidence for this. Lys3 of PLB was not cross-linked to Cys residues of SERCA2a with any of the heterobifunctional agents tested above. Likewise, when a similar series of homobifunctional NHS-ester agents were tested, no cross-linking of Lys3 of PLB to any Lys residue of SERCA2a was detected (data not shown). Moreover, after mutation of Lys3 of PLB to Cys (K3C-PLB), we detected no cross-linking of K3C-PLB to SERCA2a with any of the heterobifunctional agents depicted in Fig. 2. We therefore conclude that Lys3 of PLB is not in proximity to any endogenous Lys or Cys residue of wild-type SERCA2a. These results contradict the earlier conclusion of James et al. (11), who reported cross-linking of Lys3 of PLB at low efficiency to Lys397 or Lys400 of SERCA after detergent solubilization of the purified proteins.
For the remaining experiments, we used EMCS and KMUS for cross-linking SERCA2a to N27C-PLB and N30C-PLB, respectively, because these two compounds gave maximal cross-linking at these two PLB residues (Fig. 2).
Micromolar Ca2+ Effect on Cross-linking—Recent work suggests that micromolar Ca2+ disrupts the physical interaction between PLB and SERCA2a (7, 12), by occupying the two high affinity Ca2+ binding sites and driving the pump into the E1 conformation (Fig. 1). Consistent with this hypothesis, cross-linking of N27C-PLB to SERCA2a with EMCS and of N30C-PLB to SERCA2a with KMUS was abolished by Ca2+ at micromolar concentration (Fig. 3A). The Ki value for Ca2+ inhibition of cross-linking to SERCA2a was 0.4 µM for both N27C-PLB (black) and N30C-PLB (red) using heterobifunctional agents (Fig. 3B). Interestingly, the Ki value for Ca2+ inhibition of cross-linking of N30C-PLB to Cys318 of SERCA2a by BMH (blue) was approximately 3 times lower (0.15 µM) (inset), which confirms earlier results with BMH (7). This suggests that different cross-linking sites between PLB and SERCA2a respond differentially to conformational changes in SERCA2a associated with Ca2+ binding. Nevertheless, cross-linking results obtained with all three agents, EMCS, KMUS, and BMH, agree with the consensus that PLB binds only to SERCA2a in the Ca2+-free or E2 conformation.
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3 times more Ca2+ pump molecules to N27C-PLB and N30C-PLB, respectively, than did BMH to N30C-PLB. Essentially, virtually all of the Ca2+ pump molecules were cross-linked to PLB with use of EMCS and KMUS (see below).
Time Course of Cross-linking Using Heterobifunctional Agents—Previously, we demonstrated that cross-linking of N30C-PLB to Cys318 of SERCA2a is relatively slow, with a t
of 15 min at room temperature (7). In contrast, cross-linking with heterobifunctional agents is much faster, occurring over a time course of seconds (Fig. 4). Specifically, when measured at 37 °C in Ca2+-free buffer, the t for cross-linking N27C-PLB to SERCA2a with EMCS was 42 s, and the t for cross-linking N30C-PLB to SERCA2a with KMUS was 13 s. In the results depicted in Fig. 4, microsomes had been preincubated in Ca2+-containing buffer to place SERCA2a in the E1 conformation prior to initiation of cross-linking reactions. Cross-linking was then initiated by simultaneously shifting SERCA2a to E2 with EGTA (arrow) at the time of addition of cross-linking agents. Identical t values were obtained by preincubating membranes in the absence of Ca2+ (data not shown), however, indicating that heterobifunctional cross-linking to monitor PLB/SERCA physical interactions is not fast enough to resolve either the conformational transitions of SERCA2a from E1 to E2 (Fig. 1), or the physical association of PLB with the E2 conformation. Although the former has been shown to occur within milliseconds (31), the latter has not been previously characterized.
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values for cross-linking N27C-PLB and N30C-PLB to SERCA2a were 110 and 36 s, respectively. Cross-linking reactions were further slowed down when conducted at 4 °C (Table I). These temperature-dependent effects on cross-linking suggest that PLB binding to SERCA2a may be membrane diffusion-limited at lower temperatures (32, 33).
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Nucleotide Effects—In addition to low Ca2+ levels, cross-linking of N30C-PLB to Cys318 of SERCA2a with BMH required the presence of the nucleotide ATP or ADP (7) (Fig. 6A, lower panel). Qualitatively identical results were obtained when EMCS and KMUS were used to couple N27C-PLB and N30C-PLB to SERCA2a, respectively (Fig. 6A). For both PLB mutants, cross-linking was increased 3–4-fold when ATP or ADP was present, whereas AMP was much less effective (Fig. 6B). These results provide additional evidence that PLB interacts preferentially with the Ca2+ pump when it is in the E2, nucleotide-bound conformation, but not in the E2, thapsigargin-inhibited conformation (7).
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SERCA2a Cross-linking Site Determination—To determine the Lys residue(s) of SERCA2a cross-linked to N27C-PLB and N30C-PLB by EMCS and KMUS, respectively, we followed the strategy developed in our previous work (7). SERCA2a was cross-linked to each PLB mutant on a large scale, then individually purified from insect cell microsomes by sequential 2A7-A1 and 2D12 monoclonal antibody affinity chromatographies, to yield the exclusively cross-linked complexes (7). During the purifications, we determined that 80% of the SERCA2a molecules in insect cell microsomes were cross-linked to N27C-PLB and N30C-PLB by EMCS and KMUS, respectively. Comparison of this exceptionally high cross-linking efficiency to the 20–40% efficiency previously reported for BMH by the same method (7) corroborates results shown in Figs. 3, 6, and 7. After purification, the cross-linked complexes were subjected to endo-Asp-N and endo-Lys-C digestions and the limit dipeptides were isolated and sequenced. The same 16-kDa limit dipeptide was obtained after EMCS and KMUS cross-linking, as was previously obtained after BMH cross-linking (7) (Fig. 8). Specifically, the dipeptides began at Val4 of the PLB sequence and at Asp254 of the SERCA2a sequence. Neither peptide extended beyond Lys352 (Fig. 8). Lys262 at the cytoplasmic face of M3 was excluded as the amino acid cross-linked to N27C-PLB or N30C-PLB because readable SERCA2a sequence ran past this residue (see Fig. 8 legend). Lys297 near the lumenal border of M4 of SERCA2a could be excluded, because it is located on the opposite side of the membrane from residues 27 and 30 of PLB, which project into the cytoplasm. This left Lys residues 328, 329, and 352 of SERCA2a as the candidate amino acids cross-linked to N27C-PLB and N30C-PLB.
To identify the cross-linked lysine, we individually mutated Lys328, Lys329, and Lys352 of WT-SERCA2a to Ala, and tested for loss of cross-linking function. K328A-SERCA2a and K329A-SERCA2a retained Ca-ATPase activity, whereas K352A-SERCA2a was catalytically inactive, as predicted from earlier mutagenesis work with SERCA1a (35). Fig. 9A shows that K329A-SERCA2a and K352A-SERCA2a retained the ability to cross-link efficiently to N27C-PLB and N30C-PLB, whereas K328A-SERCA2a lost all cross-linking ability. The inability of K328A-SERCA2a to cross-link to PLB was also reflected by its absence of an upward mobility shift, when the identical cross-linked samples were probed with the anti-SERCA2a antibody, 2A7-A1 (Fig. 9B). These results thus identify Lys328 of SERCA2a as the sole lysine residue (of 56 residues) cross-linked to N27C-PLB and N30C-PLB with EMCS and KMUS, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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20–40% of SERCA2a molecules to N30C-PLB in insect cell microsomes with t of 15 min, the heterobifunctional agents cross-linked 80% or greater of SERCA2a molecules in insect cell microsomes to PLB with a t of 13–42 s. Like cross-linking at Cys318 of SERCA2a (7), PLB cross-linking at Lys328 of SERCA was inhibited by micromolar Ca2+, by the anti-PLB monoclonal antibody 2D12, and by the Ca2+ pump inhibitor, thapsigargin. Furthermore, cross-linking at both Lys328 and Cys318 was strongly potentiated by the nucleotides ATP or ADP, but not by AMP (7). Taken together, these new cross-linking data support our previous conclusion that PLB interacts only with SERCA2a in the Ca2+-free, E2 state, with a strong preference for the nucleotide-bound, E2 state.
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-adrenergic signaling pathway is a relatively fast process, occurring over a time frame of seconds (10). This sets an upper limit on the time course of association and dissociation of PLB monomers and the Ca2+ pump, which are major targets in this signaling cascade (1, 2, 4). In our previous work with the homobifunctional cross-linker BMH, we observed rapid kinetics for cross-linking of PLB monomers to each other, which occurred within seconds or faster and was independent of Ca2+ concentration (7). This was consistent with homodimer formation within preformed pentamers. On the other hand, cross-linking of PLB monomers to SERCA2a in the same study was much slower, occurring with a t of 15 min at room temperature (7). This time scale for coupling between PLB and SERCA2a was too slow to account for the dynamic regulation of contraction in intact myocardium (10). The current cross-linking results with heterobifunctional agents have resolved this discrepancy and confirm that the two molecules associate over a time course of seconds or less, the current limitation being the time resolution of the experiments. Cross-linking of residue 30 of PLB to Lys328 of SERCA2a, for example, occurred with a t of 13 s at 37 °C. Most importantly, we obtained the same short t for cross-linking of N30C-PLB to SERCA2a, whether membranes were preincubated in Ca2+-containing buffer, in which PLB is presumed to be dissociated from SERCA2a prior to cross-linking, or in Ca2+-free buffer, in which PLB is already bound to SERCA2a at the start of the cross-linking reaction (7, 12). Identical t values imply that neither dissociation of PLB monomers from pentamers nor diffusion of monomers in the membrane and binding to SERCA2a are rate-limiting. Instead, the measured rate must be intrinsic to the cross-linking mechanism, perhaps reflecting the time required for proper alignment of Lys328 on the pump with residue 30 of PLB. Whereas Lys328 is well exposed at the top of the M4/S4 helix, Cys318 is buried in the interface between M4, M5, and M6 in the structure of the thapsigargin-inhibited, E2 state (15) (Fig. 10). Thus, although the ability of BMH to cross-link Cys318 (7) indicates a dynamism in the structure of M4, the slower time scale most likely reflects constraints in exposing this particular Cys residue. We observed that the rates of cross-linking N27C and N30C of PLB to SERCA2a were strongly temperature dependent, decreasing by 20–40-fold when measured at 4 °C (Table I). One explanation for rate reduction is that diffusion of PLB monomers within the plane of the membrane becomes limiting (32, 33). This is currently under investigation. On the other hand, local domain motions within SERCA2a may be drastically slowed by temperature, as is well known from kinetic studies (3). It is clear that the cross-linking reaction itself is not rate-limiting under any of the conditions or with any cross-linkers tested. For example, all reagents tested to date cross-linked PLB monomers within pentamers (7) within a few seconds or less, even at 4 °C (data not shown). This implies that the PLB pentamer is an abundant, stable structure in insect cell microsomes, with relevant residues favorably aligned to allow very fast cross-linking. In contrast, the PLB·SERCA2a heterodimer appears to be transient, with local motions of SERCA2a structural elements required for cross-linking, and presumably also for SERCA2a inhibition by PLB.
Ca2+ Dependence of Cross-linking—Consistent with previous results (7), heterobifunctional cross-linking of PLB to Lys328 of SERCA2a was completely inhibited by micromolar Ca2+, which provides additional strong evidence that binding of Ca2+ to SERCA2a causes PLB to dissociate from the Ca2+ pump. Similar conclusions have been made by others (11, 12). X-ray crystal structures show that M4 and M2 undergo large movements during the Ca2+-induced conformational change from E2 to E1 and the movement of M2 has been specifically postulated to displace PLB from its binding site (15, 20). The importance of M4 in the PLB·SERCA2a binding interaction was first suggested by successful cross-linking of PLB to Cys318 of SERCA2a (7), which has now been reaffirmed by cross-linking of Lys328. Both Cys318 and Lys328 are on the cytoplasmic extension of M4 (sometimes referred to as S4) (Fig. 10), movements of which are likely controlled by Ca2+ binding to Glu309. Interestingly, we found different KCa values for inhibition of PLB cross-linking to Cys318 versus Lys328 of SERCA2a, indicating that there are Ca2+ concentrations at which Lys328 can still be cross-linked efficiently but Cys318 cannot. This suggests that there are movements of M4/S4 during Ca2+ binding that differentially affect interactions with residues 27 and 30 of PLB. These movements must precede the more global conformational change to E1, which would involve M2 displacements and complete disruption of the PLB binding site.
Conformational Dependence of Cross-linking—Our use of heterobifunctional cross-linking agents confirms the binding preference of PLB for the E2 conformation of SERCA2a stabilized by nucleotides (7, 28, 38). The efficiency of cross-linking of N27C and N30C of PLB to Lys328 of SERCA2a in Ca2+-free medium was increased 3–4-fold by inclusion of nucleotides; no significant cross-linking at all of N30C-PLB to Cys318 of SERCA2a was detected unless nucleotides were included (7). This suggests a substantial conformational effect of ATP or ADP on the E2 state (28, 38), which is consistent with the well characterized effects of nucleotides on the E2 to E1 transition (39). In contrast to the effects of nucleotide, the specific inhibitor thapsigargin was found to completely prevent cross-linking of N27C and N30C to either Lys328 or Cys318 (7) of SERCA2a. This is unlikely to be a direct effect of this irreversible inhibitor at the PLB binding site, given the large distance between the thapsigargin-binding site and the S4 helix containing the cross-linked PLB residues (Fig. 10B), but instead appears to be related to the inhibitory effect of thapsigargin on ATP binding to E2 (28, 29), preventing formation of the state that preferentially binds PLB (7). Given this and other evidence for global effects of both nucleotides and thapsigargin on SERCA conformation (21, 29), it may be ill advised to use the x-ray structure of nucleotide-free, thapsigargin-inhibited SERCA as a basis for detailed modeling the interaction between PLB and SERCA2a (20).
Spatial Considerations—The lengths of spacer chains for heterobifunctional cross-linking agents used here suggest atomic distances of 10 and 15 Å between N27C and N30C of PLB and Lys328 of SERCA2a, respectively (Fig. 10A). Fig. 2B indicates that the radius of cross-linking of N27C of PLB to Lys328 of SERCA2a ranges from 4 to 15 Å, albeit with lower efficiency if more or less than 10 Å. Surprisingly, Lys329 was not cross-linked in the presence or absence of Ca2+, despite being well within range of most cross-linking agents used. This may be the result of a salt bridge formed between Lys329 and Glu748, which could serve to hold Lys329 away from the cross-linking agents (Fig. 10A). Nevertheless, Lys329 and Cys318 are on the same side of the S4 helix in the thapsigargin inhibited state (15), and the dynamics that allow cross-linking of N30C on PLB to Cys318 on SERCA2a might also be expected to expose Lys329. The failure to do so may be another indication that the structure of E2 in the thapsigargin-inhibited state (15) may not be appropriate for assessing its interaction with PLB (20). It is intriguing that the optimal cross-linking distances for residues 27 and 30 of PLB differed by 5 Å, because this is the difference one would expect if they existed on a continuous helix (Fig. 10A). Furthermore, their separation by three residues would place them on the same side of this helix, which would be consistent with their interaction with the same Lys of SERCA2a. This idea contradicts recent models of PLB interacting with SERCA2a (18–20), which assumed an unstructured region between PLB residues 21 and 30, the so-called domain IB (40). Although NMR structures suggest that the transmembrane helix continues without interruption into a helical domain IB (41, 42), the collision of this structure with S4 was recently cited as a rationale for unwinding domain IB in a model for the inhibitory interaction with SERCA (20). This rationale should not be considered absolute, given the use of the thapsigargin-inhibited, nucleotide-free structure of SERCA (15) for this modeling exercise. In addition, this unwinding was necessary (20) to allow the N terminus of PLB to reach the loop on SERCA2a containing Lys397 and Lys400, residues which have been previously implicated in interacting with PLB (11, 27), but were not successfully cross-linked to PLB here.
In particular, an earlier study by James et al. (11) reported that Lys3 of PLB cross-links to Lys397 or Lys400 of SERCA at a distance of 15 Å using purified, detergent-solubilized proteins and the Denny-Jaffe reagent (11). In the current study, we found no evidence for cross-linking of Lys3 of PLB to any SERCA2a residue using a variety of different cross-linking reagents. We did observe that Lys3 of PLB was able to cross-link to N27C or N30C of another PLB monomer causing homodimer formation (Fig. 2), confirming the reactivity of Lys3 with the cross-linking reagents and suggesting a large freedom of motion in the N terminus of PLB. Furthermore, our experimental system maintains well characterized functional coupling between PLB and SERCA2a in non-perturbed microsomal membranes (7, 8, 24, 26), and allows at least 10-fold higher cross-linking efficiency at several alternative sites from that suggested by James et al. (11). We therefore believe that the conclusions of James et al. (11) on cross-linking of Lys3 of PLB to Lys397 or Lys400 of SERCA2a should be viewed with caution. Moreover, it does not seem justified at this time to constrain three-dimensional models of the PLB·SERCA2a inhibitory complex (18–20, 40) with the physical proximity of these particular residues. Although residues 397–402 of SERCA2a are apparently required for functional coupling to PLB (27), this by no means demonstrates that these SERCA residues interact physically with Lys3 of PLB, as has been often suggested (14, 20, 40). Likewise, the data provided here and previously (7) provide no evidence for the proposed interaction of residues 27 and 30 of PLB with the loop between M6 and M7 of SERCA (40), an idea that was recently withdrawn (20), and point out the questionable utility of immunoprecipitation for identifying direct residue interactions between PLB and SERCA2a (40) as well as in characterizing thapsigargin effects (12).
Finally, it is interesting to note that our anti-SERCA2a monoclonal antibody, 2A7-A1, serendipitously bound to residues 386–396 of SERCA2a, amino acids that are directly contiguous to the 397–400 loop previously proposed to bind to Lys3 of PLB (11, 27). Nevertheless, the 2A7-A1 antibody had no effect on Ca2+-ATPase activity, on functional coupling to PLB, or on cross-linking of PLB to SERCA2a at different sites. In contrast, the anti-PLB monoclonal antibody, 2D12, which recognizes residues 9–13 of PLB and disrupts the functional interaction between PLB and SERCA2a by mimicking PLB phosphorylation (8), prevented the cross-linking of residues 27 and 30 of PLB to residues 318 (7) and 328 of SERCA2a. Conformational changes in the vicinity of this epitope are likely to result from phosphorylation of Ser16 during -adrenergic stimulation (43), and it will therefore be important in future experiments to establish the sites of interaction between this important region of PLB and the Ca2+ pump to understand the mechanism of regulation in a physiological context.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Krannert Institute of Cardiology, 1800 N. Capitol Ave., Indianapolis, IN 46202. Tel.: 317-962-0095; Fax: 317-962-0113; E-mail: lrjones{at}iupui.edu.
1 The abbreviations used are: PLB, phospholamban; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; SERCA2a, cardiac isoform of SERCA; SERCA1a, fast skeletal muscle isoform of SERCA; SR, sarcoplasmic reticulum; N27C-PLB and N30C-PLB, canine PLB with Asn27 and Asn30, respectively, replaced by Cys, and Cys residues 36, 41, and 46 replaced by Ala; MOPS, 3-(N-morpholino)propanesulfonic acid; NHS, N-hydroxysuccinimide; WT, wild-type; BMH, 1,6-bismaleimidohexane;
EMCS, N-(-maleimidocaproyloxy)succinimide ester; KMUS, N-(-maleimidoundecanoyloxy)sulfosuccinimide ester; E1, high Ca2+ affinity conformation of Ca2+-ATPase; E2, low Ca2+ affinity conformation of Ca2+-ATPase.
2 We should point out that Cys-less PLB is predominately a pentamer in insect cell microsomal membranes, even though it migrates primarily as a monomer under standard SDS-PAGE conditions (7). Cys-less PLB is an unstable pentamer, which totally dissociates at SDS concentrations greater than 1% (7).
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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0.01 by one-way analysis of variance). Color code is as indicated in A.
