Spatial and Dynamic Interactions between Phospholamban and the Canine Cardiac Ca2+ Pump Revealed with Use of Heterobifunctional Cross-linking Agents

Abstract

Heterobifunctional thiol to amine cross-linking agents were used to gain new insights on the dynamics and conformational factors governing the interaction between the cardiac Ca 2؉ pump (SERCA2a) and phospholamban (PLB). PLB is a small protein inhibitor of SERCA2a that reduces enzyme affinity for Ca 2؉ and thereby regulates cardiac contractility. We found that the PLB monomer with Asn 27 or Asn 30 changed to Cys (N27C-PLB or N30C-PLB) cross-linked to lysine of SERCA2a within seconds with >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 (Ca 2؉ -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 Lys 328 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 Cys 318 of SERCA2a. In contrast, Lys 3 of PLB did not cross-link to any Lys (or Cys) of SERCA2a, suggesting that previous three-dimensional models that constrain Lys 3 near residues 397-400 of thapsigargin-inhibited SERCA2a should be viewed with caution. Furthermore, although earlier models of PLB⅐SERCA2a 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. PLB1 is a small phosphoprotein modulator of the Ca 2ϩ -pump (SERCA2a) in cardiac SR, which is critically involved in regulating the strength and duration of the heartbeat (1, 2). In the dephosphorylated state, PLB inhibits the Ca 2ϩ -ATPase by decreasing its apparent affinity for Ca 2ϩ (3). Phosphorylation of PLB at Ser 16 and Thr 17 during ␤-adrenergic stimulation of the heart reverses PLB inhibition and augments Ca 2ϩ 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, Ca 2ϩ 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 inhibit...

Superinhibitory Phospholamban Mutants Compete with Ca2+ for Binding to SERCA2a by Stabilizing a Unique Nucleotide-dependent Conformational State

“…Unfortunately, accurate measurement of Ca 2ϩ binding affinity with 45 Ca 2ϩ requires relatively high expression levels of the Ca 2ϩ -ATPase (13), which is difficult to achieve in recombinant systems (14). As an alternative, we have demonstrated that the Ca 2ϩ affinity of the enzyme is accurately estimated by assaying Ca 2ϩ inhibition of PLB cross-linking to SERCA2a (7,10). For example, N30C of PLB cross-links to Lys 328 of SERCA2a with the heterobifunctional cross-linker KMUS, and PLB cross-linking is inhibited by micromolar Ca 2ϩ concentration over the same concentration range (K i ϳ 0.3 M) as enzyme activation occurs (K Ca ϳ 0.3 M).…”

“…For consistency with previous cross-linking studies, N30C-PLB was made on the Cys-less PLB background, in which Cys residues 36, 41, and 46 were mutated to Ala (7,10). N30C-PLB has been previously well characterized, and is fully functional with an inhibitory potency similar to wild-type PLB (7,10). In control experiments, ATPase activity by forming a dead-end complex with the enzyme in E2 (E2⅐TG) (18).…”

“…PLB cross-linking studies indicate that PLB binds preferentially to E2 with bound ATP (E2⅐ATP⅐PLB). PLB does not bind to E2⅐TG or E2⅐cyclopiazonic acid (7,10) 17 , the residues phosphorylated in response to ␤-adrenergic stimulation. The point mutations N27A in domain IB (15) and L37A (3,4) and V49G (16,17) in domain II are all supershifting mutations.…”

“…cDNAs encoding PLB3 and PLB4 were generated on the wild-type PLB cDNA background inserted in the transfection vector pVL1393, using the QuikChange TM XL-Gold system (Stratagene). D351A was made similarly using canine cardiac SERCA2a cDNA as the template (10). All mutated cDNAs were confirmed by DNA sequencing of the plasmid vectors.…”

“…Unfortunately, accurate measurement of Ca 2ϩ binding affinity with 45 Ca 2ϩ requires relatively high expression levels of the Ca 2ϩ -ATPase (13), which is difficult to achieve in recombinant systems (14). As an alternative, we have demonstrated that the Ca 2ϩ affinity of the enzyme is accurately estimated by assaying Ca 2ϩ inhibition of PLB cross-linking to SERCA2a (7,10 …”