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

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 Ca2+ pump (SERCA2a) and phospholamban (PLB). PLB is a small protein inhibitor of SERCA2a that reduces enzyme affinity for Ca2+ and thereby regulates cardiac contractility. We found that the PLB monomer with Asn27 or Asn30 changed to Cys (N27C-PLB or N30C-PLB) cross-linked to lysine of SERCA2a within seconds with > or =80% efficiency. Optimal cross-linking occurred at spacer chain lengths of 10 and 15 A 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-A 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.