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 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.
Highlights
PLB1 is a small phosphoprotein modulator of the Ca2ϩ-pump (SERCA2a) in cardiac SR, which is critically involved in regu
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
Earlier models of PLB1⁄7SERCA2a 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
Summary
Materials—All of the cross-linking agents used were obtained from Pierce. The heterobifunctional cross-linkers were: N-(␣-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). Identification of Cross-linked Lys Residue of SERCA2a—SERCA2a cross-linked to N27C-PLB with EMCS, or to N30C-PLB with KMUS, was purified to homogeneity from insect cell microsomes in separate runs by sequential anti-SERCA2a (2A7-A1) and anti-PLB (2D12) monoclonal affinity chromatographies, as recently described in detail (7). Cross-linked Ca2ϩ pump was digested with endo-Asp-N and endo-Lys-C, and the limit SERCA2a fragment crosslinked to PLB was isolated by a second round of 2D12 chromatography and sequenced, as described (7). By this analysis, the cross-linked amino acid of SERCA2a was restricted to just three potential Lys residues (see Fig. 8).
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