PNNL

Abstract

We have investigated the functional role of the flexible hinge region centered near the sequence TIEMP21, which connects the N-terminal cytosolic and C-terminal membrane-spanning helical domains of phospholamban (PLB). Specifically, we ask if the conformation of this region is important to attaining optimal inhibitory interactions with the Ca-ATPase. A genetically engineered PLB mutant was constructed in which Pro21 was mutated to an alanine (P21A-PLBC); in this construct all three transmembrane cysteines were substituted with alanines to stabilize the monomeric form of PLB and a unique cysteine was introduced at position 24 near the hinge element (A24C), permitting the site-specific attachment of fluorescein-5-maleimide (FMal) to monitor structure changes. In agreement with prior measurements in cardiac SR microsomes, the calcium concentration associated with half-maximal activation (Ca1/2) of the Ca-ATPase, 290 ± 10 nM, is shifted to 580 ± 20 nM when co-reconstituted with PLBC (Pro21) as a result of a 75% reduction in the rate of formation of the second high-affinity calcium binding site associated with calcium activation. In comparison, there is a 43% reduction in ?Ca1/2 upon reconstitution of the Ca-ATPase with P21A-PLBC, which can be simulated by decreasing the rate constant associated with calcium activation by 50%. The diminished inhibitory action of P21A-PLBC is associated with alterations in the structure of the hinge element, as evidenced by the diminished solvent accessibility of FMal relative to the native structure. Likewise, increases in the a-helical content and decreases in the mobility of the carboxyl-terminal domain of P21A-PLBC are observed using circular dichroism and fluorescence spectroscopy. Collectively, these results indicate that the overall dimensions of the carboxyl-terminal domain of PLB are increased through a stabilization of secondary structural elements upon mutation in P21A-PLBC that result in a reduction in the ability of the amino-terminal cytosolic portion of PLB to productively inhibit the Ca-ATPase. Further, these results suggest that the unstructured characteristics of the flexible hinge region in PLB are critical for optimal inhibitory interactions with the Ca-ATPase and suggest its role as a conformational switch.