Intramolecular Fluorescence Resonance Energy Transfer between Fused Autofluorescent Proteins Reveals Rearrangements of the N- and C-terminal Segments of the Plasma Membrane Ca2+ Pump Involved in the Activation

Abstract

The blue and green fluorescent proteins (BFP and GFP) have been fused at the N-and C-terminal ends, respectively, of the plasma membrane Ca 2؉ pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca 2؉ -ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45 Å . The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca 2؉ -calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation.The homeostasis of the intracellular Ca 2ϩ is crucial for cell function. The Ca 2ϩ ATPases from plasma membrane (PMCAs) 2 participate in the modulation of Ca 2ϩ signals and are responsible for the long term maintenance of the low concentration of intracellular Ca 2ϩ (1). The PMCAs belong to the P2-type ATPase superfamily of ion pumps and form an aspartyl phosphate intermediate during the transport cycle (2). Another essential feature of these ATPases is their ability to switch between two different conformational states from E 2 to E 1 in the presence of the transported ion.Human PMCAs are encoded by four separate genes, and additional variants are generated via alternative splicing of primary gene transcripts. PMCA4 is found virtually in all human tissues, and the splice variant xb is the most studied isoform. Computer modeling and sequence comparisons indicate that the overall structure of the PMCAs closely resembles that of other P-ATPases. Following the domain organization proposed for the SERCA (3, 4), the PMCA would contain a transmembrane region of 10 segments (M1-M10) and three major catalytic domains exposed to the cytosol. The nucleotide-binding (N) and the phosphorylation (P) domains contain the ATP binding site and the aspartate residue that forms the acyl phosphate intermediate, respectively, whereas the actuator domain (A) plays an essential role in the long range transmission of the conformational changes occurring during the transport cycle.Despite the clear overall homology, certain amino acid segments of the PMCA protein are not found in SERCA. The existence of these segments in the PMCA molecule has been generally associated with the extensive regulatory mechanisms that are known to alter the function of the PMCA. Indeed, the major difference between the two calcium pumps is the long C-terminal segment (C region) of the PMCA following M10, and this region is in...

Hyperactivation of the Human Plasma Membrane Ca2+ Pump PMCA h4xb by Mutation of Glu99 to Lys

“…On the other hand, mutations of specific residues in the linker segments connecting the A domain with M1, M2, and M3 have been shown to generate deregulated ACA pumps, indicating that these structures are particularly important for autoinhibition (14). Previous work from our laboratory has indicated that also the N-terminal part of the PMCA molecule is involved in autoinhibition (15). Here we present results indicating that residue Glu 99 in the acidic cluster at the N-terminal portion of the M1 transmembrane segment and the C-terminal segment of the h4xb PMCA are critical for decreasing the V max in the autoinhibited state.…”

A New Conformation in Sarcoplasmic Reticulum Calcium Pump and Plasma Membrane Ca2+ Pumps Revealed by a Photoactivatable Phospholipidic Probe

“…This pump is an integral part of the Ca 2ϩ signaling mechanism (8). It is highly regulated by calmodulin, which activates this protein by binding to an auto-inhibitory region and changing the conformation of the pump from an inhibited state to an activated one (8,9). Crystallization of PMCA is particularly challenging because there is no natural source from which this protein can be obtained in large quantities.…”

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

“…This pump is an integral part of the Ca 2ϩ signaling mechanism (1) and is thus a crucial component of cell function. It is highly regulated by calmodulin (CaM), which activates this protein by binding to an auto-inhibitory region (2) and changes the conformation of the pump from an inhibited state to an activated one (2,3).…”