The Solution Structure of a Plant Calmodulin and the CaM-binding Domain of the Vacuolar Calcium-ATPase BCA1 Reveals a New Binding and Activation Mechanism

Abstract

The type IIb class of plant Ca 2؉ -ATPases contains a unique N-terminal extension that encompasses a calmodulin (CaM) binding domain and an auto-inhibitory domain. Binding of Ca 2؉ -CaM to this region can release auto-inhibition and activates the calcium pump. Using multidimensional NMR spectroscopy, we have determined the solution structure of the complex of a plant CaM isoform with the CaM-binding domain of the well characterized Ca 2؉ -ATPase BCA1 from cauliflower. The complex has a rather elongated structure in which the two lobes of CaM do not contact each other. The anchor residues Trp-23 and Ile-40 form a 1-8-18 interaction motif. Binding of Ca 2؉ -CaM gives rise to the induction of two helical parts in this unique target peptide. The two helical portions are connected by a highly positively charged bend region, which represents a relatively fixed angle and positions the two lobes of CaM in an orientation that has not been seen before in any complex structure of calmodulin. The behavior of the complex was further characterized by heteronuclear NMR dynamics measurements of the isotope-labeled protein and peptide. These data suggest a unique calcium-driven activation mechanism for BCA1 and other plant Ca 2؉ -ATPases that may also explain the action of calcium-CaM on some other target enzymes. Moreover, CaM activation of plant Ca 2؉ -ATPases seems to occur in an organelle-specific manner.The calcium ion (Ca 2ϩ ) is intimately involved as a secondary messenger in numerous signal transduction processes. Elevation of the cytoplasmic calcium levels leads to a variety of physiological responses in plants (1). Various stimuli such as abiotic stresses, hormones, wounds, and plant pathogens can trigger an influx of Ca 2ϩ into the cytoplasm. Calcium signaling is well known to play a role in plant defense signaling pathways (2). The transient Ca 2ϩ signals are translated into a physiological response by a variety of Ca 2ϩ -binding proteins including the ubiquitous Ca 2ϩ -sensor protein calmodulin (CaM) 3 (3, 4). Unlike animals, which have only one form of the CaM protein, multiple CaM proteins occur in higher plant species (3, 5). For example, the model plant Arabidopsis thaliana, harbors nine CaM genes (AtCaM1-9) coding seven different CaMs (5, 6). Likewise, the plant Glycine max (soybean) contains not less than five CaM genes (SCaM1-5) coding for four distinct CaMs (7), whereas Oryza sativa (rice) also has at least five CaMs (8). Several studies have demonstrated that each CaM isoform is utilized to control different enzymes that are involved in specific physiological reactions (9, 10). In addition to multiple CaM isoforms, plants also contain up to 50 CaM-like proteins that have so far received limited attention (8, 11).The cytosolic Ca 2ϩ concentration is restored immediately after a stimulus to a low resting level by removing Ca 2ϩ across the plasma membrane or by sequestering it into intercellular organelles, such as the vacuole, the endoplasmic reticulum, or the Golgi apparatus. Plant Ca 2ϩ -pumps, which are ...

Solution Structure of Calmodulin Bound to the Binding Domain of the HIV-1 Matrix Protein

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Structural diversity of calmodulin binding to its target sites

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Calmodulin and STIM proteins: Two major calcium sensors in the cytoplasm and endoplasmic reticulum

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