Calcium signaling plays an essential role in plant cell physiology, and chaperone-mediated protein folding directly regulates plant programmed cell death. The Arabidopsis thaliana protein AtBAG5 (Bcl-2-associated athanogene 5) is unique in that it contains both a BAG domain capable of binding Hsc70 (Heat shock cognate protein 70) and a characteristic IQ motif that is specific for Ca2+-free CaM (Calmodulin) binding and hence acts as a hub linking calcium signaling and the chaperone system. Here, we determined crystal structures of AtBAG5 alone and in complex with Ca2+-free CaM. Structural and biochemical studies revealed that Ca2+-free CaM and Hsc70 bind AtBAG5 independently, whereas Ca2+-saturated CaM and Hsc70 bind AtBAG5 with negative cooperativity. Further in vivo studies confirmed that AtBAG5 localizes to mitochondria and that its overexpression leads to leaf senescence symptoms including decreased chlorophyll retention and massive ROS production in dark-induced plants. Mutants interfering the CaM/AtBAG5/Hsc70 complex formation leads to different phenotype of leaf senescence. Collectively, we propose that the CaM/AtBAG5/Hsc70 signaling complex plays an important role in regulating plant senescence.

Store-operated Ca2+ entry (SOCE) is a critical Ca2+ signaling pathway in many cell types. After sensing Ca2+ store depletion in the endoplasmic reticulum (ER) lumen, STIM1 (STromal Interaction Molecule 1) oligomerizes and then interacts with and activates the Orai1 calcium channel. Our previous research has demonstrated that the inhibitory helix (IH) adjacent to the first coiled-coil region (CC1) of STIM1 may keep the whole C-terminus of STIM1 in an inactive state. However, the specific conformational change of CC1-IH that drives the transition of STIM1 from the resting state to the active state remains elusive. Herein, we report the structural analysis of CC1-IH, which revealed that the entire CC1-IH molecule forms a very long helix. Structural and biochemical analyses indicated that IH, and not the CC1 region, contributes to the oligomerization of STIM1. Small-angle X-ray scattering (SAXS) analysis suggested that the C-terminus of STIM1 including the IH region displays a collapsed conformation, whereas the construct without the IH region has an extended conformation. These two conformations may correspond to the conformational states of the C-terminus of STIM1 before and after activation. Taken together, our results provide direct biochemical evidence that the IH region controls the conformational switching of the C-terminus of STIM1.

The recently identified plant Bcl-2-associated athanogene (BAG) family plays an extensive role in plant programmed cell death (PCD) processes ranging from growth and development to stress responses and even cell death. In the Arabidopsis thaliana BAG (AtBAG) protein family, four members (AtBAG1-4) have a domain organization similar to that of mammalian BAG proteins. Here, crystal structures of the BAG domains (BDs) of AtBAG1-4 have been determined; they have high homology and adopt a structure comprising three short parallel α-helices, similar to some mammalian BAG proteins. The crystal structure of a complex of the AtBAG1 ubiquitin-like domain and BAG domain (UBD) with the Hsc70 nucleotide-binding domain (NBD) was also determined. The binding of the AtBAG1 BD to the Hsc70 NBD induces conformational change of the Hsc70 NBD to the open state and reduces the affinity of the NBD for ADP. In vivo studies showed that bag2-1 mutant plants are larger than wild-type plants when growing under normal conditions, indicating that the AtBAG proteins might regulate plant PCD and confer tolerance to stresses in plants. These structural and functional analyses indicate that the AtBAG proteins function as nucleotide-exchange factors for Hsp70/Hsc70 in A. thaliana and that the mechanism of regulation of chaperone-mediated protein folding is conserved in plants.

Arabidopsis thaliana BAG5 (AtBAG5) belongs to the plant BAG (Bcl-2-associated athanogene) family that performs diverse functions ranging from growth and development to abiotic stress and senescence. BAG family members can act as nucleotide-exchange factors for heat-shock protein 70 (Hsp70) through binding of their evolutionarily conserved BAG domains to the Hsp70 ATPase domain, and thus may be involved in the regulation of chaperonemediated protein folding in plants. AtBAG5 is distinguished from other family members by the presence of a unique IQ motif adjacent to the BAG domain; this motif is specific for calmodulin (CaM) binding, indicating a potential role in the plant calcium signalling pathway. To provide a better understanding of the IQ motif-mediated interaction between AtBAG5 and CaM, the two proteins were expressed and purified separately and then co-crystallized together. Diffractionquality crystals of the complex were grown using the sitting-drop vapourdiffusion technique from a condition consisting of 0.1 M Tris-HCl pH 8.5, 2.5 M ammonium sulfate. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 64.56, b = 74.89, c = 117.09 Å . X-ray diffraction data were recorded to a resolution of 2.5 Å from a single crystal using synchrotron radiation. Assuming the presence of two molecules in the asymmetric unit, a Matthews coefficient of 2.44 Å 3 Da À1 was calculated, corresponding to a solvent content of approximately 50%.

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