Structural Studies of Soybean Calmodulin Isoform 4 Bound to the Calmodulin-binding Domain of Tobacco Mitogen-activated Protein Kinase Phosphatase-1 Provide Insights into a Sequential Target Binding Mode

Abstract

The calcium regulatory protein calmodulin (CaM) binds in a calcium-dependent manner to numerous target proteins. The calmodulin-binding domain (CaMBD) region of Nicotiana tabacum MAPK phosphatase has an amino acid sequence that does not resemble the CaMBD of any other known Ca 2؉ -CaMbinding proteins. Using a unique fusion protein strategy, we have been able to obtain a high resolution solution structure of the complex of soybean Ca 2؉ -CaM4 (SCaM4) and this CaMBD. Complete isotope labeling of both parts of the complex in the fusion protein greatly facilitated the structure determination by NMR. The 12-residue CaMBD region was found to bind exclusively to the C-lobe of SCaM4. A specific Trp and Leu side chain are utilized to facilitate strong binding through a novel ''double anchor'' motif. Moreover, the orientation of the helical peptide on the surface of Ca 2؉ -SCaM4 is distinct from other known complexes. The N-lobe of Ca 2؉ -SCaM4 in the complex remains free for additional interactions and could possibly act as a calcium-dependent adapter protein.Signaling through the MAPK pathway and increases in intracellular Ca 2؉ are both hallmarks of the plant stress response, and our data support the notion that coordination of these responses may occur through the formation of a unique CaM-MAPK phosphatase multiprotein complex. Calmodulin (CaM)2 is a ubiquitous intracellular Ca 2ϩ sensor protein that plays an essential role in various Ca 2ϩ signaling pathways. Contiguous and unique CaM-binding domain (CaMBD) regions are found widely distributed in many different types of CaM target proteins including protein phosphatases and kinases, cytoskeletal proteins, ion channels, and pumps (1, 2). Even though the CaMBD from various proteins share relatively poor amino acid sequence similarity, the majority of CaMBD become ␣-helical upon binding to CaM, and they can be grouped into either the 1-5-10 or the 1-8-14 motif, where the first and the last number indicate the position of two hydrophobic anchor residues that attach the CaMBD to the two binding pockets of the bilobal Ca 2ϩ -CaM. However, several noncanonical classes of CaMBD have also been identified. For example, in the CaMBD of the MARCKS protein, the two anchor residues are separated by a single amino acid residue (3). On the other hand, in the recently determined crystal structure of Ca 2ϩ -CaM complexed with the CaMBD of the skeletal muscle ryanodine receptor RYR1, they are separated by 15 residues (1-17 motif) (4). Bulky hydrophobic side chains of residues such as Trp, Leu, Phe, and Ile are most commonly utilized as anchor residues (see Fig. 1a), and these are usually deeply inserted into the hydrophobic target-binding pocket of either the N-or C-lobe of Ca 2ϩ -CaM. However, in several cases, including the N-methyl-D-aspartate receptors (NMDAR) (5) and the voltage-gated Ca 2ϩ channels (Cav1-2) (6, 7), a polar side chain from Thr or Tyr has also been found to act as an anchor residue. In almost all Ca 2ϩ -CaM complexes studied to date, the two lobes of Ca 2ϩ -CaM bec...

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Ca2+signals: The versatile decoders of environmental cues

“…When this CAMBD forms complex with soybean Ca 2+ -CaM4 (SCaM4), the fusion protein revealed a 12-residue CaMBD region, which binds exclusively to the C-lobe of SCaM4. A novel "double anchor" motif is created by specific Trp and Leu side chain, thus facilitating much strong binding (Ishida et al, 2009).…”

Structural Insights into Calmodulin-regulated L-selectin Ectodomain Shedding

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