Calmodulin Wraps around Its Binding Domain in the Plasma Membrane Ca2+ Pump Anchored by a Novel 18-1 Motif

Nenad Juranic,Elena Atanasova,Adelaida G Filoteo,Slobodan Macura,Franklyn G Prendergast,John T Penniston,Emanuel E Strehler

doi:10.1074/jbc.m109.060491

Nenad Juranic, Elena Atanasova + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m109.060491

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Abstract

Using solution NMR spectroscopy, we obtained the structure of Ca(2+)-calmodulin (holoCaM) in complex with peptide C28 from the binding domain of the plasma membrane Ca(2+)-ATPase (PMCA) pump isoform 4b. This provides the first atomic resolution insight into the binding mode of holoCaM to the full-length binding domain of PMCA. Structural comparison of the previously determined holoCaM.C20 complex with this holoCaM.C28 complex supports the idea that the initial binding step is represented by (holoCaM.C20) and the final bound complex by (holoCaM.C28). This affirms the existing multi-step kinetic model of PMCA4b activation by CaM. The complex exhibits a new binding motif in which holoCaM is wrapped around helical C28 peptide using two anchoring residues from the peptide at relative positions 18 and 1. The anchors correspond to Phe-1110 and Trp-1093, respectively, in full-length PMCA4b, and the peptide and CaM are oriented in an anti-parallel manner. This is a greater sequence distance between anchors than in any of the known holoCaM complexes with a helical peptide. Analysis of the geometry of holoCaM-peptide binding for the cases where the target peptide adopts an alpha(D)-helix with its anchors buried in the main hydrophobic pockets of the two CaM lobes establishes that only relative sequential positions of 10, 14, 17, and 18 are allowed for the second anchor.

Highlights

In the maintenance of overall intracellular Ca2ϩ homeostasis and in local Ca2ϩ signaling [1]
A good match for the CaM C-terminal lobe was found in the x-ray structure of a complex of holoCaM with the CBD of CaM-dependent protein kinase I (Protein Data Bank entry 1mxe [24]) and for the N-terminal lobe in the x-ray structure of free holoCaM (Protein Data Bank entry 1exr [25])
The NMR solution structure of the holoCaM1⁄7 C28 complex is an excellent surrogate for the holoCaM complex with the whole C-terminal tail (C-tail) from PMCA4b

Summary

EXPERIMENTAL PROCEDURES

CaM Expression and Purification—Human CaM cDNA was subcloned into the pET-15b expression vector Protein backbone RDCs were determined for the almost complete set of 1DNH (139 of 144 for CaM and 26 of 27 for C28; the C28 Trp side chain 1DNH was included in this set), and ϳ70% of 1DC’C␣and 1DC’N, all applied with an error limit of 0.5 Hz. The 3J(HNH␣) couplings were determined for ϳ90% of the residues (128 CaM and 25 C28) and were applied with an error limit of 2 Hz. Backbone H-bonds were assigned to all ␣-helix and ␤-sheet elements of secondary structure, as identified by NOE contacts and C␣ chemical shift index (63 for CaM and 17 for C28) and applied as distance constraints. Calculation of various structural parameters (distances, angles, and dihedrals) from atomic coordinates in the Protein Data Bank structures was performed in MATLAB using home-written programs. Data Deposition—The assigned chemical shift values, constraints, and atomic coordinates for the conformers were deposited at BioMagResBank (entry ID 16465) and Protein Data Bank (entry 2kne)

RESULTS AND DISCUSSION

Experimental constraints

RMSD from idealized covalent geometry Bonds Angles

Phe anchor site

Most of these parameters are fairly constant through the series of