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GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

Exposed Loop Domains of Complexed 14-3-3 Proteins Contribute to Structural Diversity and Functional Specificity¹

Program in Plant Molecular and Cellular Biology, Department of Horticultural Science (P.C.S., B.L., R.J.F.), and Department of Chemistry (H.C., D.P.), University of Florida, Gainesville, Florida 32611

The 14-3-3 family of proteins functions through protein:phosphoprotein interactions, the nature of which has been elucidated using x-ray crystallography. However, some key structural features in nonconserved regions have yet to be fully resolved, leaving open questions regarding the functional selectivity of 14-3-3 family members for diverse clients. In an effort to study surface accessible structural features in 14-3-3 containing macromolecular complexes and to illuminate important structure/function variations among the 14-3-3 isoforms, we determined the epitopes for three unique monoclonal antibodies (mAbs) developed against the Arabidopsis (Arabidopsis thaliana) G-box DNA:protein complex. The epitopes mapped to different loops in a phylogenetically important subset of the 13 14-3-3 family members. All three epitopes were on a common exposed face of complexed 14-3-3s. Two of the mAbs recognized linear sequences within loops 5 and 6, while the third mAb recognized 14-3-3 residues surrounding the pivotal medial Gly in the divalent cation-binding domain of loop 8, together with distal residue(s) in the putative dynamic 10th helix that has yet to be determined by crystallography. Gly at this loop 8 position is unique to nonepsilon 14-3-3 isoforms of the plant kingdom, suggesting that this region constitutes a plant-specific key functional 14-3-3 feature and highlighting that the loop 8 region is functionally significant. Mutagenesis of the medial amino acid in the loop 8 domain changed the flexibility of the C terminus and altered client peptide-binding selectivity, demonstrating the functional significance of the surface accessible, evolutionarily distinct loop 8 domain.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Robert J. Ferl (robferl{at}ufl.edu).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.073916.

^* Corresponding author; e-mail robferl{at}ufl.edu; fax 352–392–4072.

Received November 7, 2005; returned for revision November 7, 2005; accepted December 6, 2005.

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