Conformational Dynamics of a Cysteine-Stabilized Plant Defensin Reveals an Evolutionary Mechanism to Expose Hydrophobic Residues.

Sugar cane defensin 5 (Sd5) is a small antifungal protein, whose structure is held together by four conserved disulfide bridges. Sd5 and other proteins sharing a cysteine-stabilized α-ß (CSαß) fold lack a regular hydrophobic core. Instead, they are stabilized by tertiary contacts formed by surface-exposed hydrophilic and hydrophobic residues. Despite excessive cross-links, Sd5 exhibits complex millisecond conformational dynamics involving all secondary structure elements. We used Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion (RD) measurements performed at different temperatures and denaturant concentrations to probe brief excursions of Sd5 to a sparsely populated "excited" state. Temperature-dependent CPMG RD experiments reveal that the excited state is enthalpically unfavorable, suggesting a rearrangement of stabilizing contacts formed by surface-exposed side chains and/or secondary structure, while the experiments performed at different denaturant concentrations suggest a decrease in accessible surface area of Sd5 in the excited state. The measured backbone 15N chemical shift changes point to a global conformational rearrangement such as a potential α- to ß-transition of the Sd5 α-helix or other major secondary structure reorganization and concomitant conformational changes in other parts of the protein. Overall, the emerging picture of Sd5 dynamics suggests this protein can populate two alternative well-ordered conformational states, with the excited conformer being more compact than the native state and having a distinct secondary structure and side-chain arrangements. The observation of an energetically unfavorable yet more compact excited state reveals a remarkable evolution of the CSαß fold to expose and reorganize hydrophobic residues, which enables the creation of versatile binding sites.