Structure-function relationships in proteins are predicated on the spatial proximity of noncovalently interacting groups of atoms. Thus, structural elements located away from a protein's active site are typically presumed to serve a stabilizing or scaffolding role for the larger structure. Here we report a functional role for a distal structural element in a PDZ domain, even though it is not required to maintain PDZ structure. The third PDZ domain from PSD-95/SAP90 (PDZ3) has an unusual additional third alpha helix (␣3) that packs in contiguous fashion against the globular domain. Although ␣3 lies outside the active site and does not make direct contact with C-terminal peptide ligand, removal of ␣3 reduces ligand affinity by 21-fold. Further investigation revealed that the difference in binding free energies between the full-length and truncated constructs is predominantly entropic in nature and that without ␣3, picosecond-nanosecond side-chain dynamics are enhanced throughout the domain, as determined by 2 H methyl NMR relaxation. Thus, the distal modulation of binding function appears to occur via a delocalized conformational entropy mechanism. Without removal of ␣3 and characterization of side-chain dynamics, this dynamic allostery would have gone unnoticed. Moreover, what appeared at first to be an artificial modification of PDZ3 has been corroborated by experimentally verified phosphorylation of ␣3, revealing a tangible biological mechanism for this novel regulatory scheme. This hidden dynamic allostery raises the possibility of as-yet unidentified or untapped allosteric regulation in this PDZ domain and is a very clear example of function arising from dynamics rather than from structure.NMR ͉ PSD-95 ͉ spin relaxation ͉ entropy P roteins owe their functionality to the 3-dimensional arrangement of atoms. A typical protein's structure stabilizes its active site, allowing for specific interactions with substrate or ligand. These basic structure-function relationships are well understood for countless types of proteins. Because most active sites are relatively small, it has been presumed that the remaining bulk of the globular structure provides a scaffolding role. Thus, even though similar domains belonging to the same family may have substrate specificity preferences, the folds of those domains are composed of invariant structural elements (1). Nonetheless, variations in tertiary fold composition, such as additional elements of secondary structure, are not uncommon. An example of this can be seen within the PDZ domain family of proteins. From this, the question of whether there is a specific role for such auxiliary structural elements remains open. In other words, how might these additional elements influence the core domain?PDZ domains (eg, PSD-95, Discs Large, Zo-1) are small, Ϸ90-aa modular structures that typically bind C-terminal tails (Ϸ4-6 residues) of target proteins (2). They are frequently found in multiple copies in proteins with diverse functions, especially those involved in signal transduction c...

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Internal motion is central to protein folding, to protein stability through the resulting residual entropy, and to protein function. Despite its importance, the precise nature of the internal motions of protein macromolecules remains a mystery. Here we report a survey of the temperature dependence of the fast dynamics of methyl-bearing side chains in a calmodulin-peptide complex using site-specific deuterium NMR relaxation methods. The amplitudes of motion had a markedly heterogeneous spectrum and segregated into three largely distinct classes. Other proteins studied at single temperatures tend to segregate similarly. Furthermore, a large variability in the degree of thermal activation of the dynamics in the calmodulin complex indicates a heterogeneous distribution of residual entropy and hence reveals the microscopic origins of heat capacity in proteins. These observations also point to an unexpected explanation for the low-temperature 'glass transition' of proteins. It is this transition that has been ascribed to the creation of protein motional modes that are responsible for biological activity.