Hidden dynamic allostery in a PDZ domain

Abstract

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...

Extensions of PDZ domains as important structural and functional elements

“…NMR relaxation measurements of the protein dynamics were apparently uninformative, showing that backbone motion remains largely unaffected after truncation. However, the key result from the dynamics study is that side-chain flexibility increases significantly for the truncated domain, but can be restored to a state like that of the extended domain upon ligand binding, suggesting that affinity reduction is mainly an effect of sidechain entropy (Petit et al, 2009). Thus, for PSD-95 PDZ3, the helical extension indirectly affects binding affinity by specific modulation of side-chain dynamics.…”

“…Isothermal titration calorimetry showed that this reduction is entirely entropic in nature-an effect which cannot be easily explained by differences in solvation because the additional helix does not form part of the ligand binding pocket (Petit et al, 2009). How then can one explain this entropy difference?…”

Long-Range Conformational Transition of a Photoswitchable Allosteric Protein: Molecular Dynamics Simulation Study

“…For example in the PSD-95 protein, three connected PDZ domains are followed by a SH3 domain and a guanylate kinase domain. 20 Nevertheless, there are examples for which a relatively well defined mechanism can be identified, such as in recent NMR experiments by Petit et al 13 for PDZ3. They showed that the affinity of ligand-binding in PDZ3 is indeed directly related to the conformational motion of a small additional α-helix attached to the C-terminus.…”

“…A well-studied example for systems showing ligand-induced allostery are modular domains for protein-protein interactions denoted as PSD95/Discs large/ZO-1 (PDZ) domains. [11][12][13][14][15][16][17][18][19][20] These domains occur in a set of proteins typically associated with cell junctions and mediate the clustering of membrane ion channels by binding to their C-termini. [15][16][17] PDZ domains share a common fold which consists of two α-helices and six β -strands, with the second α-helix and the second β -strand forming the canonical binding groove ( Figure Figure 1).…”

NMR reveals novel mechanisms of protein activity regulation

“…[33][34][35] Numerous examples are currently available highlighting the inextricable link between protein dynamics and allostery 8,36 . Long-range allosteric coupling between sites through changes in internal dynamics were seen between the nucleotide-binding cleft and the preprotein-binding site in SecA ATPase, 37 between the coordination Na þ site and exosite I in thrombin that regulates enzyme's specificity, 38 between the substrate-binding site and distal loop in dihydrofolate reductase, 39 between the mixed lineage leukemia)-and c-Myb binding sites of the KIX domain of CREB-binding protein, 40 in PDZ signaling domains, 41 the serine protease inhibitor eglin c, 42 upon binding of barstar to the RNase barnase, 43 in the interaction between the Rho GTPasebinding domain and Rac1, 44 upon cyclic nucleotide binding to the exchange protein activated by cAMP, 45,46 a in V-type allosteric enzyme, 47 and in protein kinase A, 48,49 only to mention few recent examples from a long list of systems characterized over the years. Dynamic changes in these systems were accompanied by varying extent of structural changes, ranging from minimal to substantial.…”