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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Hydrostatic pressure is used to perturb the manifold of states available to apocytochrome b562 and to examine the energetics and dynamics of the protein using hydrogen exchange monitored in real-time by heteronuclear spectroscopy at pressures ranging up to 1. 1 kbar. An analytical framework for interpreting the effects of hydrostatic pressure on the physical events leading to protein hydrogen exchange is presented. The protein is found to have three regions of subglobal cooperative stability. The most stable region, or core, is composed of the central two helices of the bundle. The dependence of the global unfolding free energy upon pressure is first order and associated with a negative volume change of -102 mL mol-1. Two additional regions of cooperative structure are identified, and both also have negative volume changes associated with their unfolding at high pressure. Surprisingly, one of the subglobal unfolding units shows a significant positive volume change at low pressures (<200 bar) suggesting the presence of a highly mispacked open state at ambient pressure. The three regions of cooperative stability are the same as identified by perturbation with chemical denaturant. The implications of these results for issues in protein folding and the form of the energy landscape of globular proteins are discussed.

The T-cell lymphoma invasion and metastasis gene 1 (Tiam1) is a guanine exchange factor (GEF) for the Rho-family GTPase Rac1 that is crucial for the integrity of adherens junctions, tight junctions, and cell-matrix interactions. This GEF contains several protein-protein interaction domains, including a PDZ domain. Earlier studies identified a consensus PDZ-binding motif and a synthetic peptide capable of binding to the Tiam1 PDZ domain, but little is known about its ligand specificity and physiological role in cells. Here, we investigated the structure, specificity, and function of the Tiam1 PDZ domain. We determined the crystal structures of the Tiam1 PDZ domain free and in complex with a “model” peptide, which revealed the structural basis for ligand specificity. Protein database searches using the consensus PDZ-binding motif identified two eukaryotic cell adhesion proteins, Syndecan1 and Caspr4, as potential Tiam1 PDZ domain binding proteins. Equilibrium binding experiments confirmed that C-terminal peptides derived from Syndecan1 and Caspr4 bound the Tiam1 PDZ domain. NMR chemical shift perturbation experiments indicated that the Tiam1 PDZ/Syndecan1 and PDZ/Caspr4 complexes were structurally distinct and identified key residues likely to be responsible for ligand selectivity. Moreover, cell biological analysis established that Syndecan1 is a physiological binding partner of Tiam1 and that the PDZ domain has a function in cell-matrix adhesion and cell migration. Collectively, our data provide insight into the structure, specificity, and function of the Tiam1 PDZ domain. Importantly, our data report on a physiological role for the Tiam1 PDZ domain and establish a novel link between two previously unrelated signal transduction pathways, both of which are implicated in cancer.

The use of side chains as catalytic cofactors for protein mediated redox chemistry raises significant mechanistic issues as to how these amino acids are activated toward radical chemistry in a controlled manner. De novo protein design has been used to examine the structural basis for the creation and maintenance of a tryptophanyl radical in a three-helix bundle protein maquette. Here we report the detailed structural analysis of the protein by multidimensional NMR methods. An interesting feature of the structure is an apparent pi-cation interaction involving the sole tryptophan and a lysine side chain. Hybrid density functional calculations support the notion that this interaction raises the reduction potential of the W degrees /WH redox pair and helps explain the redox characteristics of the protein. This model protein system therefore provides a powerful model for exploring the structural basis for controlled radical chemistry in protein.

The BID database is freely available at http://tsailab.org/BID/ To have your protein of interest entered, contact Tiffany Fischer (tiffbrink@neo.tamu.edu) or Jerry Tsai at the email below

Cytochrome b562 is a heme-binding protein consisting of four helices folded into a classic helix bundle motif. Though retaining much of the topology of the holoprotein, apocytochrome b562 displays physical features commonly associated with so-called protein molten globules. Here, the stability and dynamics of this "structured" molten globule are probed by examination of the dependence of its hydrogen exchange behavior upon the presence of a chemical denaturant. Compared to other systems studied in this manner, apocytochrome b562 displays a limited dynamic range of hydrogen exchange rates and the analysis required the development of a quantitative approach. The protein is found to have three regions of subglobal cooperative stability. The most stable region, or core, is composed of the central two helices of the bundle, with the N- and C-terminal helices being of independent and lower stability. The dependence of the global unfolding free energy upon denaturant concentration indicates the applicability of a binding model and explains the observed difference between global unfolding free energies obtained by the linear extrapolation method and those obtained by calorimetry and hydrogen exchange. These observations place a significant restraint upon the type of folding pathway that is operative for this protein and suggest that that the N- and C-terminal helices fold and unfold independently of the core of the molecule.