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Link to original content: http://www.ncbi.nlm.nih.gov/pubmed/19828436
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. 2009 Oct 27;106(43):18249-54.
doi: 10.1073/pnas.0904492106. Epub 2009 Oct 14.

Hidden dynamic allostery in a PDZ domain

Chad M Petit  1 , Jun Zhang, Paul J Sapienza, Ernesto J Fuentes, Andrew L Lee
Affiliations

Hidden dynamic allostery in a PDZ domain

Chad M Petit et al. Proc Natl Acad Sci U S A. .

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 (alpha3) that packs in contiguous fashion against the globular domain. Although alpha3 lies outside the active site and does not make direct contact with C-terminal peptide ligand, removal of alpha3 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 alpha3, 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 alpha3 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 alpha3, 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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structure of PSD-95/SAP90 PDZ3 complexed with CRIPT peptide. The α3 helix is shown in blue. The van der Waals surface of the CRIPT peptide is in purple, and the nearest approaching residues (Tyr-397 and Phe-400) of α3 are in cyan.
Fig. 2.
Fig. 2.
1H-15N HSQC spectrum of PDZ3303–402 (black) overlain with the 1H-15N HSQC spectrum of Δ7ct (red). Resonances with 1H-15N assignments for deleted residues 396–402 are indicated by arrows.
Fig. 3.
Fig. 3.
The α3 helix affects CRIPT peptide binding. ITC data for binding of PSD-95/SAP90 PDZ3303–402 (A) and Δ7ct (B) to C-terminal 9 residues of CRIPT (Ac-TKNYKQTSV-COOH) at 25 °C. Thermograms and integrated titration curves are shown in the top and bottom panels, respectively. Heats of dilution were obtained from independent buffer–buffer titration experiments (see Materials and Methods).
Fig. 4.
Fig. 4.
α3 helix-dependent changes in backbone and side-chain order parameters. (A) Backbone HN groups (ΔS2) as determined from 15N relaxation. (B) Side-chain methyl groups (ΔS2axis) as determined from 2H relaxation. S2axis values for PDZ3303–402 were previously reported by Law et al. (44).
Fig. 5.
Fig. 5.
Methyl side-chain dynamics of PDZ3303–402 as a function of removal of α3 (left to right) and CRIPT peptide binding (top to bottom). Cα atoms of the corresponding methyl-bearing residues are color-coded according to S2axis parameters: yellow, 0 ≤ S2axis ≤ 0.4; orange, 0.4 < S2axis < 0.7; red, 0.7 ≤ S2axis ≤ 1. Average S2axis values for each state are indicated by green bars.
Fig. 6.
Fig. 6.
Chemical shift perturbations in CRIPT 7-mer peptide (Ac-NYKQTSV-COOH) on addition of 7.5% and 15% molar ratios of PDZ3303–402 to peptide. Shift perturbations were observed from natural abundance 1H-13C HSQC spectra. The asterisk denotes a lack of chemical shift data due to line broadening. Δδ is a weighted vector combination of 1H and 13C chemical shift perturbations, as described in Materials and Methods.
Fig. 7.
Fig. 7.
Chemical shift perturbations of PDZ3 on peptide binding. (A) Combined 1H and 15N perturbations (defined in text) are shown for PDZ3303–402 and Δ7ct constructs. Residues that make up the binding pocket are shaded in blue. (B) Chemical shift perturbations from (A) mapped onto the structure. Residues are color coded as follows: yellow, 0 ppm ≤ Δδ ≤ 0.2 ppm; orange, 0.2 ppm < Δδ < 0.5 ppm; red, 0.5 ppm ≤ Δδ.
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