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. 2010 Nov 2;49(43):9280-91.
doi: 10.1021/bi101131f.

Crystallographic and nuclear magnetic resonance evaluation of the impact of peptide binding to the second PDZ domain of protein tyrosine phosphatase 1E

Jun Zhang  1 , Paul J Sapienza, Hengming Ke, Aram Chang, Sarah R Hengel, Huanchen Wang, George N Phillips, Andrew L Lee
Affiliations

Crystallographic and nuclear magnetic resonance evaluation of the impact of peptide binding to the second PDZ domain of protein tyrosine phosphatase 1E

Jun Zhang et al. Biochemistry. .

Abstract

PDZ (PSD95/Discs large/ZO-1) domains are ubiquitous protein interaction motifs found in scaffolding proteins involved in signal transduction. Despite the fact that many PDZ domains show a limited tendency to undergo structural change, the PDZ family has been associated with long-range communication and allostery. One of the PDZ domains studied most in terms of structure and biophysical properties is the second PDZ ("PDZ2") domain from protein tyrosine phosphatase 1E (PTP1E, also known as PTPL1). Previously, we showed through NMR relaxation studies that binding of the RA-GEF2 C-terminal peptide substrate results in long-range propagation of side-chain dynamic changes in human PDZ2 [Fuentes, E. J., et al. (2004) J. Mol. Biol. 335, 1105-1115]. Here, we present the first X-ray crystal structures of PDZ2 in the absence and presence of RA-GEF2 ligand, determined to resolutions of 1.65 and 1.3 Å, respectively. These structures deviate somewhat from previously determined NMR structures and indicate that very minor structural changes in PDZ2 accompany peptide binding. NMR residual dipolar couplings confirm the crystal structures to be accurate models of the time-averaged atomic coordinates of PDZ2. The impact on side-chain dynamics was further tested with a C-terminal peptide from APC, which showed results nearly identical to those of RA-GEF2. Thus, allosteric transmission in PDZ2 induced by peptide binding is conveyed purely and robustly by dynamics. (15)N relaxation dispersion measurements did not detect appreciable populations of a kinetic structural intermediate. Collectively, for ligand binding to PDZ2, these data support a lock-and-key binding model from a structural perspective and an allosteric model from a dynamical perspective, which together suggest a complex energy landscape for functional transitions within the ensemble.

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Figures

Fig. 1
Fig. 1
Cartoon representation of PDZ2 crystal structures. Apo (A) and RA-GEF2 bound (B) PDZ2 structures. Peptide is shown as stick model and electron density is shown as gray mesh. The density contour level is 1.5 σ. Peptide residues QVSAV have visible electron density. (C) RA-GEF2 and PDZ2 interaction network. RA-GEF2 peptide is shown by stick model and surrounding PDZ2 residues involved in peptide interaction are shown as lines. Bound water molecules involved in PDZ2 peptide interaction are shown as blue balls. The hydrogen bonds relevant to peptide binding are shown by yellow dotted lines. (D) Structural Superposition of apo (magenta) and RA-GEF2 bound PDZ2 (cyan). The RAGEF peptide is shown by green stick model. All structural graphics were prepared using PyMOL.
Fig. 2
Fig. 2
Methyl-bearing side-chain dynamics changes (ΔS2axis) induced by RA-GEF2 (A) and APC (B) binding, with respect to free PDZ2. The methyl groups with significant changes in S2axis (ΔS2axis>2σ) are shown in filled bars. Fig 2A was adapted from Fuentes et al. (16)
Fig. 3
Fig. 3
Graphical comparison of side-chain dynamic changes induced by RA-GEF2 (A) and APC binding (B). Red spheres represent residues experiencing significant (ΔS2axis>2σ) side-chain dynamic changes and peptide is shown as blue cartoon. The figures were prepared by PyMOL.
Fig. 4
Fig. 4
Two-state binding of RA-GEF2 and APC peptides based on 15N relaxation dispersion. Relaxation dispersion curves for select resonances in PDZ2 5% saturated with RA-GEF2 (A) and APC (B) peptides. Data acquired at 500 and 600 MHz (1H Larmor frequency) are shown in red and blue respectively. Data quality for these residues is typical of the entire dataset. In (C) and (D), correlation plots of fitted Δω values from relaxation dispersion and 15N Δω values from peptide titration. ΔωCPMG values are from global fits, as described in the main text. Data for RA-GEF2 and APC are in (C) and (D) respectively. The line is y = x.
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