New Conformational State of NHERF1-CXCR2 Signaling Complex Captured by Crystal Lattice Trapping

<div><p>NHERF1 is a PDZ adaptor protein that scaffolds the assembly of diverse signaling complexes and has been implicated in many cancers. However, little is known about the mechanism responsible for its scaffolding promiscuity or its ability to bind to multiple targets. Computational studies have indicated that PDZ promiscuity may be attributed to its conformational dynamics, but experimental evidence for this relationship remains very limited. Here we examine the conformational flexibility of the NHERF1 PDZ1 domain using crystal lattice trapping via solving PDZ1 structure of a new crystal form. The structure, together with prior PDZ1 structures of a different space group, reveals that 4 of 11 ligand-interacting residues undergo significant crystal packing-induced structural changes. Most of these residues correspond to the residues involved in allosteric transition when a peptide ligand binds. In addition, a subtle difference in ligand conformations causes the same peptide to bind in slightly different modes in different crystal forms. These findings indicate that substantial structural flexibility is present in the PDZ1 peptide-binding pocket, and the structural substate trapped in the present crystal form can be utilized to represent the conformational space accessible to the protein. Such knowledge will be critical for drug design against the NHERF1 PDZ1 domain, highlighting the continued need for experimentally determined PDZ1-ligand complexes.</p></div

Structural comparison of PDZ domains.

<p>(A) Superposition of the structures of PDZ1-CXCR2 (purple; PDB code: 4JL7), PDZ1-CFTR (orange; PDB code: 1I92) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076219#B12" target="_blank">12</a>], PDZ1-2AR (cyan; PDB code: 1GQ4) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076219#B13" target="_blank">13</a>], and PDZ1-PDGFR (yellow; PDB code: 1GQ5) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076219#B13" target="_blank">13</a>]. PDZ domains are represented by ribbon, while residues in the ligands are displayed as sticks. (B) Superposition of the PDZ1 ligand binding pockets. Both PDZ1 and ligand residues are depicted by sticks and colored according to the scheme in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076219#pone-0076219-g003" target="_blank">Figure 3A</a>. (C) Close-up views of structural differences of His29 (top) and Arg40 (bottom). The CXCR2 peptide is depicted by sticks overlaid with 2Fo − Fc omit map calculated at 1.16 Å and contoured at 2.0 σ. (D) Superposition of NHERF1 PDZ1 (purple) and PDZ2 (pink; PDB code: 2OZF) peptide binding pockets. CXCR2 peptide is shown in green and PDZ residues are depicted by balls-and-sticks.</p

Structural similarities of two crystal forms.

<p>(A) Ribbon view of overall <i>P</i>2₁-PDZ1 structure. PDZ1 is shown in green and the CXCR2 peptide shown in blue. Secondary structures of PDZ1, α-helices and β-strands, are labeled and numbered according to their position in the sequence. (B) Superposition of <i>P</i>2₁-PDZ1 (green) and <i>P</i>3₁21-PDZ1 (magenta). (C) Stereo view of the PDZ1-CXCR2 interaction in <i>P</i>2₁-PDZ1. The PDZ1 residues are represented by sticks with their carbon atoms colored in green. The CXCR2 peptide is depicted by sticks overlaid with 2F_o−F_c omit map calculated at 1.1 Å and contoured at 1.5 σ. Hydrogen bonds are illustrated as orange broken lines. (D) Superposition of the Leu0 and (E) Thr-1 recognition regions. Both <i>P</i>2₁-PDZ1 and <i>P</i>3₁21-PDZ1 are depicted by sticks and colored according to the scheme in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081904#pone-0081904-g002" target="_blank">Figure 2B and 2C</a>.</p

Different Arg40 conformations of two crystal forms.

<p>(A) Overall view of conformational differences in the peptide-binding pocket. <i>P</i>2₁-PDZ1 and <i>P</i>3₁21-PDZ1 are superimposed and colored according to the scheme in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081904#pone-0081904-g002" target="_blank">Figure 2</a>. (B) Arg40 crystal contacts in <i>P</i>2₁-PDZ1 (top) and in <i>P</i>3₁21-PDZ1 (bottom). Symmetry-related molecules are represented by ribbons and sticks with their carbon atoms colored in cyan. (C) Thermal ellipsoid representation of Arg40 of <i>P</i>2₁-PDZ1 (top) and <i>P</i>3₁21-PDZ1 (bottom). Carbon atoms are colored gray, nitrogen atoms blue, and oxygen atoms red. Thermal ellipsoids are contoured at the 50% probability level.</p

Structure of NHERF1 PDZ1 in complex with the CXCR2 C-terminal sequence TSTTL.

<p>(A) Ribbon diagram of the PDZ1-CXCR2 structure, front view on the left and side view on the right. PDZ1 is shown in purple and the CXCR2 peptide shown in green. Secondary structures of PDZ1, α-helices and β-strands, are labeled and numbered according to their position in the sequence. Side chains of putative PDZ1 lipid-binding residues are depicted by balls-and-sticks in the side view of the structure. (B) Sequence alignment of selected PDZ domains. The alignment was performed by ClustalW [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076219#B45" target="_blank">45</a>], including human NHERF1, human NHERF2 and mouse PDZK1. Identical residues are shown as white on black, and similar residues appear shaded in cyan. Secondary structure elements are displayed above the sequences and labeled according to the scheme in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076219#pone-0076219-g001" target="_blank">Figure 1A</a>. Sequence numbering is displayed to the left of the sequences, with every 10th residue marked by a dot shown above the alignment. (C) Sequence alignment of the last five residues of natural NHERF binding targets. The alignment includes CXCR2, CFTR, 2AR, PDGFR, PTHR, Npt2a (type 2 sodium-phosphate cotransporter), purinergic receptor P2Y1, CCR5 (C-C chemokine receptor type 5), and AQP9 (aquaporin 9). Protein names are shown at the left of the sequences. Position numbering is displayed above the alignment, with position 0 referring to the very C-terminal residue.</p

Isotropic B-factor and anisotropy.

a<p>Numbers in parentheses refer to standard deviation.</p>b<p>The mean anisotropy <i>A</i> is defined as the ratio of the smallest to the largest eigenvalue of <i>U</i>, where <i>U</i> is ADP tensor.</p>c<p>Ser-3∶1 conformation 1; Ser-3∶2 conformation 2.</p

Crystal packing differences between two crystal forms.

<p>(A) Section of the crystal lattice of <i>P</i>3₁21-PDZ1 and (B) <i>P</i>2₁-PDZ1. The unit cell is shown as a red box, with the origin and axes labeled. PDZ1 is shown as a Cα trace, with red standing for a reference molecule and green the symmetry-related molecules. (C) Surface representation of crystal contacts around the ligand-binding site of <i>P</i>3₁21-PDZ1 and (D) <i>P</i>2₁-PDZ1. The surface is colored in blue if the distance to symmetry-related molecules is 3.5 Å or less and is colored in green otherwise. PDZ1 is depicted as ribbon and the bound CXCR2 peptide is labeled and represented by sticks.</p

Distinct modes of CXCR2 peptide interaction.

<p>(A) Double conformation of Ser-3 in <i>P</i>2₁-PDZ1. Residues of the CXCR2 peptide are represented by sticks with their carbon atoms colored in blue. PDZ1 residues are shown in green, while its symmetry-related residue is shown in cyan. Orange broken lines depict hydrogen bonds, and the labels I and II indicate individual conformers of Ser-3 double conformation. (B) Superposition of the CXCR2 peptides of <i>P</i>2₁-PDZ1 (blue) and <i>P</i>3₁21-PDZ1 (magenta). (C) Ser-3 crystal contacts in <i>P</i>3₁21-PDZ1. (D) Thermal ellipsoid representation of Ser-3 interaction in <i>P</i>2₁-PDZ1 (top) and <i>P</i>3₁21-PDZ1 (bottom).</p