Binding site plasticity in viral PPxY Late domain recognition by the third WW domain of human NEDD4

The recognition of PPxY viral Late domains by the third WW domain of the HECT-E3 ubiquitin ligase NEDD4 (hNEDD4-WW3) is essential for the completion of the budding process of numerous enveloped viruses, including Ebola, Marburg, HTLV1 or Rabies. hNEDD4-WW3 has been validated as a promising target for the development of novel host-oriented broad spectrum antivirals. Nonetheless, finding inhibitors with good properties as therapeutic agents remains a challenge since the key determinants of binding affinity and specificity are still poorly understood. We present here a detailed structural and thermodynamic study of the interactions of hNEDD4-WW3 with viral Late domains combining isothermal titration calorimetry, NMR structural determination and molecular dynamics simulations. Structural and energetic differences in Late domain recognition reveal a highly plastic hNEDD4-WW3 binding site that can accommodate PPxY-containing ligands with varying orientations. These orientations are mostly determined by specific conformations adopted by residues I859 and T866. Our results suggest a conformational selection mechanism, extensive to other WW domains, and highlight the functional relevance of hNEDD4-WW3 domain conformational flexibility at the binding interface, which emerges as a key element to consider in the search for potent and selective inhibitors of therapeutic interest.This research has been financed by grants BIO2009-13261-C02, BIO2012-39922-CO2 and BIO2016-78746-C2-1-R from the Spanish Ministry of Education and Science (I.L.) including AEI/FEDER EU funds, by CTQ2017-83810-R grant (F.J.B) and by BFU2014-53787-P, the IRB Barcelona and the BBVA Foundation (M.J.M)

Post-Translational Modifications Modulate Ligand Recognition by the Third PDZ Domain of the MAGUK Protein PSD-95

The relative promiscuity of hub proteins such as postsynaptic density protein-95 (PSD-95) can be achieved by alternative splicing, allosteric regulation, and post-translational modifications, the latter of which is the most efficient method of accelerating cellular responses to environmental changes in vivo. Here, a mutational approach was used to determine the impact of phosphorylation and succinimidation post-translational modifications on the binding affinity of the postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ3) domain of PSD-95. Molecular dynamics simulations revealed that the binding affinity of this domain is influenced by an interplay between salt-bridges linking the α3 helix, the β2–β3 loop and the positively charged Lys residues in its high-affinity hexapeptide ligand KKETAV. The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein. This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects. Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.This research was supported by grants CVI-05915, from the Andalusian Regional Government (http://www.juntadeandalucia.es), BIO2009-13261-C02 and BIO2012-39922-C02, from the Spanish Ministry of Science and Innovation (http://www.idi.mineco.gob.es/portal/site​/MICINN/) and FEDER. JMC received a postdoctoral contract from the Spanish Ministry of Science and Innovation. CCV was a recipient of a Formación de Personal Investigador fellowship from the Spanish Ministry of Science and Innovation

Two-state dynamics of the SH3–SH2 tandem of AbI kinase and the allosteric role of the N-cap

The regulation and localization of signaling enzymes is often mediated by accessory modular domains, which frequently function in tandems. The ability of these tandems to adopt multiple conformations is as important for proper regulation as the individual domain specificity. A paradigmatic example is Abl, a ubiquitous tyrosine kinase of significant pharmacological interest. SH3 and SH2 domains inhibit Abl by assembling onto the catalytic domain, allosterically clamping it in an inactive state. We investigate the dynamics of this SH3–SH2 tandem, using microsecond all-atom simulations and differential scanning calorimetry. Our results indicate that the Abl tandem is a two-state switch, alternating between the conformation observed in the structure of the autoinhibited enzyme and another configuration that is consistent with existing scattering data for an activated form. Intriguingly, we find that the latter is the most probable when the tandem is disengaged from the catalytic domain. Nevertheless, an amino acid stretch preceding the SH3 domain, the so-called N-cap, reshapes the free-energy landscape of the tandem and favors the interaction of this domain with the SH2-kinase linker, an intermediate step necessary for assembly of the autoinhibited complex. This allosteric effect arises from interactions between N-cap and the SH2 domain and SH3–SH2 connector, which involve a phosphorylation site. We also show that the SH3–SH2 connector plays a determinant role in the assembly equilibrium of Abl, because mutations thereof hinder the engagement of the SH2-kinase linker. These results provide a thermodynamic rationale for the involvement of N-cap and SH3–SH2 connector in Abl regulation and expand our understanding of the principles of modular domain organization

Peptide ligand freedom during MD simulations.

<p>The upper panels display peptide ligand (orange) freedom during MD simulations of the PDZ3/KKETAV (upper left panel) and P³⁹⁷-PDZ3/KKETAV (upper right panel) complexes. The lower panels display different moments of the MD simulations with the PDZ3/KKETAV (lower left panel) P³⁹⁷-PDZ3/KKETAV (lower right panel) complexes, showing the displacement of the KKETAV ligand towards phosphorylated Tyr397 residue.</p

Thermodynamic parameters of the interaction between PSD-95-PDZ3 and various ligands determined by ITC^a.

a<p>Examples of the ITC experiments are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090030#pone-0090030-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090030#pone.0090030.s001" target="_blank">S1</a>. The variability in the experimental values was estimated to be approximately 1% for the number of binding sites, 5% for the binding enthalpy, and 10% for the binding affinity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090030#pone.0090030-Palencia1" target="_blank">[29]</a>.</p>b<p>The experimental conditions were 50 mM potassium phosphate (pH 7.5) at 25°C.</p>c<p>The experimental conditions were 20 mM MES (pH 6.0) and 10 mM NaCl, at 25°C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090030#pone.0090030-Saro1" target="_blank">[19]</a>.</p>d<p>The experimental conditions were 50 mM potassium phosphate (pH 7.5) at 25°C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090030#pone.0090030-Chi1" target="_blank">[28]</a>.</p>e<p>The experimental conditions were 20 mM sodium phosphate (pH 6.8), 50 mM NaCl, and 1 mM EDTA, at 25°C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090030#pone.0090030-Zhang2" target="_blank">[11]</a>.</p

Frequencies of formation of some relevant salt-bridges after 400 ns of MD simulations of the PSD-95-PDZ3 constructs in complex with KKETAV.

<p>Frequencies of formation of some relevant salt-bridges after 400 ns of MD simulations of the PSD-95-PDZ3 constructs in complex with KKETAV.</p

The ‘supertertiary’ structure of PSD-95 protein.

<p>The ‘supertertiary’ structure of PSD-95 protein.</p

The structure and sequence of the PDZ3 domain of PSD-95.

<p>The panel on the upper left shows a structural representation of the PDZ3 domain of PSD-95 in complex with the hexapeptide KKETAV (orange), modelled from the X-ray structure of the PDZ3-CRIPT complex (Protein Data Bank ID: 1BE9). The α1, α2, and α3 helices are shown in green, light blue and blue, respectively. The β2–β3 loop is shown in red and the β2 chain is shown in yellow. The dashed line indicates the binding pocket. The panel on the upper right is a detailed view of the interface of the α3 helix at the C-terminus of PDZ3 showing the spatial arrangement of the Phe, Tyr, Asp, and Glu residues. The lower panel shows the sequence of the PDZ3 domain and its secondary structures. Numbering of the protein residues is relative to their positions in the full-length PSD-95 protein. Numbering of the KKETAV peptide residues is from 0 (C-terminal Val residue) to −5 (N-terminal Lys residue).</p

Different moments of the MD simulation of the PSD-95-PDZ3/KKETAV complex at pH 7.5.

<p>The upper panels show models of the interactions of Lys-4 in KKETAV (orange) with Glu331 and Glu373 in PSD-95-PDZ3. The lower panels show models of the interactions between Lys-5 in KKETAV and Glu331, Glu334, and Glu401 in PSD-95-PDZ3.</p

Calorimetric titration of the PSD-95-PDZ3 domain with the KKETAV ligand.

<p>Example of a calorimetric titration of KKETAV to PSD-95-PDZ3 at 25°C in 50 mM potassium phosphate (pH 7.5). The upper panel shows the net heat effects (after dilution subtraction) associated with the injection of KKETAV. The lower panel shows the ligand concentration dependence of the heat released upon binding after normalisation and correction for the heats of dilution. The trend line shows the best fit to a model considering one set of binding sites.</p