Phage display identification of nanomolar ligands for human NEDD4-WW3: Energetic and dynamic implications for the development of broad-spectrum antivirals

This research has been financed by grants BIO2016-78746-C2-1-R and PID2020-112895RB-100 from the Spanish Ministry of Education and Science (I.L.) including AEI/FEDER EU funds. R.N.H. was funded in part by National Institutes of Health grants AI138052 and AI138630. M. I.B. and J.M.C. were recipients of a research contract from the Spanish Ministry of Education and Science. F.C. was funded by a predoctoral fellowship from the Andalusian Government P10-CVI-5915. J.M.C. ac-knowledges a reincorporation research contract from the University of Granada. We thank Dr. Sachdev Sidhu for his invaluable assistance setting up the phage display techniques in our laboratory. We also thank the support of the C.I.C. of the University of Granada.The recognition of PPxY viral Late domains by the third WW domain of the human HECT-E3 ubiquitin ligase NEDD4 (NEDD4-WW3) is essential for the budding of many viruses. Blocking these interactions is a promising strategy to develop broad-spectrum antivirals. As all WW domains, NEDD4-WW3 is a challenging therapeutic target due to the low binding affinity of its natural interactions, its high conformational plasticity, and its complex thermodynamic behavior. In this work, we set out to investigate whether high affinity can be achieved for monovalent ligands binding to the isolated NEDD4-WW3 domain. We show that a competitive phage-display set-up allows for the identification of high-affinity peptides showing inhibitory activity of viral budding. A detailed biophysical study combining calorimetry, nuclear magnetic resonance, and molecular dynamic simulations reveals that the improvement in binding affinity does not arise from the establishment of new interactions with the domain, but is associated to conformational restrictions imposed by a novel C-terminal -LFP motif in the ligand, unprecedented in the PPxY interactome. These results, which highlight the complexity of WW domain interactions, provide valuable insight into the key elements for high binding affinity, of interest to guide virtual screening campaigns for the identification of novel therapeutics targeting NEDD4-WW3 interactions.Spanish Government BIO2016-78746-C2-1-R PID2020-112895RB-100 AEI/FEDER EU funds AI138052 AI138630United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA P10-CVI-5915German Research Foundation (DFG)University of Granad

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

Motif mediated protein-protein interactions as drug targets

Abstract Protein-protein interactions (PPI) are involved in virtually every cellular process and thus represent an attractive target for therapeutic interventions. A significant number of protein interactions are frequently formed between globular domains and short linear peptide motifs (DMI). Targeting these DMIs has proven challenging and classical approaches to inhibiting such interactions with small molecules have had limited success. However, recent new approaches have led to the discovery of potent inhibitors, some of them, such as Obatoclax, ABT-199, AEG-40826 and SAH-p53-8 are likely to become approved drugs. These novel inhibitors belong to a wide range of different molecule classes, ranging from small molecules to peptidomimetics and biologicals. This article reviews the main reasons for limited success in targeting PPIs, discusses how successful approaches overcome these obstacles to discovery promising inhibitors for human protein double minute 2 (HDM2), B-cell lymphoma 2 (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP), and provides a summary of the promising approaches currently in development that indicate the future potential of PPI inhibitors in drug discovery

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

The ‘supertertiary’ structure of PSD-95 protein.

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