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

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

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

Semi-supervised Learning Predicts Approximately One Third of the Alternative Splicing Isoforms as Functional Proteins

Alternative splicing acts on transcripts from almost all human multi-exon genes. Notwithstanding its ubiquity, fundamental ramifications of splicing on protein expression remain unresolved. The number and identity of spliced transcripts that form stably folded proteins remain the sources of considerable debate, due largely to low coverage of experimental methods and the resulting absence of negative data. We circumvent this issue by developing a semi-supervised learning algorithm, positive unlabeled learning for splicing elucidation (PULSE; http://www.kimlab.org/software/pulse), which uses 48 features spanning various categories. We validated its accuracy on sets of bona fide protein isoforms and directly on mass spectrometry (MS) spectra for an overall AU-ROC of 0.85. We predict that around 32% of “exon skipping” alternative splicing events produce stable proteins, suggesting that the process engenders a significant number of previously uncharacterized proteins. We also provide insights into the distribution of positive isoforms in various functional classes and into the structural effects of alternative splicing