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)

Structural basis of a redox-dependent conformational switch that regulates the stress kinase p38α

Many functional aspects of the protein kinase p38α have been illustrated by more than three hundred structures determined in the presence of reducing agents. These structures correspond to free forms and complexes with activators, substrates, and inhibitors. Here we report the conformation of an oxidized state with an intramolecular disulfide bond between Cys119 and Cys162 that is conserved in vertebrates. The structure of the oxidized state does not affect the conformation of the catalytic site, but alters the docking groove by partially unwinding and displacing the short αD helix due to the movement of Cys119 towards Cys162. The transition between oxidized and reduced conformations provides a mechanism for fine-tuning p38α activity as a function of redox conditions, beyond its activation loop phosphorylation. Moreover, the conformational equilibrium between these redox forms reveals an unexplored cleft for p38α inhibitor design that we describe in detail.© 2023. The Author(s)

Effect of Systemic Hypertension With Versus Without Left Ventricular Hypertrophy on the Progression of Atrial Fibrillation (from the Euro Heart Survey).

Hypertension is a risk factor for both progression of atrial fibrillation (AF) and development of AF-related complications, that is major adverse cardiac and cerebrovascular events (MACCE). It is unknown whether left ventricular hypertrophy (LVH) as a consequence of hypertension is also a risk factor for both these end points. We aimed to assess this in low-risk AF patients, also assessing gender-related differences. We included 799 patients from the Euro Heart Survey with nonvalvular AF and a baseline echocardiogram. Patients with and without hypertension were included. End points after 1 year were occurrence of AF progression, that is paroxysmal AF becoming persistent and/or permanent AF, and MACCE. Echocardiographic LVH was present in 33% of 379 hypertensive patients. AF progression after 1 year occurred in 10.2% of 373 patients with rhythm follow-up. In hypertensive patients with LVH, AF progression occurred more frequently as compared with hypertensive patients without LVH (23.3% vs 8.8%, p = 0.011). In hypertensive AF patients, LVH was the most important multivariably adjusted determinant of AF progression on multivariable logistic regression (odds ratio 4.84, 95% confidence interval 1.70 to 13.78, p = 0.003). This effect was only seen in male patients (27.5% vs 5.8%, p = 0.002), while in female hypertensive patients, no differences were found in AF progression rates regarding the presence or absence of LVH (15.2% vs 15.0%, p = 0.999). No differences were seen in MACCE for hypertensive patients with and without LVH. In conclusion, in men with hypertension, LVH is associated with AF progression. This association seems to be absent in hypertensive women

Progression From Paroxysmal to Persistent Atrial Fibrillation. Clinical Correlates and Prognosis

Objectives: We investigated clinical correlates of atrial fibrillation (AF) progression and evaluated the prognosis of patients demonstrating AF progression in a large population. Background: Progression of paroxysmal AF to more sustained forms is frequently seen. However, not all patients will progress to persistent AF. Methods: We included 1,219 patients with paroxysmal AF who participated in the Euro Heart Survey on AF and had a known rhythm status at follow-up. Patients who experienced AF progression after 1 year of follow-up were identified. Results: Progression of AF occurred in 178 (15%) patients. Multivariate analysis showed that heart failure, age, previous transient ischemic attack or stroke, chronic obstructive pulmonary disease, and hypertension were the only independent predictors of AF progression. Using the regression coefficient as a benchmark, we calculated the HATCH score. Nearly 50% of the patients with a HATCH score >5 progressed to persistent AF compared with only 6% of the patients with a HATCH score of 0. During follow-up, patients with AF progression were more often admitted to the hospital and had more major adverse cardiovascular events. Conclusions: A substantial number of patients progress to sustained AF within 1 year. The clinical outcome of these patients regarding hospital admissions and major adverse cardiovascular events was worse compared with patients demonstrating no AF progression. Factors known to cause atrial structural remodeling (age and underlying heart disease) were independent predictors of AF progression. The HATCH score may help to identify patients who are likely to progress to sustained forms of AF in the near future. \ua9 2010 American College of Cardiology Foundation

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

The structure of Prp40 FF1 domain and its interaction with the crn-TPR1 motif of Clf1 gives a new insight into the binding mode of FF domains.

The yeast splicing factor Prp40 (pre-mRNA processing protein 40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5' end of U1 small nuclear RNA and to interact directly with two proteins, the Clf1 (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). In this work we reported the solution structure of the first FF domain of Prp40 and the identification of a novel ligand-binding site in FF domains. By using chemical shift assays, we found a binding site for the N-terminal crooked neck tetratricopeptide repeat of Clf1 that is distinct and structurally separate from the previously identified CTD-RNAPII binding pocket of the FBP11 (formin-binding protein 11) FF1 domain. No interaction, however, was observed between the Prp40 FF1 domain and three different peptides derived from the CTD-RNAPII protein. Indeed, the equivalent CTD-RNAPII-binding site in the Prp40 FF1 domain is predominantly negatively charged and thus unfavorable for an interaction with phosphorylated peptide sequences. Sequence alignments and phylogenetic tree reconstructions using the FF domains of three functionally related proteins, Prp40, FBP11, and CA150, revealed that Prp40 and FBP11 are not orthologous proteins and supported the different ligand specificities shown by their respective FF1 domains. Our results also revealed that not all FF domains in Prp40 are functionally equivalent. We proposed that at least two different interaction surfaces exist in FF domains that have evolved to recognize distinct binding motifs

The structure of Prp40 FF1 domain and its interaction with the crn-TPR1 motif of Clf1 gives a new insight into the binding mode of FF domains.

The yeast splicing factor Prp40 (pre-mRNA processing protein 40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5' end of U1 small nuclear RNA and to interact directly with two proteins, the Clf1 (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). In this work we reported the solution structure of the first FF domain of Prp40 and the identification of a novel ligand-binding site in FF domains. By using chemical shift assays, we found a binding site for the N-terminal crooked neck tetratricopeptide repeat of Clf1 that is distinct and structurally separate from the previously identified CTD-RNAPII binding pocket of the FBP11 (formin-binding protein 11) FF1 domain. No interaction, however, was observed between the Prp40 FF1 domain and three different peptides derived from the CTD-RNAPII protein. Indeed, the equivalent CTD-RNAPII-binding site in the Prp40 FF1 domain is predominantly negatively charged and thus unfavorable for an interaction with phosphorylated peptide sequences. Sequence alignments and phylogenetic tree reconstructions using the FF domains of three functionally related proteins, Prp40, FBP11, and CA150, revealed that Prp40 and FBP11 are not orthologous proteins and supported the different ligand specificities shown by their respective FF1 domains. Our results also revealed that not all FF domains in Prp40 are functionally equivalent. We proposed that at least two different interaction surfaces exist in FF domains that have evolved to recognize distinct binding motifs