Shedding Light on the Interaction of Human Anti-Apoptotic Bcl-2 Protein with Ligands through Biophysical and in Silico Studies

Abstract

Bcl-2 protein is involved in cell apoptosis and is considered an interesting target for anti-cancer therapy. The present study aims to understand the stability and conformational changes of Bcl-2 upon interaction with the inhibitor venetoclax, and to explore other drug-target regions. We combined biophysical and in silico approaches to understand the mechanism of ligand binding to Bcl-2. Thermal shift assay (TSA) and urea electrophoresis showed a significant increase in protein stability upon venetoclax incubation, which is corroborated by molecular docking and molecular dynamics simulations. An 18 °C shift in Bcl-2 melting temperature was observed in the TSA, corresponding to a binding affinity multiple times higher than that of any other reported Bcl-2 inhibitor. This protein-ligand interaction does not implicate alternations in protein conformation, as suggested by SAXS. Additionally, bioinformatics approaches were used to identify deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) of Bcl-2 and their impact on venetoclax binding, suggesting that venetoclax interaction is generally favored against these deleterious nsSNPs. Apart from the BH3 binding groove of Bcl-2, the flexible loop domain (FLD) also plays an important role in regulating the apoptotic process. High-throughput virtual screening (HTVS) identified 5 putative FLD inhibitors from the Zinc database, showing nanomolar affinity toward the FLD of Bcl-2.

Neutron reflectometry and NMR spectroscopy of full-length Bcl-2 protein reveal its membrane localization and conformation

“…Since all of these helical structures have affinities for the insides of hydrophobic membrane environments, to various degrees 29 , 42 , a location inside a lipid bilayer with only the loop region sticking out into the aqueous exterior is consistent with expectations. MD simulations comparing truncated versions with our physiologically intact Bcl-2 protein indicate that it is slightly less compact with the large flexible loop region, but still properly folded 36 .…”

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure

“…Moreover, the additives Gly-Gly-Gly ( T m of 59.72 °C), PEG3350 ( T m of 59.64 °C), DNA Library ( T m of 59.53 °C), Biotin ( T m of 59.31 °C), and TCEP ( T m of 59.29 °C) exhibit higher T m values than the control ( Figure 4 C). The observed slight positive melting temperature shift (Δ T m) might contribute to increase the protein stability reducing the respective conformational flexibility favoring further structural studies [ 57 , 58 ]. Hence, the referred additives could be considered promising candidates to be included in (i) the early stages of STEAP1 production and expression to promote a proper folding and to prevent aggregation, and (ii) the final protein buffer as putative crystallization additives.…”

“…Several other additives noticeably decreased the STEAP1 thermal stability, in particular Fos Choline 12 ( T m of 36.09 °C), K + Sulfate ( T m of 44.08 °C), Na + Phosphate (dibasic) ( T m of 46.60 °C), Mg 2+ Sulfate ( T m of 48.08 °C) and Na + Phosphate (monobasic) ( T m of 50.18 °C). These negative Δ T m values potentially indicate important structural changes towards a more disordered conformation or even protein misfolding [ 57 , 58 ]. A ranking of the best and the worst additives and their respective Tm is summed up in Figure 5 .…”

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

“…The effect of deuteration on reduced thermal stability and activity is noted. The structural analyses highlight subtle but important differences that are related to the decrease in the thermal stability of D-HEWL EC ; these differences are of significance for protein folding (Biter et al, 2016), enzymatic activity (Lea & Simeonov, 2012), crystallization (Geders et al, 2012;Reinhard et al, 2013) and protein-ligand interactions (Bai et al, 2019;Forneris et al, 2009;Holdgate et al, 2010;Ramos et al, 2019). The improved yield (by a factor of more than three compared with that found using P. pastoris) paves the way for a wide range of studies that can exploit H/D isotopic substitution in this protein.…”