How Electrostatic Coupling Enables Conformational Plasticity in a Tyrosine Kinase

Protein kinases are important cellular signaling molecules involved in cancer and a multitude of other diseases. It is well-known that inactive kinases display a remarkable conformational plasticity; however, the molecular mechanisms remain poorly understood. Conformational heterogeneity presents an opportunity but also a challenge in kinase drug discovery. The ability to predictively model various conformational states could accelerate selective inhibitor design. Here we performed a proton-coupled molecular dynamics study to explore the conformational landscape of a c-Src kinase. Starting from a completely inactive structure, the simulations captured all major types of conformational states without the use of a target structure, mutation, or bias. The simulations allowed us to test the experimental hypotheses regarding the mechanism of DFG flip, its coupling to the αC-helix movement, and the formation of regulatory spine. Perhaps the most significant finding is how key titratable residues, such as DFG-Asp, αC-Glu, and HRD-Asp, change protonation states dependent on the DFG, αC, and activation loop conformations. Our data offer direct evidence to support a long-standing hypothesis that protonation of Asp favors the DFG-out state and explain why DFG flip is also possible in simulations with deprotonated Asp. The simulations also revealed intermediate states, among which a unique DFG-out/α-C state formed as DFG-Asp is moved into a back pocket forming a salt bridge with catalytic Lys, which can be tested in selective inhibitor design. Our finding of how proton coupling enables the remarkable conformational plasticity may shift the paradigm of computational studies of kinases which assume fixed protonation states. Understanding proton-coupled conformational dynamics may hold a key to further innovation in kinase drug discovery

Constant pH Molecular Dynamics Reveals pH-Modulated Binding of Two Small-Molecule BACE1 Inhibitors

Targeting β-secretase (BACE1) with small-molecule inhibitors offers a promising route for treatment of Alzheimer’s disease. However, the intricate pH dependence of BACE1 function and inhibitor efficacy has posed major challenges for structure-based drug design. Here we investigate two structurally similar BACE1 inhibitors that have dramatically different inhibitory activity using continuous constant pH molecular dynamics (CpHMD). At high pH, both inhibitors are stably bound to BACE1; however, within the enzyme active pH range, only the iminopyrimidinone-based inhibitor remains bound, while the aminothiazine-based inhibitor becomes partially dissociated following the loss of hydrogen bonding with the active site and change of the 10s loop conformation. The drastically lower activity of the second inhibitor is due to the protonation of a catalytic aspartate and the lack of a propyne tail. This work demonstrates that CpHMD can be used for screening pH-dependent binding profiles of small-molecule inhibitors, providing a new tool for structure-based drug design and optimization

Predicting Catalytic Proton Donors and Nucleophiles in Enzymes: How Adding Dynamics Helps Elucidate the Structure–Function Relationships

Despite the relevance of understanding structure–function relationships, robust prediction of proton donors and nucleophiles in enzyme active sites remains challenging. Here we tested three types of state-of-the-art computational methods to calculate the p<i>K</i>_a’s of the buried and hydrogen bonded catalytic dyads in five enzymes. We asked the question what determines the p<i>K</i>_a order, i.e., what makes a residue proton donor vs a nucleophile. The continuous constant pH molecular dynamics simulations captured the experimental p<i>K</i>_a orders and revealed that the negative nucleophile is stabilized by increased hydrogen bonding and solvent exposure as compared to the proton donor. Surprisingly, this simple trend is not apparent from crystal structures and the static structure-based calculations. While the generality of the findings awaits further testing via a larger set of data, they underscore the role of dynamics in bridging enzyme structures and functions

Mcl-1 expression is localized to the lysosomes, and forced Mcl-1 expression prevents ceramide- or etoposide-induced lysosomal-mitochondrial apoptosis.

<p>(A) Lysosomes and mitochondria were first isolated from untreated 10I or A549 cells. We used Western blotting to determine the expression of Mcl-1 in the different fractions of lysosome extracts. LAMP-1 and COX IV were detected as lysosomal and mitochondrial markers, respectively. Fractions obtained from sucrose-density gradient are lysosomes (<12% or <19%) and mitochondria (>27%). Total cell lysates were used as the positive control. (B) overexpression of human Mcl-1 in A549 cells. Mcl-1 expression was detected by Western blotting (top). β-actin served as an internal control. Cells treated with ceramide (25 μM) or etoposide (50 μM) for 24 h were stained with AO (second), rhodamine 123 (third), and PI (bottom), followed by flow cytometric analysis. The percentages of cells with LMP, MTP reduction, and apoptosis are shown (means ± S.D. of three individual experiments). *, <i>P</i> < 0.05 compared with the untreated group. #, <i>P</i> < 0.05 compared with the ceramide-treated or etoposide-treated group. (C) A schematic diagram of the pro-apoptotic role of GSK-3β in ceramide- or etoposide-induced lysosomal-mitochondrial axis-mediated apoptosis.</p

Ceramide or etoposide causes cathepsin D translocation followed by mitochondrial apoptosis.

<p>(A) 10I cell MTP reduction and apoptosis induced by 25 μM C₂-ceramide or 50 μM etoposide for 6 h were determined in the presence or absence of the cathepsin D inhibitor pepstatin A (25 μM) using rhodamine 123 (top) and PI (bottom) staining, respectively, followed by flow cytometric analysis (means ± S.D. of three individual experiments). A representative histogram is shown. *, <i>P</i> < 0.05 compared with the untreated group. #, <i>P</i> < 0.05 compared with the ceramide-treated or etoposide-treated group. (B) Blockage of ceramide- or etoposide-induced cell apoptosis by cathepsin D siRNA. A549 cells were transfected with siRNA (50 nM) against cathepsin D or a scrambled control. Cathepsin D expression was detected by Western blotting (left). β-actin served as an internal control. Transfected cells were treated with ceramide (25 μM) or etoposide (50 μM) for 24 h and stained with PI followed by flow cytometric analysis (right). The percentages of apoptotic cells are shown (means ± S.D. of three individual experiments). *, <i>P</i> < 0.05 compared with the untreated group. #, <i>P</i> < 0.05 compared with the ceramide-treated or etoposide-treated group. (C) A549 cells were treated with 25 μM C₂-ceramide or 50 μM etoposide for 24 h. After fixation, cells were incubated with a cathepsin D-specific antibody followed by an Alexa Fluor 488-labeled secondary antibody and DAPI nuclear staining. Cathepsin D translocation from lysosomes (characterized by punctate staining) to the cytoplasm (characterized by diffuse staining) is shown. The scale bar is 10 μm.</p

Inhibiting cathepsin D inhibits the ceramide- or etoposide-induced activation of caspase-8, but not caspase-2, and mitochondrial apoptosis.

<p>(A) 10I cells were treated with 25 μM C₂-ceramide or 50 μM etoposide for 6 h in the presence or absence of the cathepsin D inhibitor pepstatin A (25 μM). (B) A549 cells were treated with 25 μM C₂-ceramide for 24 h with or without cathepsin D siRNA (50 nM) pretreatment. (C) To further determine whether cathepsin D affects caspase-8 activation, recombinant human cathepsin D was incubated together with lysates from untreated A549 cells for 0.5 h at 37°C with or without 25 μM pepstatin A or 10 μM of the caspase-8 inhibitor z-IETD-fmk. We used enzymatic cleavage of the specific substrates benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-pNA and benzyloxycarbonyl-Ile-Glu(-OMe)-Thr-Asp(-OMe)-pNA to determine the activities of caspase-2 and caspase-8, respectively. OD, optical density. Data are given as the average of three individual experiments (means ± S.D.). *, <i>P</i> < 0.05 compared with the untreated group. #, <i>P</i> < 0.05 compared with ceramide-treated, etoposide-treated, or cathepsin D-treated group. We used Western blotting to determine the activation of caspase-2, caspase-8, truncated Bid (tBid), caspase-3, PARP, and cathepsin D. β-actin served as an internal control.</p

Ceramide or etoposide causes lysosomal destabilization and mitochondrial apoptosis.

<p>Mouse T hybridoma 10I cells were treated with 25 μM C₂-ceramide for the indicated time periods (A) or with different doses of C₂-ceramide (B) for 6 h. 10I cells and human epithelial A549 cells were treated with 25 μM C₂-ceramide or 50 μM etoposide for the indicated time periods (C). Using AO (top), rhodamine 123 (middle), and PI (bottom) staining, respectively, followed by flow cytometric analysis, the percentages of cells with lysosomal membrane permeabilization (LMP), the reduction of MTP (ΔΨ_m), and apoptosis are shown (means ± S.D. of three individual experiments). C₂-dihydroceramide was used as a negative control. A representative histogram obtained from 10I cells and shown in A. *, <i>P</i> < 0.05 compared with the untreated group.</p

Inhibiting caspase-2 or GSK-3β blocks ceramide- or etoposide-induced LMP and cathepsin D translocation.

<p>(A) 10I cells were treated with 25 μM C₂-ceramide or 50 μM etoposide for 6 h in the presence or absence of 10 μM of the caspase-2 inhibitor z-VDVAD-fmk or 10 mM of the GSK-3β inhibitor lithium chloride (LiCl). Using AO staining followed by flow cytometric analysis, the percentages of cells with LMP are shown (means ± S.D. of three individual experiments). A representative histogram is shown. *, <i>P</i> < 0.05 compared with the untreated group. #, <i>P</i> < 0.05 compared with the ceramide-treated or etoposide-treated group. (B) The translocation of cathepsin D in 25 μM C₂-ceramide- or 50 μM etoposide-treated A549 cells for 24 h was determined in the presence or absence of 10 μM z-VDVAD-fmk or 10 mM LiCl using a cathepsin D-specific antibody followed by an Alexa Fluor 488-labeled secondary antibody and DAPI nuclear staining. The scale bar is 10 μm. (C) Blockage of ceramide- or etoposide-induced LMP and apoptosis by knockdown of caspase-2 or GSK-3β expression. 10I cells were transfected with shRNAs (five clones in total) against caspase-2 (shCasp-2) or GSK-3β (shGSK-3β) or a negative-control shRNA (shLuc). Caspase-2 and GSK-3β expression was detected by Western blotting. β-actin served as an internal control. Ceramide (25 μM)- or etoposide (50 μM)-treated cells transfected with effective clones as indicated for 6 h were stained with AO or PI followed by flow cytometric analysis. The fold-increase of cells with LMP and the percentages of apoptotic cells are shown (means ± S.D. of three individual experiments). *, <i>P</i> < 0.05 compared with the untreated group. #, <i>P</i> < 0.05 compared with the ceramide-treated shLuc or etoposide-treated shLuc group.</p